Biological / chemical analysis device and method with simplified steps, small sample, rapidity and ease of use

Abstract

The present invention relates to the fields of biological / chemical sampling, sensing, detection and application. Specifically, the present invention relates to how to make sampling / detection / determination simple to use, or to obtain results quickly, or to be highly sensitive, or to be easy to use, or to use a tiny sample volume, such as 0.5 microliters or less, or to not require any professional operation, or to be read by a mobile phone, or to be low-cost, or a combination thereof.

Description

[0001] Cross-references

[0002] This application is a divisional application filed based on the following original application:

[0003] Original application: Application number 201680056780.9; Application date August 10, 2016; Name of invention: Simplified steps, small sample, fast and easy-to-use biological / chemical analysis device and method; Authorization notice issuance date: February 4, 2021.

[0004] This application claims the benefit of the following provisional applications as its parent, Serial Nos. 62 / 202,989, filed on August 10, 2015, 62 / 218,455 filed on September 14, 2015, 62 / 293,188 filed on February 9, 2016, 62 / 305,123 filed on March 8, 2016, and 62 / 369,181 filed on July 31, 2016, all of which are hereby incorporated by reference into the present invention for all purposes. Field of the Invention

[0005] The present invention relates to the fields of biological / chemical sampling, sensing, detection and application.

[0006] summary

[0007] The present invention relates to the fields of biological / chemical sampling, sensing, detection and application. Specifically, the present invention relates to how to make sampling / detection / determination simple to use, or quickly obtain results, or highly sensitive, or easy to use, or use a tiny sample amount (e.g., 0.5 microliters or less), or do not require any professional operation, or read the results by a mobile phone, or low cost, or a combination thereof. Background Art

[0008] In many biological / chemical sensing and detection (e.g., immunoassays, nucleotide assays, blood cell counts, etc.), chemical reactions, and other processes, there is a need for methods and devices that can speed up the process (e.g., binding reactions, reagent mixing, etc.) while quantifying various parameters (e.g., analyte concentration, sample volume, etc.), simplify the sample collection and measurement process, process small volume samples, allow a complete assay to be completed in less than a minute, allow testing via smartphones (e.g., mobile phones, etc.), allow non-professionals to perform self-tests, and allow test results to be communicated to relevant parties by local communication, remote communication, or wireless network methods, etc. The methods, devices, and systems of the present invention address these needs. Summary of the invention

[0009] The following brief overview is not intended to include all features and aspects of the present invention. The methods, devices and systems of the present invention make biological / chemical sensing (including, but not limited to, immunoassays, nucleic acid detection, electrolyte analysis, etc.) faster, more sensitive, require fewer steps, easier to implement, require less sample volume, reduce (or even eliminate) the need for professional assistance, and / or consume lower costs than many current detection methods and devices.

[0010] The goal of many current assays is to accurately determine the absolute concentration of an analyte in a sample. For example, a red blood cell count assay involves measuring the number of red blood cells in a volume of whole blood and then calculating the number of red blood cells per microliter of whole blood. However, such measurements can be challenging if not performed in a specialized testing center (i.e., a "home test," "pharmacy test," or "point of care test" environment) because such tests typically require specialized instrumentation and / or precise measuring devices that can accurately measure relatively small volumes of biological fluid samples (such as a precise pipette or the like).

[0011] Measurement of relevant volumes

[0012] Many assays are able to provide the absolute concentration of an analyte in a sample. However, when only a small volume of sample (e.g., 100 nanoliters to 10 microliters, for example) is analyzed, the results of these assays become quite inaccurate. This is because small volume samples are difficult to handle and / or measure accurately.

[0013] In some assays, a liquid sample is placed between two plate structures separated by a spacer for analysis. In theory, the volume of the sample being analyzed can be calculated by multiplying the thickness of the sample being analyzed by the area of ​​the sample being analyzed. In practice, however, such estimates are not easy to obtain and can be quite inaccurate for a variety of reasons. For example, some devices use beads as spacer structures, and either the beads or the plates are deformable. Such devices may tend to be inaccurate for the following reasons:

[0014] There is only a much smaller contact area (almost a point) between the spherical spacer and the plate structure. In such a device, because of this much smaller contact area, for each unit of compressive force applied, there will be a much larger pressure acting on the contact surface of both the plate structure and the spherical spacer. This much larger pressure causes the spherical spacer and / or the plate structure (if they are flexible structures) to deform, thus corrupting any measurement.

[0015] Spherical spacers usually end up in a random distribution between the two plates. Because the spherical spacers are randomly distributed, the spacing between the spacers varies greatly, and some of the spacings are quite large. This causes the spacers and / or the plate structure (if they are flexible) to deform more in some areas than in other areas, which also corrupts the measurement results.

[0016] • Randomly placed spacers, if too close together, may act as obstacles blocking the movement of analytes (eg, cells), potentially creating "clumps" of analytes or cells, causing further difficulties.

[0017] Significant deformation of one of the plate structures may result in cell lysis, which may lead to errors in cell counting.

[0018] • Because the number of spherical spacers in the analyzed area and the degree to which one of the spacers and / or the plate structure is deformed is not certain, the calculation of the volume will be inaccurate.

[0019] • The time required for the deformation to cause the molecules to diffuse to the surface of one of the plate structures is not constant.

[0020] For devices using spherical spacers, the volume of the sample portion being analyzed may be estimated by a) counting the spheres in the sample portion being analyzed; b) estimating the thickness of the sample layer by experiment (e.g., adding an internal standard, such as an immiscible liquid containing a known concentration of a calibrant, to calculate the distance between the plates). However, the additional steps are inconvenient to perform, and measurements obtained with such devices are still not very precise due to significant deformation of the top plate and / or spacers during use.

[0021] In contrast, some embodiments of the methods and apparatus provided by the present invention rely on spacers of generally uniform height, nearly uniform cross-section, and planar (i.e., "flat") tops affixed in a regular pattern to one or more plate structures, wherein the spacers are spaced at a consistent, predetermined distance from one another (i.e., not randomly placed in a Poisson distribution). During use of some embodiments of the methods and apparatus of the present invention, the spacers and plate structures are not significantly compressed or deformed in any dimension, at least when the plate structures are in a closed configuration and drawn together by capillary forces. The apparatus may have a number of advantages, in that the volume of the data acquisition portion of the sample (i.e., the "volume of interest" or the portion of the sample being analyzed) may be easily and very accurately calculated when the apparatus of the present invention is used, and in some cases may even be calculated prior to the assay being performed, even if the sample is placed in the apparatus in unknown quantities. Because, in the closed configuration, the plate structures are substantially flat (meaning that the thickness of the sample is uniform) and the number and size of the spacers in the analysis region are known, the volume of the region in the sample may be easily and very accurately calculated. The volume of the sample volume of interest can be determined without counting spacers or estimating the sample layer thickness. It is also necessary to place a specific amount of sample in the device. In addition, at the beginning of the assay incubation, the analyte molecules should be evenly distributed throughout the volume of interest (within the limits allowed by Poisson statistics) rather than relatively concentrated in certain areas.

[0022] Shortened reaction time

[0023] It is known that the diffusion constant of many analytes in aqueous environments is very low, and, therefore, many assays require lengthy incubation times (usually several hours, in some cases, even days), require stirring and use of agents that promote or force mixing. Such assays are designed so as to allow the analyte to diffuse laterally from an initial position to a plate structure on a remote target (see, for example, Wei et al, Nucl. Acids Res. 33: e78 and Toegl et al, J. Biomol. Tech. 2003 14: 197–204). Such systems have great limitations because they may take several hours to obtain results. In addition, even if the results are obtained, it is often difficult to determine with certainty whether the reaction has reached a reaction equilibrium when it is terminated. This uncertainty, along with other matters, makes it impossible to estimate the absolute concentration of the analyte in the sample.

[0024] As will be explained in more detail below, in some embodiments of the present methods and apparatus, the height of the spacer and the assay endpoint can be selected to limit the amount of lateral diffusion of the analyte during the assay. In these cases, such assays (typically binding assays) can be run in a very short time. In addition, the concentration of the analyte in the sample can be estimated very accurately, even though the entire sample may not be analyzed in its entirety, or may have an unknown volume.

[0025] In these embodiments, an assay may be stopped, and / or assay results read, at 1) a time equal to or longer than the time required for the target analyte to diffuse across the thickness of the uniform layer in the closed configuration (i.e., no shorter than the time required for the target analyte to diffuse longitudinally from one plate to the other); and 2) a time shorter than the time required for the target analyte to diffuse transversely across the linear dimension of a given binding site area (i.e., shorter than the time required for the target analyte to diffuse transversely from one side of the binding site to the other). In such a "local binding" configuration, the volume of sample for which data is acquired (the "volume of interest") can be estimated with reasonable accuracy because it is the volume of the sample immediately above the analysis area. In fact, the volume of sample for which data is acquired is known prior to initiating the assay. Such "local binding" embodiments also have the advantage that the sample, and possibly any detection reagent, is compressed into a thin layer over the binding sites, and therefore any binding between analyte and / or detection reagent should reach equilibrium more quickly than in embodiments where the sample is not compressed into a thin layer, for example, if a drop of sample is simply placed on the plate with the binding sites. As such, in many cases binding equilibrium can be reached in seconds rather than minutes, and thus many assays, particularly binding assays, can be completed very rapidly, e.g., in less than a minute.

[0026] Multiple parallel

[0027] In addition, the "local binding" configuration allows one to perform multiple parallel assays without the need for fluid isolation of different reactions. In other words, multiple assays can be performed in an open environment without being separated from each other by walls (i.e., without fluid isolation). For example, in a local binding embodiment, two different analyses can be performed side by side in the same sample simultaneously because the assay for the analyte has been stopped and / or the assay results have been read before diffusion from one assay area to another, and the absolute concentrations of the analytes in the sample can be determined separately from each other, even if they are not isolated from each other by fluid.

[0028] By simply sandwiching the sample between two plate structures and performing the assay in a diffusion-restricted manner, multiple assays can be performed on a single sample without fluid isolation, which has many advantages. For example, these assays can be performed by simply placing a few drops of an unknown amount of sample (e.g., blood), pressing the plate structures together to spread the sample across the plate, incubating the sample reaction for a certain period of time, and reading data from multiple stations of the device. When implementing the method, there is no need to transfer a specified amount of sample to multiple chambers, which is difficult to achieve without precise fluid transfer and / or measurement devices. In addition, the method is very fast for the reasons mentioned above. In addition, since the plate structures here do not require the production of "walls", the manufacture of the device is simple and easy. Finally, there is no requirement for ports for any of the plate structures, that is, ports that may be used to add or remove samples or reagents when the device is in a closed phase.

[0029] Enhanced Surface

[0030] Additionally, in some embodiments of the present devices and methods, the device may include an "enhanced surface," e.g., a surface that enhances a signal, e.g., fluorescence or cold light emitted by a detection agent. In some cases, the signal may be enhanced by nanoplasmon effects (e.g., surface enhanced Raman scattering). Embodiments of signal enhancement by an enhanced surface are described in, e.g., Li et al, Optics Express 2011 19:3925-3936 and patent application WO2012 / 024006, which are incorporated herein by reference. In some cases, the enhanced surface may be a "disk affinity post dot antenna array" (D2PA), which is described in U.S. Pat. No. 9,013,690. In use, a device comprising an enhanced surface may be able to enhance the signal by 10% relative to a device not configured for signal enhancement. 3 times or more, thereby allowing the analyte to be detected with very high sensitivity. In some embodiments, the amount of analyte in the sample-related volume, especially non-cellular analytes detected using sandwich methods, can be digitally counted using the methods described in WO2014144133. The use of enhanced surfaces, in some cases, allows the reading of the assay using a smartphone or the like.

[0031] Other Features

[0032] In embodiments of the present apparatus, spacers affixed to one or more plates are not capable of changing position or being washed away if the plates are immersed in an aqueous environment. The spacers are not spherical and they are not affixed to the surface of the plates via weak forces, such as electrostatic forces, gravity, etc. In some embodiments, the plates with spacers may be a single piece. In many embodiments, the spacers are not prefabricated and affixed to the plates (e.g., glued or the like). Instead, the spacers may be grown and / or etched from the plates using embossing and / or micromachining (e.g., photolithography) processes.

[0033] The parameters of the spacers (e.g., their cross-section, spacing, and density, etc.) can be optimized so that in the closed phase, the top plate (which can be flexible) does not deform significantly above the portion of the sample being analyzed (the "relevant volume" of the sample). In some cases, the parameters of the spacers can be adjusted depending on the flexibility of the top plate. For example, if the top plate is more flexible, the spacers may be closer. Similarly, if the top plate is not flexible enough, the spacers can be further apart.

[0034] Furthermore, in many embodiments using the present device, the analyte migrates through the device in a non-directional manner after the device is closed. Thus, in the closed phase, there is no need for sorting or fractionation of the analyte, and the analyte is forced to flow through the device in a non-directional manner (e.g., by gravity or electrophoresis), as described in Austin (US 6632652). In many cases, it is not necessary to connect the device to a power source to generate an electromotive force. In many embodiments, there is no "obstacle" to hinder the analyte (cell) when the sample is unfolded, so that the analyte is evenly distributed throughout the relevant volume (within the range allowed by Poisson statistics) rather than being more concentrated in a certain area. In addition, in other devices, the function of the cover plate is to seal the device to prevent liquid leakage, and therefore, the cover plate is placed on top of the substrate at a time, while there is no sample on any plate. This device does not push the liquid into a thin layer that can be analyzed on an open plate surface. In addition, in other devices, the main function of the support is to "filter" or sort nano-matter (such as cells or the like). Thus, the distance between the pillars is determined by the nanomaterial being sorted, rather than being used to provide uniform spacing between the cover and substrate plates. Finally, in an apparatus such as the Austin apparatus, the sorting accuracy is primarily controlled by the distance between the pillars rather than the spacing between the plates, and controlling the spacing between the plates is not considered important. Thus, such disclosure would not lead one to adjust the uniformity of the plate spacing by varying the size, shape, spacing between the pillars, etc.

[0035] In view of the above, the present apparatus and method provide an easy-to-use, inexpensive, simple to operate, and very rapid way to determine the absolute concentration of an analyte (or multiple analytes if the apparatus and method are configured in a multiplexed parallel manner) in a liquid sample.

[0036] One aspect of the present invention is the use of a pair of relatively movable plates to manipulate small volumes of sample or one or more reagents or both to provide a simpler, faster and / or better assay. Such manipulations include, but are not limited to, reshaping the sample, forcing sample flow, making contact between sample and reagent, measuring sample volume, reducing diffusion distances, increasing collision frequency, etc. - each of which is beneficial in certain assays. In the present invention, the specific functions and properties of the plates, the specific methods of manipulating the plates, and the specific methods of handling reagents and samples all provide advantages for assay analysis.

[0037] One aspect of the present invention is a device for making at least a portion of a droplet of a liquid sample deposited on a plate into a thin film of controlled and predetermined thickness and uniform over a large area. The uniform thickness of the film can be as thin as less than 1 micron. In addition, the present invention allows the same uniform thickness to be maintained for a long period of time without suffering from evaporation into the environment.

[0038] Another aspect of the present invention is to utilize the predetermined uniform sample thickness formed by the present invention to determine the volume of a portion or all of a sample without the use of any pipette or similar device.

[0039] Another aspect of the present invention is an embodiment of a spacer (for controlling the spacing between two plates) having a flat top and a columnar structure with a nearly uniform cross-section. This spacer provides many advantages in controlling sample thickness over a spacer in the shape of a ball (bead).

[0040] Another aspect of the invention is a device for causing certain chemical reactions (or mixing) to occur primarily in a small portion of a sample and not in other portions of the sample without using a fluid barrier between the two portions of the sample.

[0041] Another aspect of the present invention is a device that allows multiple chemical reactions (or mixing) to occur primarily in small portions of a sample, rather than in other portions of the sample, without the need for fluid isolation between the two portions of the sample. Thus, the present invention allows multiple parallel assays to be performed using a small drop of sample without fluid isolation between different reaction sites.

[0042] Another aspect of the invention is a device that makes assays (e.g., immunoassays, nucleic acid assays, etc.) faster. For example, the saturation incubation time (the time it takes for the binding between molecules to reach equilibrium) is shortened from several hours to less than 60 seconds.

[0043] Another aspect of the invention is a device that significantly improves detection sensitivity by one or more methods, including the combination of an amplification surface, or larger or brighter labels, etc.

[0044] Another aspect of the invention is a device for performing assays using very small amounts of sample, for example as little as 0.5 microliters.

[0045] Another aspect of the invention is a device that simplifies assays by allowing placement of minute amounts of bodily fluid from a subject directly into a test or sample area.

[0046] Another aspect of the present invention is a device that simplifies and speeds up the assay by pre-coating the plate with reagents. For example, the capture agent and the detection agent are pre-coated on the plate and dried. In another example, all necessary sensing reagents are pre-coated on the plate, and sensing is achieved by depositing the sample on the pre-coated plate without placing other reagents.

[0047] Another aspect of the present invention is an apparatus for reading measurement results performed by a mobile phone.

[0048] Another aspect of the present invention is a device that allows a person to test his / her own biomarkers in 60 seconds by placing his / her body fluid (such as saliva) directly between a pair of plastic devices and taking a picture with a mobile phone.

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The skilled artisan will appreciate that the accompanying drawings, as described below, are for illustrative purposes only. The accompanying drawings are not intended to limit the scope of the invention in any way. The accompanying drawings may not be to scale. In the accompanying drawings presenting experimental data points, the lines connecting the data points are only used to guide the observation of the data and have no other meaning.

[0051] Figure 1 is an illustration of a CROF (Compression Regulable Unobstructed Flow) embodiment. Inset (a) shows a first plate and a second plate, wherein the first plate has a spacer. Inset (b) shows a sample deposited on a first plate (shown), or a second plate (not shown), or both (not shown) in an open phase. Inset (c) shows (i) using two plates to extend the sample (the sample flows between the plates) and reduce the thickness of the sample, and using spacers and plates to adjust the sample thickness in a closed phase, and (ii) the inner surface of each plate may have one or more bonding points and / or storage locations (not shown).

[0052] Figure 2Plates with binding sites or storage sites are shown. Panel (a) shows a plate with binding sites. Panel (b) shows a plate with reagent storage sites. Panel (c) shows a first plate with one binding site and a second plate with reagent storage sites. Panel (d) shows a plate with multiple sites (binding sites and / or storage sites).

[0053] Figure 3 Flow chart and schematic diagram of a method for reducing the incubation time of an assay by reducing the thickness of a sample. Inset (a) represents a first plate having at least one binding point on a substrate surface. Inset (b) shows a second plate (which may have a different size from the first plate). Inset (c) represents a deposited sample (containing a target binding entity) on a substrate surface (as shown) or a cover plate (not shown), or both (not shown). Inset (d) represents reducing the thickness of the sample by reducing the spacing of the internal space between the plates so that they face each other and by moving the first and second plates. The sample with reduced thickness is incubated. The reduced sample thickness speeds up the incubation time. Some embodiments of the method use spacers to adjust the spacing, not shown in the (spacer) figure.

[0054] Figure 4 Shows the reduction of binding or mixing time by using two plates, a spacer, and compression (shown in cross section). Inset (a) shows the reduction of time to bind entities in a sample to binding sites on a solid surface (X-(volume to surface)). Inset (b) shows the reduction of time to bind entities (e.g., reagents) stored on the surface of one plate to binding sites on the surface of another surface (X-(surface to surface)). Inset (c) shows the reduction of time for adding a reagent stored on one surface of a plate to a sample clamped between a plate and another plate (X-(surface to volume)).

[0055] Figure 5 Shows how to avoid or reduce local bending of a flexible board. Inset (a) shows that if the distance between the spacers is too large for the flexible board (second board, such as a plastic film) under a given set of samples and compression conditions, assuming that the first board is rigid, the board has a local depression (i.e., inward bending) between two adjacent spacers in the closed phase. The sample between the boards is not shown. Inset (b) shows that the local bending (depression) of the flexible board in inset (a) is reduced or almost avoided by using an appropriate distance between the spacers and an appropriate compression force. The sample between the boards is not drawn.

[0056] Figure 6Figure 2 shows the effect of reducing large dust on the adjustment of plate spacing (sample thickness). Panel (a) shows that when two rigid plates are used, dust thickness greater than the spacer height can disrupt the expected plate spacing adjusted by the spacer (and therefore disrupt the expected sample thickness adjustment). The sample between the plates is not plotted. Panel (b) shows that using appropriate flexible plates and appropriate inter-spacer distance, the effect of dust is isolated to a small area around the dust, while in other areas, the plate spacing (and therefore the sample thickness) is adjusted by the spacer rather than the dust. In this illustration, the first plate is rigid, the second plate is flexible, and the spacer is initially fixed to the first plate. Panel (c) shows that using appropriate flexible plates and appropriate inter-spacer distance, the effect of dust is isolated to a small area around the dust, while in other areas, the plate spacing (and therefore the sample thickness) is adjusted by the spacer rather than the dust. In this illustration, the first plate is rigid, the second plate is flexible, and the spacer is initially fixed to the second plate.

[0057] Figure 7 Figure 1 shows how the effects of variations in the surface flatness of the plates can be reduced by using an appropriate spacer arrangement and flexible plates. Panel (a) shows that variations in surface flatness can be significantly larger than the expected sample thickness, leading to errors in determining sample thickness. In this figure, only one plate has a large variation in flatness (in reality, both plates may have large variations in flatness). The sample between the plates is not plotted. Panel (b) shows the variation in surface flatness of the plates as the distance from one local maximum to the adjacent local minimum of the surface height. Panel (c) shows how smaller variations in surface flatness can be achieved by using one or both plates to be flexible and using an appropriate inter-spacer distance and an appropriate compressive force in the closed phase to correct for the original surface flatness variation of the plate in the open phase. The sample between the plates is not plotted. Panel (d) shows how the sample thickness variation is less than the initial surface flatness variation of the plate by using a flexible second plate and an appropriate inter-spacer distance. The flexible plate has the profile of the rigid plate. The sample between the plates is not plotted.

[0058] Figure 8 Plates and closed spacers (wells) for sample thickness adjustment are shown. Panel (a) shows a first plate and a second plate, wherein the first plate has a closed spacer (well). Panel (b) shows a sample deposited on a first plate (shown), or a second plate (not shown), or both (not shown) in an open phase. Panel (c) shows (i) using two plates to expand the sample (sample flow between the plates) and reduce the thickness of the sample, and (ii) using spacers and plates to adjust the sample thickness in a closed phase.

[0059] Fig. 9Another embodiment of adjusting the sample thickness using a closed spacer (well) is shown. Inset (a) shows a first plate and a second plate, wherein the first plate has a closed spacer (well) and at least one spacer in the well. Inset (b) shows a sample deposited on a first plate (shown), or a second plate (not shown), or both (not shown) in an open phase. Inset (c) shows (i) using two plates to expand the sample (the sample flows between the plates) and reduce the thickness of the sample, and (ii) using spacers and plates to adjust the sample thickness in a closed phase. Inset (d) shows another embodiment of a first and a second plate, wherein the first plate does not have a spacer in the well.

[0060] Fig.10 Schematically illustrates an exemplary embodiment of the present invention, multiplexing detection in a single CROF device using one binding site on one plate and multiple storage sites on another plate. Panels (a) and (b) are perspective and cross-sectional views of an exemplary device, respectively.

[0061] Fig.11 Schematically illustrates another exemplary embodiment of the present invention, multiplexed detection in a single CROF device using one storage site on one plate and multiple binding sites on another plate. Panels (a) and (b) are perspective and cross-sectional views of an exemplary device, respectively.

[0062] Fig.12 A further exemplary embodiment of the invention is schematically shown, multiplexing detection in a single CROF device using multiple binding sites on one plate and multiple storage sites on another plate. Panels (a) and (b) are perspective and cross-sectional views of an exemplary device, respectively.

[0063] Fig.13A A QMAX assay using a CROF device comprising a spacer array with a 30 micron spacer height to achieve an assay with a saturation incubation time of less than 30 seconds is schematically shown.

[0064] Fig. 13B is a measure of the capture tag signal relative to the incubation time, indicating Fig.13A The saturation incubation time for the QMAX assay shown did not exceed 30 seconds.

[0065] Fig.14 Represents experimental measurements of LoD (limit of detection) for QAX & QMAX assays using a 30 micron gap (for CROF device), with elution (heterogeneous assay) and without elution (homogeneous assay).

[0066] Fig.15 Shown are (i) a top view and a cross-sectional view of a small volume of sample dropped onto a glass substrate, and (ii) an area of ​​the sample enlarged in the closed phase of CROF.

[0067] Fig.16 The meanings of some of the terms used herein are shown.

[0068] Fig.17 Spacers on the plate are shown. Photo top view: (a) 46 micron x 46 micron pillar spacer size and 54 micron inter-pillar distance, (b) 10 micron x 70 micron pillar spacer size and 10 um inter-pillar distance; and scanning electron microscope perspective view: (c) 30 micron x 40 micron pillar spacer size and 2 micron spacer height, and (d) 30 micron x 40 micron pillar spacer size and 30 micron spacer height.

[0069] Fig.18 Effect of IDS and plate thickness and material on sample thickness. Measured sample thickness deviation and uniformity vs. inter-spacer distance (IDS) for different plate and spacer materials, different plate thicknesses, and different samples. The substrate of the CROF device is an untreated 250 μm thick planar PMMA (25.4 mm x 25.4 mm in size. The X-plate includes a periodic array of pillar spacers, where the spacers have a height of 5 μm, a rectangular shape (10×10 μm pillar lateral dimensions, nearly uniform cross-section, and rounded corners), and an inter-spacer distance of 20 um, 50 um, 100 um, 200 um, and 500 um. The X-plate size is 25.4 mm x 25.4 mm and is made of PMMA or PS. The sample is 2 μl of blood (placed by direct finger contact), saliva, or PBS (pipette drop), and the CROF device is pressed by squeezing and wiping on an area of ​​1 inch by 1 inch, and is self-retaining after pressing. In the figure, the label Refers to the 175 μm thick PMMA plate used for the blood sample, marked Refers to the 175 μm thick PMMA plate used for the saliva sample, marked Refers to the 125 μm thick PMMA plate using PBS, marked Refers to the 50 μm thick PMMA plate using blood samples, marked Refers to the 25 μm thick PS plate using blood samples.

[0070] Fig.19 Determine the thickness deviation and uniformity of the sample vs. the ISD of the X-plate 4 / (h x E) (in the figure, x = 1). ISD is the distance between the spacers, h is the height (thickness) of the material, E is the Young's modulus of the material, and x is the A fitting parameter of a typical range of . In the test, the substrate of the CROF device was untreated 250 μm thick PMMA (size 25.4 mm x 25.4 mm), the X-plate was 175 μm thick untreated PMMA, 125 μm thick untreated PS, 50 μm thick untreated PMMA and 25 μm thick untreated PS (size 25.4 mm x 25.4 mm), the X-plate included a periodic array of pillar spacers, where the spacers had a height of 5 μm, a rectangular shape (10×10 μm pillar lateral dimension, nearly uniform cross-section, and rounded corners), and 20 μm, 50 μm, 100 μm, 200 μm, and 500 μm inter-spacer distances, and the X-plate size was 25.4 mm x 25.4 mm. The sample was 2 μl of blood (placed by direct finger contact), saliva or PBS (pipette drop). The CROF device was pressed by being squeezed and rubbed on an area of ​​1 inch by 1 inch, and was self-retaining after pressing. At ISD 4 / h x＝1 In the calculation of ISD / E, the Young's modulus of PMMA is 2.5 GPa and that of PS is 3.3 GPa. 4 / (hE) value is greater than 10 6 um 3 / Gpa, the performance of the CROF device deteriorates. In the figure, the label Refers to the 175 μm thick PMMA plate used for the blood sample, marked Refers to the 175 μm thick PMMA plate used for the saliva sample, marked Refers to the 125 μm thick PMMA plate using PBS, marked Refers to the 50 μm thick PMMA plate used for the blood sample, marked Refers to the 25 μm thick PS plate using blood samples.

[0071] Fig. 20 The thickness deviation and uniformity of the samples were determined vs. the inter-spacer distance for different pillar spacer sizes and heights on the X-plate. The base plate of the CROF device was untreated 1 mm thick glass (size 25.4 mm x 25.4 mm), and the X-plate was 125 μm thick untreated PS (size 25.4 mm x 25.4 mm), including a periodic pillar spacer array, where the pillars had a rectangular shape with 5 μm spacer height and 10×10 μm lateral dimensions (near uniform cross-section and rounded corners), with inter-spacer distances of 20 um, 50 um, 100 um, 200 um, and 500 um (marked ); 40×40 micron lateral dimensions, with 60um, 150um, and 200um spacer distances (marked ); a periodic pillar-spacer array, where the pillars have a 12 micron spacer height and a rectangular shape with 40×40 micron lateral dimensions, and inter-spacer distances of 150um and 200um (marked ); a periodic pillar-spacer array, where the pillars have a 22 micron spacer height and a rectangular shape with 40×40 micron lateral dimensions, and inter-spacer distances of 150um and 200um (marked ); 2 microliters of PBS for the 5 micron CROF device sample, 5 microliters of PBS for the 12 micron CROF device, and 9 microliters of PBS for the 22 micron CROF (dropped by a pipette), the CROF device was pressed by squeezing and wiping on an area of ​​1 inch by 1 inch, and was self-retaining after being pressed. (The lines in the figure are for guiding observation.)

[0072] Fig.21 The thickness deviation and uniformity of the samples were measured vs. different ratios of pillar width to pillar height while maintaining ISD for all samples less than 150 microns. The substrate of the CROF device is untreated 1 mm thick glass (size 25.4 mm x 25.4 mm). The CROF device is pressed by being squeezed and rubbed on an area of ​​1 inch by 1 inch and is self-retaining after being pressed. The samples in the figure are labeled as follows:

[0073] A. X-plate made of 125 μm thick PS (marked ), from left to right: 1: X-plate column size 10×10 μm, height 22 μm, ISD 100 μm, 9 μl PBS buffer, ratio (w / h) = 0.45; 2: X-plate column size 10×10 μm, height 12 μm, ISD 100 μm, 5 μl PBS buffer, ratio (w / h) = 0.83; 3: X-plate column size 40×40 μm, height 22 μm, ISD 150 μm, 9 μl PBS buffer, ratio (w / h) = 1.81; 4: X-plate column size 40×40 μm, height 5 μm, ISD 100 μm, 2 μl PBS buffer, ratio (w / h) = 2; 5: X-plate column size 40×40 μm, height 12 μm, ISD 150 μm, 5 μl PBS buffer, ratio (w / h) = 3.33; 6: X-plate column size 40×40 μm, height 5 μm, ISD 150 μm, 2 μl PBS buffer, ratio (w / h) = 8; 7: X-plate column size 70×70 μm, height 5 μm, ISD 150 μm, 2 μl PBS buffer, ratio (w / h) = 14

[0074] B. X-plate made of 175 μm thick PMMA (marked ), from left to right: 1: X-plate pillar size 10×10 μm, height 22 μm, ISD 100 μm, 5 μl blood, ratio (w / h) = 0.45; 2: X-plate pillar size 10×10 μm, height 5 μm, ISD 50 μm, 2 μl blood, ratio (w / h) = 2; 3: X-plate pillar size 30×30 μm, height 30 μm, ISD 80 μm, 12 μl blood, ratio (w / h) = 1; 4: X-plate pillar size 30×30 μm, height 10 μm, ISD 80 μm, 1 μl blood, ratio (w / h) = 3; 5: X-plate pillar size 30×30 μm, height 2 μm, ISD 80 μm, 1 μl blood, ratio (w / h) = 15.

[0075] C. X-plate made of 50 μm thick PMMA (marked ), from left to right: 1: X-plate column size 10×10 μm, height 5 μm, ISD 50 μm, 2 μl blood, ratio (w / h)=2.

[0076] D. X-plate made of 25 μm thick PS (marked ), from left to right: 1: X-plate column size 10×10 μm, height 5 μm, ISD 50 μm, 2 μl blood, ratio (w / h)=2.

[0077] Fig. 22 The thickness deviation and uniformity of the obtained samples were measured vs. inter-spacer distance and pillar size / X-plate height. The substrate of the CROF device was untreated 1 mm thick glass (size 25.4 mm x 25.4 mm), and the X-plate was 125 μm thick untreated PS (size 25.4 mm x 25.4 mm), including a periodic pillar spacer array, where the pillars had a rectangular shape with 5 μm spacer height and 10×10 μm lateral dimensions (near uniform cross-section and rounded corners), with inter-spacer distances of 20 um, 50 um, 100 um, 200 um, and 500 um (marked ); 40×40 micron lateral dimensions, with 60um, 150um, and 200um spacer distances (marked ); a periodic pillar spacer array, where the pillars have a 12 micron spacer height and a rectangular shape with 40×40 micron lateral dimensions, and an inter-spacer distance of 150um and 200um (marked ); a periodic pillar-spacer array, where the pillars have a 22 micron spacer height and a rectangular shape with 40×40 micron lateral dimensions, and inter-spacer distances of 150um and 200um (marked ); 2 microliters of PBS for the 5 micron CROF device sample, 5 microliters of PBS for the 12 micron CROF device, and 9 microliters of PBS for the 22 micron CROF (dropped by a pipette). The CROF device is pressed by squeezing and wiping on an area of ​​1 inch by 1 inch, and is self-retaining after being pressed. (The lines in the figure are for guiding observation.)

[0078] Fig.23 The sample thickness deviation and uniformity were measured vs. different X-plate thicknesses (25 μm to 525 μm), fixed post dimensions (30×38 μm), post height (at 2 μm) and pitch distance (80×82 μm), made of untreated PMMA, where the substrate was a 1 mm thick untreated glass slide (size 25.4 mm x 25.4 mm), the sample was 1 μL of blood dropped by direct contact with a finger, and the CROF device was pressed by squeezing and wiping on a 1 inch by 1 inch area and self-retained after pressing.

[0079] Fig.24 Measured pitch size deviation / uniformity of CROF device (marking of different combinations: hydrophilic-hydrophilic , hydrophilic-hydrophobic ), 0.1 microliter to 0.5 microliter blood sample volume, the same X-plate column size (30×38um), column height (2um) and spacing distance (80×82 microns), wherein the substrate is a glass sheet with a size of 1 mm thick (25.4 mm X 25.4 mm), and the X-plate is made of 175um thick PMMA (size 25.4 mm X 25.4 mm). The sample is dropped by direct contact with a finger, while the CROF device is pressed by being squeezed and rubbed on an area of ​​1 inch by 1 inch, and is self-retaining after pressing.

[0080] Fig.25 The thickness deviation and uniformity of the samples were measured vs. an untreated 1 mm thick glass substrate (marked ) or untreated 250 μm thick PMMA substrate (marked ) (size 25.4 mm x 25.4 mm), where the X-plate is a 175 μm thick untreated PMMA, including a periodic array of pillar spacers, where the spacers have a 5 μm height, a rectangular shape (10×10 μm pillar lateral dimensions, nearly uniform cross-section, and rounded corners), and inter-spacer distances of 50 μm, 100 μm, 200 μm, and 500 μm. The sample is 2 μL of blood dropped by direct contact with a finger, and the CROF device is pressed by squeezing and wiping on an area of ​​1 inch by 1 inch, and is self-retaining after pressing

[0081] Fig.26 .The sample thickness deviation and uniformity were measured vs. different hand pressing times from 0 to 60 seconds, where the substrate of the CROF device was made of untreated 250 micron thick PMMA (size 25.4 mm x 25.4 mm), where the X-plate was a 175 micron thick untreated PMMA (size 25.4 mm x 25.4 mm), including a periodic array of pillar spacers, where the spacers had a 2 micron height, a rectangular shape (30×38 micron pillar lateral dimensions, nearly uniform cross-section, and rounded corners), and an 80um inter-spacer distance. The sample was 1 microliter of blood dropped by direct contact with a finger, and the CROF device was pressed by being squeezed and rubbed on a 1 inch by 1 inch area, and was self-retaining after pressing. Fig. 27 . Thickness deviation and uniformity of samples were measured vs. average IDS for samples using random ball spacers or conventional pillar spacers (X-plate), where the substrate of the CROF device was made of untreated 1 mm thick PMMA (size 25.4 mm x 25.4 mm), where the X-plate was a 175 μm thick untreated PMMA including a periodic array of pillar spacers, where the spacers had 5 μm height, rectangular shape (10×10 μm pillar lateral dimensions, nearly uniform cross-section, and rounded corners), and inter-spacer distances of 20 um, 50 um, and 100 um. The sample was 2 μl PBS, and the CROF device was pressed by squeezing and wiping on an area of ​​1 inch by 1 inch, and was self-retaining after pressing. The ball spacers were soda lime microspheres with an average diameter of 4 μm in PBS (5% size variation). The microspheres were 4x10 5 / uL,0.9x10 5 / uL, and 0.2x10 5 / uL concentration distribution in PBS, which corresponds to 20 microns, 50 microns and an average of 100 microns inter-spacer distances after pressing. Two cover plates were used, untreated 220 micron thick glass (size 25.4 mm x 25.4 mm) and untreated 175um thick PMMA (size 25.4 mm x 25.4 mm). All devices were pressed by being squeezed and rubbed on an area of ​​1 inch by 1 inch and were self-retaining after pressing. Marking Refers to the use of X-board, marking Refers to the use of ball spacers as spacers and 220 μm thick glass sheets as cover plates, marked Refers to the use of ball spacers as spacers and 175 micron thick PMMA as cover.

[0082] Fig.28. Measured sample thickness deviation and uniformity vs. different X-plate thicknesses (25 microns to 350 microns) and substrate thicknesses (25 microns to 750 microns). The X-plate has fixed pillar size (30×38 microns), pillar height (10 microns), spacer distance (80×82 microns), and is made of untreated PMMA with thicknesses of 25 microns, 175 microns and 350 microns, while the substrate is made of untreated PMMA (size 25.4 mm X 25.4 mm) with thicknesses of 25 microns, 50 microns, 175 microns, 250 microns and 750 microns. The sample was 4uL of blood dropped by direct contact with a finger, while the CROF device was pressed by being squeezed and rubbed on an area of ​​1 inch by 1 inch, and was self-retaining after pressing. In the figure, the marker Refers to the use of 25 micron thick X-plate, marked Refers to the use of 175 micron thick X-plate, marked Refers to the use of 350 micron thick X-plate. .

[0083] Fig.29 Microscope photos (40x) showing (a) blood cells in X-devices with plate spacing (i.e., sample thickness) of 1 micron, 2 microns, 3 microns, and 5 microns. The 1 micron spacing X-device lyses most (99%) of the red blood cells without lysing platelets. The 2 micron spacing X-device has good separation of each red blood cell and the red blood cells form a monolayer. Some stacked red blood cells are visible in the 3 micron spacing X-device, while more stacked red blood cells are visible in the 5 micron spacing X-device. The monolayer cell count (2 micron spacing X-device) is optimal. And (b) the ratio of the red blood cell area (measured from the 2D top view image) to the total lateral area of ​​the CROF plate. This ratio is the largest at a 2 micron plate spacing (i.e., sample thickness) because below 2 microns some red blood cells are lysed and above 2 microns the red blood cells overlap and rotate, all of which give a smaller red blood cell area in the 2D image.

[0084] Fig.30 . Schematic diagram of BCI (counting blood cells by CROF and imaging) performed by a smartphone device (a), and (b) a photo of the device. When using the smartphone BCI for blood testing, one first has a card (1) and then pricks his / her finger (2) and deposits a small amount of blood directly from the finger onto the CROF card (2) by touching the card (2), closes the card (3) and presses it with the finger (4), then releases it (5), inserts the card into the optical adapter (5), and finally takes a photo of the card using the smartphone (6), and from the captured picture, the blood volume and blood cell count and other parameters are measured by the software (6). (b) A photo of an actual smartphone and adapter used for p-BCI.

[0085] Fig.31. Bright field optical microscopy images of fresh blood (a) and stored undiluted whole blood (b) in CROF cards with different plate spacings, and illustrations of red blood cell behavior under different confinement gaps. Fresh blood does not contain anticoagulant and is obtained from a finger prick, while stored blood has anticoagulant and is obtained from a commercial supplier. (a-1 to a-6) and (b-1 to b-6): g equals 2, 2.2, 2.6, 3, 5, and 10 μm, respectively. (c) Cross-sectional view and top view showing that (1) in CROF with a spacing of 2 μm, red blood cells are separated from each other with no observable overlap; (2) in CROF with a spacing greater than 2 μm, red blood cells overlap each other.

[0086] Fig.32 Brightfield (1) and fluorescence (2) images of the same sample (fresh blood in a CROF card) acquired using (a) a smartphone with an optical adapter and (b) a high-resolution microscope with a DSLR camera. The images show that the smartphone with the optical adapter has a similar quality of blood cell pictures as a high-resolution microscope and camera.

[0087] Fig.33 Shows the measured light intensity of a typical WBC and platelet vs. the position of these separate cells. The diameter (FWHM) of a WBC is around 12 microns, while the diameter (FWHM) of a platelet is around 2 microns. The maximum intensity of a WBC is about 3 times greater than that of a platelet. Both the intensity and area give values ​​that are about 108 times greater than that of a platelet. Therefore, if a lower magnification (such as 4X) is used, the area of ​​the WBC becomes smaller and its overall intensity becomes lower. The signal of the platelet will be negligible in this case.

[0088] Fig.34 Shows (a) a scatter plot of green channel light intensity vs. red channel light intensity; and (b) a histogram of the red / green channel light intensity ratio of 594 white blood cells in the image. From this image, we can clearly see that these cells cluster into three different areas (the shaded areas are for guidance), corresponding to the three main white cell subpopulations.

[0089] Detailed Description of Exemplary Embodiments

[0090] The following detailed description describes some embodiments of the invention by way of example and not by way of limitation. The section headings and all subheadings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. The content under a section heading and / or subheading is not limited to the section heading and / or subheading, but applies to the entire description of the invention.

[0091] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0092] The present invention relates to, among other things, methods, devices, and systems that can improve and / or accelerate the quantification, binding, and / or sensing of analytes and / or entities in a sample.

[0093] definition

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are described herein.

[0095] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. The term "amino acid" as used herein refers to natural and / or unnatural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0096] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence" and "oligonucleotide" are used interchangeably and may also include the plural form of each respective one depending on the context of the term. They refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment defined from linkage analysis, exons, introns, messenger RNA (mRNA), locus (locus), transfer RNA (tRNA), ribosomal RNA, ribozymes, small interfering RNA (siRNA), micro RNA (miRNA), small nuclear RNA (snRNA), cDNA, recombinant polynucleotides of any sequence, branched polynucleotides, plasmids, vectors, isolated DNA (A, B and Z structures), PNA, locked nucleic acid (LNA), TNA (treose nucleic acid), isolated RNA of any sequence, nucleic acid probes and primers. LNAs, often referred to as inaccessible RNAs, are a type of modified RNA nucleotide. The ribose portion of an LNA nucleotide is modified with an additional bridge connecting the 2' and 4' carbons. The bridge "locks" the ribose in a 3'-endo conformation, which is typically found in the A-form of DNA or RNA, which can significantly increase thermal stability.

[0097] As used herein, the term "capture agent" refers to a binding member, such as a nucleic acid molecule, a polypeptide molecule, or any other molecule or compound that can specifically bind to its binding partner, for example, a second nucleic acid molecule sequence containing nucleotides complementary to a first nucleic acid molecule, an antibody that specifically recognizes an antigen, an antigen specifically recognized by an antibody, a nucleic acid aptamer that can specifically bind to a target molecule, etc. A capture agent can concentrate a target molecule from a heterogeneous mixture of different molecules by specifically binding to the target molecule. Binding can be non-covalent or covalent. The affinity between a binding member and its binding partner is determined by K. D (dissociation constant) describes the K D For 10 -5 M or smaller, 10 -6 M or smaller, 10 -7 M or smaller, including 10 -8 M or smaller, e.g. 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -13 M or smaller, 10 -14 M or smaller, 10 -15M or smaller, including 10 -16 M or less. “Affinity” refers to the strength of binding, and increased binding affinity is associated with a lower K D Related.

[0098] The term "secondary capture agent", which may also be referred to as a "probe agent", refers to a group of biomolecules or chemical compounds that have a highly specific affinity for an antigen. The secondary capture agent can be linked to an optically detectable label, such as an enzyme, a fluorescent label, or can itself be detected by another probe agent that is linked to an optically detectable label through biocoupling (Hermanson, "Bioconjugation Technology" Academic Press, 2nd edition, 2008).

[0099] The term "capture agent reactive group" refers to a chemically functional portion in a molecule that is reactive with a capture agent, that is, can react with a group of a capture agent (e.g., hydroxyl, sulfhydryl, carboxyl or amine) to produce a stable strong, e.g., covalent bond.

[0100] The terms "specific binding" and "selective binding" refer to the ability of a capture agent to preferentially bind to a specific target analyte present in a heterogeneous mixture of different target analytes. Specific or selective binding interactions will discriminate between desired (e.g., mobile) and undesired (e.g., inactive) target analytes in a sample, typically by more than about 10 to 100 times or more (e.g., greater than about 1000 or 10,000 times).

[0101] The term "antibody", as used herein, refers to a protein consisting of one or more polypeptides encoded by all or part of a recognized immunoglobulin gene. Recognized immunoglobulin genes, such as in humans, include kappa (κ), lamda (λ) and heavy chain gene loci, which together constitute numerous variable region genes, and constant region genes mu (μ), delta (δ), gamma (γ), sigma (σ), and alpha (α), which encode IgM antibodies, IgD, IgG, IgE and IgA antibody "isotypes" or "classes", respectively. Antibodies herein include full-length antibodies and antibody fragments, and may refer to antibodies from any organism, engineered antibodies, or antibodies and natural antibodies produced recombinantly for experimental, therapeutic or other purposes. The term "antibody" includes full-length antibodies and antibody fragments, such as those known in the art, such as Fab, Fab', F(ab')2, FV, scFv, or other antigen-binding subsequences of antibodies, or produced by modification of intact antibodies or those synthesized de novo using recombinant DNA technology.

[0102] The terms "antibody epitope", "epitope", and "antigen" are used interchangeably herein to refer to a biological molecule bound by an antibody. Antibody epitopes may include proteins, carbohydrates, nucleic acids, hormones, receptors, tumor markers, and the like, as well as mixtures thereof. An antibody epitope may also be an epitope of a group of antibodies, such as a specific portion of a protein eluted from a size exclusion chromatography column. Furthermore, an antibody epitope may also be determined from a specified clone of an expression library or a random epitope pool.

[0103] As used herein, an "allergen" is a substance that causes an allergic, inflammatory reaction in an individual when the individual is exposed to the substance, e.g., by skin contact, ingestion, inhalation, eye contact, etc. Allergens can be found in sources from the following groups: natural and artificial fibers (cotton, linen, wool, silk, teak, etc., wood, straw and other dusts); tree pollens (alder, birch, hazel, oak, poplar, palm and others); weeds, flowers (ragweed, wormwood, etc.); grasses and corn (fescue, timothy, rye, wheat, corn, bluegrass, etc.); drugs (antibiotics, antimicrobials, analgesics and nonsteroidal anti-inflammatory drugs, anesthetics and muscle relaxants, hormones, and other substances); epidermal and animal allergens (epithelial cells of animals, feathers of birds, serum and others); molds and yeasts (Penicillium, Cladosporium , Aspergillus fumigatus, Mucor, etc. ); insect venom; preservatives (butylparaben, sorbic acid, benzoic acid, etc.); semen (semen); parasitic and mite allergens (roundworms, house dust mites, dust mites, Euroglyphusmaynei, and others); occupational and hobby allergens (coffee beans, formaldehyde, latex, chloramines, dyes, and others); food allergens (egg products, dairy products and cheese, meat products, fish and seafood, soy products, mushrooms, flour and cereals, vegetables, melon crops, beans, herbs and spices, nuts, citrus and other fruits, berries, tea and herbs, nutritional supplements and other products), etc.

[0104] The term "hybridization" refers to a reaction in which one or more polynucleotides react to form a stable complex by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding or any other sequence-specific manner. The complex can include a double-stranded structure formed by two strands, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. As is well known to those skilled in the art, hybridization can be performed under various stringent conditions. Suitable hybridization conditions are such that the recognition interaction between a capture sequence and a target nucleic acid is both sufficiently specific and sufficiently stable. Conditions that increase the stringency of hybridization reactions are widely known and published. See, for example, Green et al., (2012), hereinafter the same.

[0105] The term "protein" refers to any length of amino acids, i.e., greater than 2 amino acids, greater than about 5 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, greater than about 50 amino acids, greater than about 100 amino acids, greater than about 200 amino acids, greater than about 500 amino acids, greater than about 1000 amino acids, or a polymeric form of greater than about 2000 amino acids, usually not greater than about 10,000 amino acids, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids and polypeptides with modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions of heterologous and homologous leader sequences, with or without an N-terminal methionine residue; immunologically labeled proteins; fusion proteins with detectable fusion partners, for example, fusion proteins including as fusion partners fluorescent proteins, beta-galactosidase, luciferase, etc.; and the like. These terms also include those that are modified post-translationally in the cell, e.g., glycosylation, cleavage, secretion, prenylation, carboxylation, phosphorylation, etc., and polypeptides with secondary or tertiary structure, and polypeptides that are strongly bound, e.g., the polypeptide covalently or non-covalently, to other moieties, e.g., other polypeptides, atoms, cofactors, and the like.

[0106] As used herein, the term "complementary" refers to a nucleotide sequence that base pairs with a target nucleic acid of interest through hydrogen bonds. In canonical Watson-Crick base pairing, adenine (A) base pairs with thymine (T) and guanine (G) base pairs with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). Thus, A is complementary to T and G to C. In general, "complementary" refers to a nucleotide sequence that is completely complementary to the target of interest, such that the nucleotides in each sequence are complementary to each nucleotide in the target nucleic acid at corresponding positions. When a nucleotide sequence is not completely complementary (100% complementary) to a non-target sequence, but can still base pair with the non-target sequence, due to the complementarity of some stretches of the nucleotide sequence to the non-target sequence, the percent complementarity can be calculated to assess the possibility of non-specific (off-target) binding. In general, a complementarity of 50% or less will not result in non-specific binding. In addition, a complementarity of 70% or less may not result in non-specific binding under stringent hybridization conditions. The term "ribonucleic acid" and "RNA" as used herein refers to a polymer composed of ribonucleotides. The term "deoxyribonucleic acid" and "DNA" as used herein refers to a polymer composed of deoxyribonucleotides.

[0107] The term "oligonucleotide" as used herein refers to a single-stranded nucleotide polymer of about 10 to 200 nucleotides, up to 300 nucleotides in length, or longer, for example, up to 500 nucleotides in length or longer. Oligonucleotides can be synthetic, and in certain embodiments, less than 300 nucleotides long.

[0108] As used herein, the term "attached" refers to a strong, e.g., covalent or non-covalent bond that joins one molecule to another. As used herein, the term "surface attached" refers to a molecule that is firmly attached to a surface.

[0109] As used herein, the term "sample" refers to a material or material mixture containing one or more analytes or entities of interest. In a specific embodiment, the sample can be obtained from a biological sample such as a cell, tissue, body fluid, and feces. The body fluid of interest includes, but is not limited to, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), milk, cerebrospinal fluid (CSF), cerumen (ear wax), chyle, calculus, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneum, pleural fluid, pus, rhubarb, saliva, sebum (skin oil), semen, saliva, sweat, synovial fluid, tears, vomitus, urine, and exhaled condensed water. In a specific embodiment, the sample can be obtained from a subject, such as a human being, and can be processed before use in the subject determination. For example, before analysis, protein / nucleic acid can be extracted from a tissue sample before use in a known manner. In particular embodiments, the sample can be a clinical sample, eg, a specimen collected from a patient.

[0110] The term "analyte" refers to molecules (e.g., proteins, peptides, DNA, RNA, nucleic acids or other molecules), cells, tissues, viruses and nanoparticles having different shapes. The term "assay" / "determination" refers to testing a sample to detect the presence and / or abundance of an analyte.

[0111] As used herein, the terms "determine," "measure," and "assess" and "assay" are used interchangeably and include both quantitative and qualitative measurements.

[0112] As used herein, the term "luminescent label" refers to a label that is capable of emitting light upon external stimulation. This may be photoluminescence. Fluorescent labels, which include dye molecules or quantum dots, and luminescent labels, such as electro- or chemiluminescent labels, are different types of luminescent labels. The external stimulus is light (photons) for fluorescence, an electric current for electroluminescence, and a chemical reaction for chemiluminescence. The external stimulus may be a combination of the above.

[0113] The term "labeled analyte" refers to an analyte that is detectably labeled with a luminescent label, such that the analyte can be detected by evaluating the presence of the label. A labeled analyte can be directly labeled (i.e., the analyte itself can be directly coupled to a label, for example, via a strong bond, such as a covalent or non-covalent bond), or a labeled analyte can be indirectly labeled (i.e., the analyte is detected by a secondary capture agent that is directly labeled).

[0114] The terms "hybridization" and "binding" are used interchangeably with respect to nucleic acids. The term "capture agent / analyte complex" refers to a complex produced by a specific binding of an analyte to a capture agent. Capture agents and analytes for capture agents will typically bind specifically to each other under "specific binding conditions" or "binding suitable for specific conditions", where such conditions are those conditions (salt concentration, pH value, eluent, protein concentration, temperature, etc.) that allow the binding between the capture agent and the analyte to occur in solution. Such conditions are well known, particularly for antibodies and their antigens and nucleic acid hybridization techniques. (See, for example, Harlow and Lane (Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and Ausubel et al., Short Protocols in Molecular Biology, 5th ed., Wiley & Sons, 2002).

[0115] With respect to the binding of the capture agent to the analyte, e.g., biomarker, biomolecule, synthetic organic compound, inorganic compound, etc., the terms "conditions for specific binding" and "appropriate combination of conditions" as used herein refer to conditions that produce nucleic acid duplexes, or protein / protein (e.g., antibody / antigen) complexes, protein / compound complexes, aptamer / target complexes containing pairs of molecules that specifically bind to each other, while at the same time, not favoring the formation of complexes between pairs of molecules that do not specifically bind to each other. The conditions for specific binding are the sum or combination (overall) of hybridization and elution conditions, and may include elution and blocking steps if necessary. For nucleic acid hybridization, specific binding conditions can be achieved by incubating at 42°C in a solution of 50% formamide, 5×SSC (150 mM sodium chloride, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), diluted in 5×Denhardt's solution, 10% dextran sulfate and 20 μg / ml denatured, sheared salmon sperm DNA, followed by elution of the filter with 0.1×SSC at about 65°C. For antibody binding to an antigen, specific binding conditions can be achieved by blocking a first plate containing the antibody with a sample diluted with the analyte in a blocking solution (e.g., 3% BSA or skim milk in PBS), followed by incubation in the blocking solution. After this incubation, the first plate is eluted in an elution solution (e.g., PBS+Tween 20) and incubated with a secondary capture antibody (a detection antibody that recognizes a second site of the antigen). The secondary capture antibody can be coupled with an optically detectable label, for example, a fluorophore such as IRDye800CW, Alexa 790, Dylight 800. After another wash, the presence of the bound secondary capture antibody can be detected. Those skilled in the art understand the parameters that can be modified to increase the detection signal and reduce the background noise.

[0116] The subject can be any human or non-human animal. The subject can be a person performing the method, a patient, a client at a testing center, etc. As used herein, "analyte" is any substance suitable for testing by the method of the present invention.

[0117] As used herein, "diagnostic sample" refers to any biological sample that is a bodily byproduct, such as a body fluid that has been obtained from a subject. A diagnostic sample can be obtained directly from a subject in liquid form, or can be obtained from a subject by first placing the body byproduct in a solution, such as a buffer. Exemplary diagnostic samples include, but are not limited to, saliva, serum, blood, sputum, urine, sweat, tears, semen, feces, breath, biopsy, mucus, and the like.

[0118] As used herein, "environmental sample" refers to any sample obtained from the environment. Environmental samples include liquid samples from rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, etc.; solid samples from soil, compost, sand, rocks, concrete, wood, bricks, sewage, etc.; and gas samples from air, water vents, industrial exhaust, automobile exhaust, etc. Typically, samples that are not in liquid form are converted to liquid form before analysis by the method of the present invention.

[0119] As used herein, "food sample" refers to any sample suitable for animal consumption, e.g., human consumption. Food samples may include raw materials, cooked foods, foods of plant and animal origin, pre-processed foods, and partially or completely processed foods, etc. Typically, samples that are not in liquid form are converted to liquid form prior to analysis as described in the methods of the present invention.

[0120] The term "diagnosis", as used herein, refers to methods used or for identifying an analyte, predicting an outcome, and / or predicting a response to a treatment of a disease or condition of interest. Diagnosis can include predicting the likelihood or susceptibility to having a disease or condition, estimating the severity of a disease or condition, determining the risk of progression of a disease or condition, assessing the clinical response to treatment, and / or predicting a response to treatment.

[0121] As used herein, a "biomarker" is any molecule or compound found in a sample of interest and known to be associated with the diagnosis or presence or predisposition of a disease or condition of interest in the subject from which the sample was derived. Biomarkers include, but are not limited to, polypeptides or complexes (e.g., antigens, antibodies), nucleic acids (e.g., DNA, miRNAs, mRNAs), metabolites of drugs, lipids, carbohydrates, hormones, vitamins, etc., known to be associated with a disease or condition of interest.

[0122] "Condition" as used herein with respect to diagnosing a health condition refers to a physiological state of mind or body, as distinguished from other physiological states. A health condition may not be diagnosed as a disease in some cases. Exemplary health conditions of interest include, but are not limited to, nutritional health; aging; exposure to environmental toxins, pesticides, herbicides, synthetic hormone analogs; pregnancy; menopause; andropause; sleep; stress; prediabetes; exercise; fatigue; chemical balance; etc. The term "biotin moiety" refers to an affinity agent comprising a biotin moiety bound to streptavidin or a biotin analog such as desthiobiotin, oxybiotin, 2'-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc. at an affinity of at least 10-8M. The biotin affinity agent may also include a linker, for example, ─LC biotin, ─LC-LC-biotin, ─SLC biotin or ─PEGn biotin, where n is 3-12.

[0123] The term "streptavidin" refers to streptavidin and avidin, and any variants thereof that bind biotin with high affinity. The term "marker," as used in describing a biological sample, refers to an analyte whose presence or abundance in a biological sample is associated with a disease or condition.

[0124] The term "bond" includes covalent and non-covalent bonds, including hydrogen bonds, ionic bonds and bonds resulting from van der Waals forces.

[0125] The term "enhancement" refers to an increase in the amplitude of a signal, for example, at least a 10-fold increase, at least a 100-fold increase, at least a 1000-fold increase, at least a 10,000-fold increase, or at least a 100,000-fold increase in the signal.

[0126] The term "entity" refers to, but is not limited to, proteins, peptides, DNA, RNA, nucleic acids, molecules (large or small), cells, tissues, viruses, nanoparticles of different shapes, which will bind to the "binding site". The entity includes capture agents, probes and blocking agents. "Entity" includes "analyte", and the two terms can be used interchangeably.

[0127] The term "binding site" refers to a location on a solid surface that can immobilize an "entity" in a sample.

[0128] The term "entity partner" refers to, but is not limited to, proteins, peptides, DNA, RNA, nucleic acids, molecules (large or small), cells, tissues, viruses, nanoparticles of different shapes, which are at the "binding site" and will bind to the entity. Entity, including but not limited to capture agent, probe agent, secondary probe agent, or "capture agent / analyte complex".

[0129] The term "target analyte" or "target entity" refers to a specific analyte that is to be specifically analyzed (ie, detected), or a specific entity that is to be specifically bound to a binding site.

[0130] The term "smartphone" or "mobile phone," which may be used interchangeably, refers to a cell phone that has a camera and communications hardware and software that can capture images using the camera, process images captured by the camera, and transmit data to a remote location. In some embodiments, a smartphone has a flash. The term "light" refers, unless otherwise specified, to electromagnetic radiation of various wavelengths.

[0131] The term "average linear dimension" of a region is defined as being equal to the area of ​​the region multiplied by 4 divided by the length of the perimeter of the region. For example, if the region is a rectangle with a width w and a length L, the average linear dimension of the rectangle is 4*W*L / (2*(L+W)) (where "*" means multiplication and " / " means division). According to this definition, the average linear dimension, W is the square of the width W, and d is a circle with a diameter d. The region includes, but is not limited to, the region of a binding point or storage location.

[0132] The term "period" of a periodic array of structures refers to the distance from the center of one structure to the center of the nearest adjacent identical structure.

[0133] The term "storage spot" refers to an area on a plate, wherein the spot contains a reagent to be added to a sample, which reagent is capable of being dissolved into the sample in contact with the reagent and diffused in the sample.

[0134] The term "associated" means that it is related to analyte detection, quantification and / or control of an analyte or entity in a sample or on a plate, control or quantification of a reagent added to a sample or plate.

[0135] The terms "hydrophilicity", "wetness" or "wetness" of a surface mean that the contact angle of a sample on the surface is less than 90 degrees.

[0136] The terms "hydrophobic," "non-wetting," or "non-wetting" of a surface mean that the contact angle of a sample on the surface is equal to or greater than 90 degrees.

[0137] The term "change" in a quantity refers to the difference between the actual value and the desired value or average of a quantity. The term "relative change" in a quantity refers to the ratio of the change to the desired value or average of the quantity. For example, if the desired value of a quantity is Q and the actual value is (Q+), then the change and / (Q+) is the relative change. The term "relative sample thickness change" refers to the ratio of the sample thickness change to the average sample thickness. The term "optically transparent" refers to a material that allows the transmission of an optical signal, wherein the term "optical signal" means, unless otherwise specified, an optical signal used to detect a property of a sample, plate, spacer, scale mark, any structure used, or any combination thereof.

[0138] The term "non-sample volume" refers to the volume between the plates in a closed configuration of a CROF process that is not occupied by the sample but by other objects that are not part of the sample. These objects include, but are not limited to, spacers, bubbles, dust, or any combination thereof. The non-sample volume is often mixed with the sample.

[0139] The term "saturated incubation time" refers to the time required for the binding between two molecules to reach equilibrium (e.g., capture agent and analyte). For surface immobilization assays, "saturated incubation time" refers to the time required for the binding between the target analyte (entity) in the sample and the binding sites on the plate to reach equilibrium, that is, the time after which the average number of target molecules (entities) captured and immobilized by the binding sites is statistically almost constant.

[0140] In some cases, "analyte" and "binding entity" are used interchangeably.

[0141] "Processor", "communication device", "mobile device", refers to a computer system containing basic electronic components (including one or more memories, input-output interfaces, central processing units, instructions, network interfaces, power supplies, etc.) to perform computing tasks. The computer system can be a general-purpose computer containing instructions to perform specific tasks, or it can be a special-purpose computer. "Site" or "location" used in describing signal or data communication refers to a local area where a device or subject is located. A site can refer to a room within a building structure, such as a hospital, or a smaller geographically defined area within a larger geographically defined area. A remote site or remote location refers to a first site that is remote from a second site, a first location that is physically separated from the second site by distance and / or by physical barriers. The remote site can be a first site in a different room of a building structure than the second location, a first site in a different building structure than the second site, a first site in a different city than the second site, and the like.

[0142] As used herein, the term "sample collection site" refers to a location at which a sample can be obtained from a subject. The sample collection site may be, for example, a retailer location (e.g., a chain store, drug store, supermarket, or department store), a supplier's office, a doctor's office, a hospital, a subject's home, a military location, an employer site, or other site or combination of sites. As used herein, the term "sample collection site" may also refer to the owner or representative of a business, service, or institution associated with the site.

[0143] As used herein, "raw data" includes signals and direct readouts from sensors, cameras and other components and instruments that detect or measure properties or characteristics of a sample. For example, raw data includes voltage or current output from a sensor, detector, counter, camera or other component or device; raw data includes digital or analog digital output from a sensor, detector, counter, camera or other component or device; and raw data may include digitized or filtered output from a sensor, detector, counter, camera or other component or device. For example, raw data includes the output of a photometer, which may include an output in "relative light units" relative to the number of photons detected by the photometer. Raw data may include a JPEG, bitmap or other image file generated by a camera. Raw data may include cell counts; light intensity (at a specific wavelength, or at or within a wavelength range); rate of change of detector output; difference between two similar measurements; number of events detected; number of events detected within a predetermined range or meeting a predetermined criterion; minimum value measured within a certain period of time, or within a field of view; maximum value measured within a certain period of time, or within a field of view; and other data. When sufficient, raw data can be used without further processing or analysis. In other cases, the raw data may be further processed or used for further analysis related to the sample, subject, or for further analysis for other purposes.

[0144] "Representative of a sample" refers to an output signal or raw data representative of a sample, and refers to an output signal or raw data reflecting a measured property of the sample or a portion thereof, for example, reflecting the amount of an analyte of interest present in the sample. For example, the intensity of the fluorescent signal of a representative sample may be greater in a fluorescently labeled sample containing more analyte of interest than in a fluorescently labeled sample containing less analyte of interest.

[0145] As used herein, "Clinical Laboratory Improvement Amendments" and "CLIA" refer to Part F of 42 U.S.C., e.g., subpart 2, sections 263A to 263A7, 42 CFR Chapter W (sections 493.1 to 493.2001), and related laws, regulations, and amendments. Regulations related to CLIA are administered by the Centers for Medicare and Medicaid Services (CMS) of the U.S. Department of Health and Human Services.

[0146] As used herein, "process management" refers to a set of methods and systems used to plan and / or monitor process performance, such as a sample analysis process.

[0147] As will be apparent to those skilled in the art who read this disclosure, each of the various embodiments described and illustrated herein has discrete elements and features that may be separated or combined with the features of several other embodiments without departing from the scope or spirit of the present teachings. Any of the methods described may be performed in the order of events recited or in any other order that is logically possible.

[0148] It will be understood by one skilled in the art that the present invention is not limited in its application to the details of construction, arrangement of components, class selection, weighting, predetermined signals, or steps arranged or listed in the specification or drawings herein. The present invention can be implemented or carried out in many different ways.

[0149] The practice of various embodiments of the present disclosure uses, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA within the skill of the art. See Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., ed., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: Practical Techniques (MJ MacPherson, BD Hames and GR Taylor, ed. (1995)), Harlow and Lane, ed. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (RI Freshney, ed. (1987)).

[0150] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise, such as when the word "single" is used. For example, reference to "an analyte" includes a single analyte and multiple analytes, reference to "a capture agent" includes a single capture agent and multiple capture agents, reference to "a probe agent" includes a single probe agent and multiple probe agents and reference to "a reagent" includes one reagent and multiple reagents.

[0151] Compression Controlled Open Flow

[0152] In an assay, manipulation of samples or reagents can result in improvements in the assay. Manipulation includes, but is not limited to, manipulation of the geometry and position of a sample and / or reagents, mixing or combining of samples and reagents, and contact area of ​​the sample or reagents with the plate.

[0153] Many embodiments of the present invention utilize a method for manipulating sample and / or reagent geometry, location, contact area, and mixing, a method referred to as "Compression Controlled Open Flow (CROF)", and devices for performing CROF.

[0154] The term "compressed open flow (COF)" refers to the process of compressing a sample between two plates by (i) placing at least a portion of the sample above the other plate and (ii) then compressing the sample by pushing the two plates toward each other; wherein the compression reduces the thickness of at least a portion of the sample, causing the sample to flow into the open space between the plates, thereby changing the shape of the flowable sample deposited on the plates. The term "compressed controlled open flow" or "CROF" (or "self-calibrated compressed open flow" or "SCOF" or "SCCOF") refers to a specific type of COF in which the final thickness of a portion or all of the sample after compression is "adjusted" by a spacer, wherein the spacer is placed between the two plates.

[0155] In a CROF the term "the final thickness of a portion or all of the sample is adjusted by the spacers" means that during a CROF, once a specific sample thickness is reached, the relative motion of the two plates ceases and the sample thickness changes thereby, wherein the specific thickness is determined by the spacers.

[0156] One embodiment of the CROF method, such as Figure 1 As shown, including:

[0157] (a) obtaining a flowable sample;

[0158] (b) obtaining a first plate and a second plate, the plates being movable relative to each other into different configurations, wherein each plate has a sample contacting surface that is substantially flat, and one or both plates include spacers, and wherein the spacers have a predetermined substantially uniform height, the spacers being above the respective sample contacting surfaces;

[0159] (c) depositing a sample on one or both plates when the two plates are in an open phase, wherein the open phase is a phase in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by a spacer; and

[0160] (d) after completing (c), by moving the plates to a closed phase, wherein, in the closed phase, the plates face each other, the spacer and a related volume of the sample are between the plates, the thickness of the sample related volume is adjusted by the plates and the spacer, wherein the related related volume is at least a portion of the sample, and wherein when the sample expands, the sample flows laterally between the two plates.

[0161] The term "plate" refers, unless otherwise specified, to a plate used in a CROF process, ie a solid body having a surface that can be used with another plate to compress a sample located between the two plates to reduce the thickness of the sample.

[0162] The term "plate" or "a pair of plates" refers to the two plates in a CROF process.

[0163] The term "first plate" or "second plate" refers to the plate used in a CROF process.

[0164] The term "plates facing each other" refers to a situation in which at least parts of a pair of plates face each other.

[0165] The term "spacer" or "stopper" refers, unless otherwise specified, to a mechanical object that is placed between two plates to set the minimum value of the spacing between the two plates that can be achieved when the two plates are pressed together. That is, when compressed, the spacer will stop the relative movement of the two plates to prevent the spacing between the plates from becoming less than a preset (i.e., predetermined) value. There are two types of spacers: "open spacers" and "closed spacers."

[0166] The term "open spacer" means that the spacer has a shape that allows liquid to flow around the entire perimeter of the spacer and through the spacer. For example, a post is an open spacer.

[0167] The term "closed spacer" refers to a spacer having a shape in which a liquid cannot flow around the entire perimeter of the spacer and cannot flow through the spacer. For example, an annular spacer is a closed spacer for liquid within the ring, wherein the liquid within the annular spacer remains within the ring and cannot go to the outside (periphery).

[0168] The terms "spacers have a predetermined height" and "spacers have a predetermined inter-spacer distance" mean that the values ​​of the spacer height and the inter-spacer distance are known before the CROF process. If the values ​​of the spacer height and the inter-spacer distance are unknown before the CROF process, it is not predetermined. For example, beads sprayed on a plate are used as spacers, wherein the positions of the beads landing on the plate are random, and the inter-spacer distance is not predetermined. Another example of unspecified inter-spacer distance is that the spacer moves during the CROF process.

[0169] The term "spacer is fixed to its corresponding board" during a CROF process means that the spacer is attached to a position on the board and the attachment is maintained during the CROF (i.e., the position of the corresponding board on the spacer does not change). An example of "spacer is fixed to its corresponding board" is that a spacer is made integrally from a material of a board and the position of the spacer relative to the board surface does not change during the CROF. An example of "spacer is not fixed to its corresponding board" is that a spacer is glued to the board by an adhesive, and during the process of using the board, during the CROF, the adhesive cannot maintain the spacer in its original position on the board surface and the spacer moves away from the original position on the board surface.

[0170] The term "spacer is integrally fixed to the plate" means that the spacer and the plate act like a single piece during use and the spacer will not move or be displaced from its original position on the plate.

[0171] The term "open phase" in the CROF process refers to a phase in which two plates are partially or completely separated and the spacing between the plates is not regulated by a spacer.

[0172] The term "closed phase" in the CROF process refers to a phase in which the plates face each other, the spacer and the relevant volume of the sample are between the two plates, the thickness of the relevant volume of the sample is the thickness of the plates and the spacer, and wherein the relevant volume is at least a part of the whole of the sample.

[0173] The term "sample thickness is adjusted by the plate and spacer" in the CROF process means that under given conditions of the plate, sample, spacer, and the plate compression method, the thickness of at least a portion of the sample in the closed phase of the plate can be predetermined from the properties of the spacer and the plate.

[0174] The term "inner surface", or "sample side" of a plate in a CROF apparatus refers to the surface of the plate that contacts the sample, while the other surface of the plate (ie, not contacting the sample) is referred to as the "outer surface".

[0175] The term "X-plate" of a CROF apparatus refers to a plate comprising spacers on a sample surface of the plate, wherein the spacers have a predetermined inter-spacer distance and spacer height, and wherein at least one of the spacers is within a sample contacting area.

[0176] The term "CROF apparatus" refers to an apparatus that performs a CROF process. The term "CROFed" means that a CROF process has been used. For example, the term "a sample CROFed" means that the sample was placed in a CROF apparatus, subjected to CROF treatment, and that the sample was maintained, unless otherwise specified, in the final phase of CROF.

[0177] The term "CROF plates" refers to the two plates used in performing the CROF process.

[0178] The term "surface smoothness" or "surface smoothness variation" of a planar surface refers to the average deviation of a plane from a perfect plane over short distances of a few microns or less. Surface smoothness is not the same as surface flatness variation. A planar surface can have a good surface flatness and also have poor surface smoothness.

[0179] The terms "surface flatness" or "surface flatness variation" of a planar surface refer to the average deviation of a planar surface from a perfect plane over longer distances of about or greater than 10 microns. Variations in surface flatness are distinct from variations in surface smoothness. A planar surface can have a good surface smoothness but poor surface flatness (i.e., large surface flatness deviations).

[0180] The term "relative surface flatness" of a plate or sample is the ratio of the change in plate surface flatness to the final sample thickness.

[0181] In the CROF process, the term "final sample thickness" refers, unless otherwise stated, to the thickness of the sample in the closed phase of the panel during the CORF process.

[0182] The CROF term "compression method" refers to the method of bringing two plates from an open phase to a closed phase.

[0183] The term "region of interest" or "area of ​​interest" of a board refers to an area of ​​a board that is relevant to the function that the board performs.

[0184] The term “at most” means “equal to or less than.” For example, a spacer height of at most 1 micrometer means that the spacer height is equal to or less than 1 micrometer.

[0185] The term "sample area" refers to an area of ​​the sample that is approximately parallel to the space between the plates and perpendicular to the thickness of the sample.

[0186] The term "sample thickness" refers to the dimension of the sample in a direction perpendicular to the surfaces of two plates facing each other (eg, in the direction of the spacing between the plates).

[0187] The term "plate spacing" refers to the distance between the inner surfaces of two plates.

[0188] The CROF term "deviation in final sample thickness" refers to the difference between the predetermined spacer height (determined from the manufacture of the spacers) and the thickness of the final sample, where the average final sample thickness is the average over a given area (e.g., the average of 25 different points (4 mm apart) over an area of ​​more than 1.60 cm by 1.6 cm).

[0189] The term "uniformity of the measured final sample thickness" for the CROF process refers to the standard deviation of the measured final sample thickness over a given sample area (eg, standard deviation relative to the mean).

[0190] The terms "relevant volume of sample", and "relevant area of ​​sample" in the CROF process refer to the volume and area of ​​the whole or part of the sample deposited on the plate during the CROF process, which are related to the function performed by the corresponding method or device, wherein the function includes, but is not limited to, reducing the time required for analyte or entity binding, detecting analyte, quantifying volume, quantifying concentration, mixing reagents, or controlling the concentration of (analyte, entity or reagent).

[0191] The terms "some embodiments", "in some embodiments", "in the present invention, in some embodiments", "example", "one example", "another embodiment", "certain embodiments", "in multiple embodiments", or the like refer to the embodiment(s) disclosed in their entirety (i.e., the entire invention) unless otherwise stated.

[0192] The term "height" or "thickness" of an object processed in CROF refers, unless otherwise specified, to the dimension of the object in the direction perpendicular to the surface of the board. For example, the height of a spacer is the dimension of the spacer in the direction perpendicular to the surface of the board, and spacer height and spacer thickness refer to the same thing.

[0193] The term "area" of an object in the CROF process refers to the area of ​​the object parallel to the surface of the plate unless otherwise specified. For example, the area of ​​a spacer is the area of ​​the spacer parallel to the surface of the plate.

[0194] The terms "lateral" or "laterally" in the CROF process refer, unless otherwise specified, to a direction parallel to the surface of the sheet.

[0195] The term "width" of a spacer in the CROF process refers to, unless otherwise specified, the lateral dimension of the spacer.

[0196] The term "spacer within the sample" means that the spacer is surrounded by the sample (eg, a pillar spacer within the sample).

[0197] The term "critical bending span" of a board in the CROF process means that for a given flexible board, sample, and compressive force, the span of the board between two supports at which the board bends is equal to its allowed bending. For example, if for a given flexible board, sample, and compressive force, the allowed bending is 50 nanometers and the critical bending span is 40 microns, the bending of the board between two adjacent spacers will be 40um apart from 50 nanometers, and the bending will be less than 50 nanometers if the two adjacent spacers are less than 40 microns.

[0198] The term "flowable" sample refers to a sample that increases in transverse dimension as the thickness of the sample decreases. For example, a stool sample is considered flowable.

[0199] In some embodiments of the invention, a CROF-processed sample does not have to be flowable to benefit from the process, as long as the thickness of the sample can be reduced during the CROF process. For example, by placing a dye on the surface of a CROF plate to stain a tissue, the CROF process can reduce tissue thickness and thereby speed up the saturation incubation time of the dye staining.

[0200] 1. Reduce (shorten) the combination or mixing time (X)

[0201] Reducing the incubation / reaction time in performing an assay or other chemical reaction is. For example, in a surface immobilization assay where the target analyte in a sample is captured by a capture agent (i.e., solid phase) immobilized on a plate surface, it is often desirable to have short saturation incubation times for capturing the target analyte on the plate surface, or for both the sample in solution, or for immobilization of both the capture agent and the detection agent. Another example is the need to shorten the time it takes to coat the capture agent on the plate surface. And yet another example is the need to shorten the time it takes for the reagent to be mixed into the sample.

[0202] The present invention provides methods and designs that reduce (i.e., shorten) the incubation time required to saturate the binding sites of a bound entity on a solid surface (i.e., time to go from one volume to one entity on a surface). Another aspect of the invention is to reduce the time required to bind to binding sites stored on a disk surface on another disk surface (i.e., time to go from one surface to another). Another aspect of the invention is to reduce the time required to convert a volume of reagent stored on a surface / mixed sample (i.e., time to add / mix the volume of sample from the reagent on the surface).

[0203] The present invention reduces binding and / or mixing by using a device and method for spreading a sample (or liquid) with a thinner thickness, thereby reducing the saturation incubation time when measuring mixing when diffusing an entity in the sample thickness. The diffusion time of an entity in a material (such as a liquid or solid or semi-solid) is proportional to the diffusion distance in the direction, therefore, reducing the thickness of the sample can reduce the diffusion distance, resulting in a sharp reduction in diffusion time and saturation incubation time. Thinner thickness (for example, a tightly closed space) also increases the frequency of physical collisions of one material with other entities, thereby further improving binding and mixing. Themeans in the present invention also enable the reduction of sample thickness to be precise, uniform, rapid, simple (fewer operating steps) and applicable to reduce the thickness of the sample to micrometers or nanometers thick. The invention has great utilities for rapid, low-cost, PoC, diagnostics and chemical / biological analysis. Several embodiments of the present invention are Figure 1-4 Shown in.

[0204] 1.1 Reduce the saturation incubation time required for binding entities to binding sites on the solid surface by reducing the sample thickness.

[0205] X1. A method for reducing the saturation of binding sites on the plate surface for binding target entities in a sample by incubation time. Figure 1-2 , Figure 3 a, and as shown in 4a, including:

[0206] (a) obtaining a sample that is flowable and contains a target entity that is capable of diffusing with the sample;

[0207] (b) obtaining a first plate and a second plate movable relative to each other into different configurations, wherein the first plate has, on its surface, binding points configured for binding of a target entity, wherein one or both of the plates include spacers, and each of the spacers is secured to its corresponding plate and has a predetermined height;

[0208] (c) depositing, when the plates are constructed in an open configuration, on a sample of one or two plates; the open configuration is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by a spacer;

[0209] (d) after (c), spreading the sample by bringing the plates into a closed configuration, wherein in the closed configuration: the plates are facing each other, the spacer and a relevant volume of the sample are between the plates, the bonding site is in contact with the relevant volume, and the thickness of the relevant volume of the sample is regulated by the plates and the spacer, which is thinner than the maximum thickness of the sample when the plates are in the open configuration;

[0210] The relevant volume part or the whole volume of the sample and

[0211] Therein, a decrease in the thickness of the sample reduces the saturation incubation time for the binding of the target entity at the binding sites of the relevant volume.

[0212] For a given sample volume, a CROF reduces sample thickness but increases the lateral dimensions of the sample. The present invention exploits the fact to perform (a) local binding or mixing in a portion of the sample, and (b) multiplexing of multiple binding or mixing networks without fluid barriers to separate the sample fluid into different pockets of isolation fluid.

[0213] X2. A device for reducing the saturation incubation time for the volume of a target entity bound to the surface of a sample. Figure 1-2 , Figure 3 a, and as shown in 4a, including:

[0214] A first plate and a second plate which (a) are movable relative to each other into different configurations, (b) each plate having a sample contact area for contacting a sample with a target entity having an associated volume in the sample, (c) one of the plates having a bound target entity, and (d) at least one of the plates includes spacer binding sites having a predetermined distance and height between spacers and affixed to its respective surface, wherein at least one of the spacers is within the sample contact area;

[0215] wherein one of the configurations is an open structure, wherein: the two plates are partially or completely separated, and the spacing between the plates is not adjusted by a spacer,

[0216] Another configuration is a closed structure, which is configured after sample deposition in an open phase; and in the closed phase: the plates face each other, the spacer and the relevant volume of the sample are between the plates, the binding point is in contact with the relevant volume, and the thickness of the relevant volume of the sample is adjusted by the plates and the spacer, and is thinner than the maximum thickness of the sample when the plates are in the open phase; wherein a portion of the relevant volume or the entire volume of the sample and wherein the sample thickness is reduced reduces the saturation incubation time for a target entity bound to the binding point in the relevant volume.

[0217] 1.2 Reduce the saturation incubation time to store binding sites on one surface of the binding entities on other surfaces

[0218] X3. A method of reducing the saturation incubation time to bind an entity stored at a storage site on one plate to a corresponding binding site on another plate. Figure 1 , as shown in 3c and 4b, including:

[0219] (a) obtaining a first plate and a second plate movable relative to each other into different configurations, wherein a surface of the first plate has a binding site, and a surface of the second plate has a storage site comprising a solid body bonded to the binding site; wherein an area of ​​the binding site and an area of ​​the storage site are smaller than those of the corresponding plates; and wherein one or both of the plates include spacers and each spacer is fixed to its corresponding plate and has a predetermined height;

[0220] (b) obtaining a transfer medium, wherein the entity at the storage point is capable of being dissolved into the transfer medium and diffusing into the transfer medium;

[0221] (c) depositing, when the plates are configured in an open configuration, a transfer medium on one or both plates; the open configuration being a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by a spacer;

[0222] (d) after (c), spreading the transfer medium by bringing the plates into a closed phase, wherein in the closed configuration: the plates face each other, the spacers, the bonding points, the storage sites and at least a portion of the transfer medium are between the plates, the bonding points and the storage sites are at least partially on top of each other, the transfer medium contacts at least a portion of the bonding points and the storage sites, and the thickness of the transfer medium is adjusted by the plates and the spacers and is thinner than the maximum thickness of the transfer medium when the plates are in an open phase;

[0223] The reduced thickness of the transfer medium reduces the time of gluttony of the entities of the first sheet stored at the junctions of the second sheet.

[0224] X4. A physical device for reducing the saturation incubation time of a binding site stored in a disk at a binding site on another disk. Figure 1 , as shown in 3c and 4b, including:

[0225] A first plate and a second plate move relative to each other to form different configurations, wherein a surface of the first plate has a bonding site; and a surface of the second plate has a storage site including a body bonded to the bonding site; wherein the area of ​​the bonding site and the storage site are smaller than the area of ​​the corresponding plate; and wherein one or both of the plates include spacers and each spacer is fixed to its corresponding plate and has a predetermined height;

[0226] wherein one of the configurations is an open structure wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by a spacer, a transfer medium is capable of being deposited on one or both plates, wherein the entity at the storage point is capable of being dissolved into the transfer medium and the transfer medium diffuses,

[0227] Another configuration is a closed structure, which is configured after the transfer medium is deposited in the open phase; and in the closed phase: the plates face each other, the spacers, the bonding sites, the storage sites and at least a portion of the transfer medium are between the plates, the bonding sites and the storage sites are at least partially on top of each other, at least a portion of the bonding sites and the storage sites, the thickness of the transfer medium is regulated by the plates and the spacers when the transfer medium contacts, is thinner than the maximum thickness of the transfer medium when the plates are in the open phase;

[0228] The incubation time in which the thickness of the transfer medium is reduced reduces the saturation for the entity to overeat the storage sites of the second plate to the binding sites of the first plate.

[0229] In the method of paragraph X3 and the device of paragraph X4, in some embodiments, the transfer medium comprises a liquid that allows diffusion of the entity or the agent or both.

[0230] In the method of paragraph X3 and the device of paragraph X4, in some embodiments, the transfer medium is a sample, wherein the sample contains an analyte (also called a target analyte) bound to the binding site.

[0231] In the method of paragraph X3 and the device of paragraph X4, in some embodiments, the transfer medium is a sample of a binding site, wherein the sample contains an analyte (also called a target analyte) and a reagent is a detection agent that binds the analyte.

[0232] 1.3 When adding (mixing) reagents stored on the surface to reduce the liquid sample

[0233] Many assays require reagents (including liquids) to be added to a sample. Often the concentration of the reagent to be added to the sample or liquid must be controlled. There is a need for new methods to perform such reagent addition and concentration control simply and / or at low cost. Two examples where reagent addition is required are: (a) blood cell counting, where an anticoagulant and / or staining reagent(s) may be added to a blood sample, and (b) immunoassays where a probe is added to bind to a target analyte in solution.

[0234] One aspect of the present invention is a method, apparatus and system that makes reagent addition and reagent concentration control simple and / or low cost. In the current invention, an embodiment of a reagent layer (e.g., a dried reagent layer) is first placed on a CROF device, and then the sample is deposited onto the plate surface of the CROF device, and a CROF process makes the sample contact with the reagent and the thickness of the sample is greater than the thickness of the sample when it is thinner in the open configuration of the CROF plate. By reducing the thickness of the sample, this will reduce the diffusion time of the reagent to diffuse from the surface to the entire sample, and thus reduce the time for the reagent to mix with the sample.

[0235] X5. A method for reducing the time it takes for a reagent stored on the plate surface to be mixed with a sample. Figure 1 , as shown in 3b and 4c, including:

[0236] (a) obtaining a first plate and a second plate in different configurations relative to each other, wherein the first plate has a movable storage site on its surface which contains a reagent to be added to the sample, and the reagent is capable of being dissolved into the sample and diffused into the sample; and wherein one or both of the plates include spacers and each spacer is fixed to its corresponding plate and has a predetermined height;

[0237] (b) the sample obtained;

[0238] (c) depositing, when the plates are constructed in an open configuration, on a sample of one or two plates; the open configuration is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by a spacer;

[0239] (d) after (c), spreading the sample by bringing the plates into a closed configuration, wherein in the closed configuration: the plates are facing each other, the spacers, the storage points, and at least a portion of the sample is between the plates, at least a portion of the storage points are in contact with the sample, the thickness of the sample at the storage points is adjusted by the plates and the spacers, and is thinner than the maximum thickness of the sample when the plates are in the open configuration;

[0240] Among other things, the reduced thickness of the sample reduces the time required for reagents to mix with the sample storage site.

[0241] The method of paragraph X5, further comprising the step of incubating while the plate is in the closed phase, wherein the incubation time is selected so as to result in a significant number of reagents dissolved in the sample being contained in a volume associated with the sample, wherein the volume associated with the sample is in a storage location and the incubation is a process to allow the reagents to dissolve and diffuse in the sample.

[0242] In the method of paragraph X5, it further includes a step of, after (d) and, while the plate is in the closed phase, incubating the sample for a time equal to or less than a factor of the diffusion time of the reagent throughout the sample, the thickness of the sample being adjusted by the plate in the closed phase, and then stopping the incubation wherein the incubation allows the reagent to diffuse into the sample; and wherein the factor is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000, 10,000 or any range between values. For example, if the factor is 1.1 and the diffusion time is 20 seconds, then the incubation time is equal to or less than 22 seconds. In a preferred embodiment, the factor is 0.1, 1, 1.5 or any range between values.

[0243] X6. A device for reducing the time required to add reagents stored on the surface of a plate to a sample, such as Figure 1 , as shown in 3b and 4c, including:

[0244] A first plate and a second plate are movable relative to each other into different configurations, wherein the first plate has storage sites on its surface which contain reagents to be added to the sample, the reagents being capable of being dissolved into the sample and diffusing in the sample; and wherein one or both of the plates include spacers and each spacer is fixed with its corresponding plate and has a predetermined height;

[0245] One of the configurations is an open structure, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by a spacer, and the sample is deposited on one or both plates;

[0246] Another configuration is a closed structure, which is configured after the transfer medium is deposited in the open phase; and in the closed phase: the plates face each other, the spacer, the storage point, and at least a portion of the sample are between the plates, the sample contacts at least a portion of the storage point, and the thickness of the sample at the storage point is adjusted by the plates and the spacer and is thinner than the maximum thickness of the sample when the plates are in the open phase;

[0247] Among other things, the reduced thickness of the sample reduces the time required for reagents to mix with the sample storage site.

[0248] M1. In the method or apparatus X1-6 of any paragraph, in some embodiments, the associated volume of the sample is a binding point or storage point seated in the volume of the sample (ie, on top).

[0249] M2. In the method or apparatus X1-6 of any paragraph, in some embodiments, the relevant volume of the sample is the entire area or partial area of ​​the binding point located on (ie, at the top) the volume or storage site of the sample.

[0250] M3. In the method or device X1-6 of any paragraph, in some embodiments, the ratio of the lateral dimension of the binding point or the storage site to the thickness of the sample in the closed phase is 1.5 3 or greater, 3 or greater, 5 or greater, 10 or greater, 20 or greater, 30 or greater, 50 or greater, 100 or greater, 200 or greater, 1000 or greater, 10,000 or greater, or within a range between any two of these values.

[0251] In the method or any device of paragraphs X1-6, the ratio of the lateral dimension of the binding point or storage site to the sample thickness in the closed phase is 3 and 20 in one preferred embodiment, between 20 and 100 in another preferred embodiment, between 100 and 1000 in another preferred embodiment, and between 1000 and 10000 in another preferred embodiment.

[0252] In the methods of any of paragraphs X1 and X3, in some embodiments, the area of ​​the binding site is significantly reduced compared to the sample thickness, so that entities in the sample area that are outside the binding site will bind to the binding site for a longer time. With the appropriate choice of incubation time, the entities bound to the binding site will be mainly at the binding site sites on the entities in the sample volume (i.e., the sample volume, which is just the binding area described above). The calculation of the concentration of the entity in the sample will then be based on the thickness of the sample and the area of ​​the binding site.

[0253] In the method of paragraph X5, in some embodiments, the final reduced sampling thickness is significantly smaller than the area of ​​the storage site, thereby making the entity

[0254] Areas in the sample outside of the binding sites will take longer to bind to the binding sites. With proper choice of incubation time, entities bound to the binding sites will be primarily at the binding site sites of the entity in the sample volume (i.e., the sample volume, which is just the binding area described above). Calculation of the concentration of the entity in the sample will then be based on the thickness of the sample and the area of ​​the binding sites.

[0255] In any of the methods of paragraphs X2, X4, and X6, the method further includes converting the plate from the open phase to a closed structure of the compression device. In some embodiments, the compression device is one or any combination of the embodiments described in the disclosure.

[0256] In the method of any of paragraphs X2, X4, and X6, it further includes a compression device that brings the plate from an open phase to a closed configuration, and a holding device configured to hold the plate in the closed configuration. In some embodiments, the holding device is one or any combination of the embodiments described in the disclosure.

[0257] In the method of any of paragraphs X2, X4, and X6, it further includes a compression device that brings the plate from an open phase to a closed configuration, and is configured to hold the plate in a closed phase for a period of 0.001 seconds or less, 0.01 seconds or less, 0.1 seconds or less, 1 second or less, 5 seconds or less, 10 seconds or less, 20 seconds or less, 30 seconds or less, 40 seconds or less, 1 minute or less, 2 minutes or less, 3 minutes or less, 5 minutes or less, 10 minutes or less, 20 minutes or less, 30 minutes or less, 60 minutes or less, 90 minutes or less, 120 minutes or less, 180 minutes or less, 250 minutes or less, or a range between any two of these values.

[0258] In the method of any of paragraphs X2, X4, and X6, it further includes a compression device that brings the plate from an open phase to a closed configuration, and a holding device configured to maintain the plate in a closed phase for a time that, in a preferred embodiment, is less than 0.001 seconds, less than 0.01 seconds, 0.1 seconds or less, 1 second or less, 5 seconds or less, 10 seconds or less, 20 seconds or less, 30 seconds or less, 40 seconds or less, 1 minute or less, 2 minutes or less, 3 minutes or less, or a range between any two of these values.

[0259] Final sample thickness. The final sample thickness at the closed phase of the plate can be a significant factor in reducing the saturation incubation time. The thickness reduction / final sample thickness after deformation depends on the physical properties and sample as well as the application, as discussed with respect to the adjustment spacing of the plate.

[0260] In some embodiments, the final sample thickness is less than about 0.5 micrometers (micrometers), less than about 1 micrometer, less than about 1.5 micrometers, less than about 2 micrometers, less than about 4 micrometers, less than about 6 micrometers, less than about 8 micrometers, less than about 10 micrometers, less than about 12 micrometers, less than about 14 micrometers, less than about 16 micrometers, less than about 18 micrometers, less than about 20 micrometers, less than about 25 micrometers, less than about 30 micrometers, less than about 35 micrometers, less than 40 micrometers, less than 45 micrometers, less than 50 micrometers, less than 55 micrometers, less than 60 micrometers, less than 70 micrometers, less than 80 micrometers, less than about 90 micrometers, less than about 100 micrometers, less than about 110 micrometers, less than about 120 micrometers, less than about 140 micrometers, less than about 160 micrometers, less than about 170 micrometers, less than about 180 micrometers, less than about 190 micrometers, less than about 200 micrometers, less than about 210 micrometers, less than about 220 micrometers, less than about 230 micrometers, less than about 240 micrometers, less than about 250 micrometers, less than about 260 micrometers, less than about 270 micrometers, less than about 280 micrometers, less than about 290 micrometers, less than about 300 micrometers, less than about 310 micrometers, less than about 320 micrometers, less than about 330 micrometers, less than about 350 micrometers, less than about 360 micrometers, less than about 3 less than about 180 microns, less than about 200 microns, less than about 250 microns, less than about 300 microns, less than about 350 microns, less than about 400 microns, less than about 450 microns, less than about 500 microns, less than about 550 microns, less than about 600 microns, less than about 650 microns, less than about 700 microns, less than about 800 microns, less than about 900 microns, less than about 1000 microns (1 mm), less than about 1.5 mm, less than about 2 mm, less than about 2.5 mm, less than about 3 mm, less than about 3.5 mm, less than about 4 mm, less than about 5 mm, less than about 6 mm, less than about 7 mm, less than about 8 mm, less than about 9 mm, less than about 10 mm, or in a range between any two of these values.

[0261] In some embodiments, the final sample thickness in the closed configuration is less than 0.5 microns (micrometers), less than 1 micron, less than 5 microns, less than 10 microns, less than 20 microns, less than 30 microns, less than 50 microns, less than 100 microns, less than 200 microns, less than 300 microns, less than 500 microns, less than 800 microns, less than 200 microns, less than 1 millimeter (millimeter), less than 2 mm (millimeter), less than 4 millimeters (millimeters), less than 8 millimeters (millimeters), or a range between any two of these values.

[0262] In certain embodiments, the Q-method allows for uniform final sample thickness and flat surfaces of the first plate and the second plate are used.

[0263] In the present invention, sample incubation is accomplished at various temperatures, humidities, gas environments, and for different durations, with or without shaking.

[0264] Incubation time. The method of any of paragraphs X1 and X3, further comprising a step of, after (d) and while the plate is in the closed phase, incubating the sample for a time equal to or less than a factor multiplied by the diffusion time for the entity to diffuse across the sample thickness conditioned by the plate in the closed phase, and then stopping the incubation wherein the incubation allows the entity to bind to the binding sites; and wherein the factor is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000, 10,000 or any range therebetween. For example, if the factor is 1.1 and the diffusion time is 20 seconds, then the incubation time is equal to or less than 22 seconds. In a preferred embodiment, the factor is 0.1, 1, 1.5 or any range therebetween.

[0265] The method of paragraph X5, further comprising the step of, after (d) and, while the plate is in the closed phase, incubating the sample at a time equal to or less than a factor times the diffusion time of the reagent in the sample thickness by conditioning the plate in the closed phase, and then stopping the incubation wherein the incubation allows binding of entities to the binding sites; and wherein the factor is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000, 10,000 or any range therebetween. For example, if the factor is 1.1 and the diffusion time is 20 seconds, then the incubation time is equal to or less than 22 seconds. In a preferred embodiment, the factor is 0.1, 1, 1.5 or any range therebetween.

[0266] M4. The method of paragraph 1 of any of paragraphs X1, X3 and X5, or any of paragraphs X2, X4 and X6, wherein at least one of the spacers is within the sample contact region.

[0267] M5. The device of any of paragraphs X1, X3 and X5, or any of paragraphs X2, X4, and the method of paragraph X6, wherein the spacers have a predetermined inter-spacer distance.

[0268] M6. In the method of any of paragraphs X1, X3, and X5, further comprising the step of incubating while the plate is in the closed phase, the saturation incubation time is 0.001 seconds or less, 0.01 seconds or less, 0.1 seconds or less, 1 second or less, 5 seconds or less, 10 seconds or less, 20 seconds or less, 30 seconds or less, 40 seconds or less, 1 minute or less, 2 minutes or less, 3 minutes or less, 5 minutes or less, 10 minutes or less, 20 minutes or less, 30 minutes or less, 60 minutes or less, 90 minutes or less, 120 minutes or less, 180 minutes or less, 250 minutes or less, or a range between any two of these values.

[0269] M7. In the method of any of paragraphs X1, X3, and X5, the saturation incubation time at the reduced sample thickness in the closed phase is 0.001 seconds or less, 0.01 seconds or less, 0.1 seconds or less, 1 second or less, 5 seconds or less, 10 seconds or less, 20 seconds or less, 30 seconds or less, 40 seconds or less, 1 minute or less, 2 minutes or less, 3 minutes or less, 5 minutes or less, 10 minutes or less, 20 minutes or less, 30 minutes or less, 60 minutes or less, 90 minutes or less, 120 minutes or less, 180 minutes or less, 250 minutes or less, or a range between any two of these values.

[0270] In some embodiments, first the capture agent is immobilized on the binding site, then the sample is placed in contact with the binding site and the entity in the sample is captured by the capture agent, and finally the detection agent is added to bind with the captured entity and a signal from the detection agent will be read out (e.g., by optical methods or electrical methods or a combination). In some embodiments, other reagents are added in addition to the capture agent and the detection agent (e.g., blocking agents).

[0271] In many applications, such as PoC, it is desirable to have a simple and / or low cost device and method to add additional reagents to a sample. One aspect of the invention relates to a simple and / or low cost device and method to add additional reagents to a sample. The additional reagents added include probes, adhesives, optical signal enhancers, optical signal quenchers, or others. In some embodiments of the invention, it controls the determination of the process by using different release times of reagents stored in the same location. Different release times can be connected by adding other materials with different dissolution rates.

[0272] In certain embodiments, the concentration of the reagents mixed in the sample can be controlled by controlling the sample thickness (eg, controlling the ratio of sample thickness to storage site area and / or mixing).

[0273] 2. Plates, spacers, scale markings, sample thickness adjustment

[0274] 2.1 Plate structure and sample thickness adjustment

[0275] In some embodiments, in the open configuration, the two panels (i.e., the first panel and the second panel) are separated from each other. In some embodiments, the two panels have one side connected together during all operations of the panels (including the open and closed phases), and the two panels are similar to a book opening and closing. In some embodiments, the two panels have a rectangular (or square) shape, and the two sides of the rectangle are connected together during all operations of the panels.

[0276] In some embodiments, the open phase includes a configuration where the plates are spaced apart from each other such that a sample is deposited into one pair of plates without interfering with the other plate in another pair of plates.

[0277] In some embodiments, the open configuration includes a configuration in which the plates are apart such that the sample is deposited directly onto one plate as if the other plate were not present.

[0278] In some embodiments, the open phase comprises a configuration where a pair of plates are separated by at least 10 nm, at least 100 nm, at least 1000 nm, at least 0.01 cm, at least 0.1 cm, at least 0.5 cm, at least 1 cm, at least 2 cm, or at least 5 cm, or a range of any two values.

[0279] In some embodiments, the open phase comprises a configuration in which a pair of plates are oriented in different orientations. In some embodiments, the open phase comprises a configuration defining an access gap between a pair of plates configured to allow addition of a sample.

[0280] In some embodiments, the open configuration includes a configuration wherein each plate has a sample contacting surface, and wherein at least one contacting surface of the plates is exposed when the plates are in an open phase.

[0281] Closed phase and sample thickness adjustment. In the present invention, the closed phase of two plates is a configuration in which the spacing (i.e., distance) between the inner surfaces of the two plates is adjusted by the spacer between the two plates. Since the inner surfaces of the plates (also referred to as "sample surfaces") are in contact with the sample during the compression step of the CROF process, the sample thickness is adjusted by the spacer in the closed configuration.

[0282] In the process of bringing the plates from the open phase to the closed phase, the plates face each other (at least a portion of the plates face each other) and a force is applied to bring the two plates together. When the two plates change from the open phase to the closed phase, the inner surfaces of the two plates compress the sample deposited on the plates to reduce the sample thickness (while the sample has lateral open flow between the plates), and the thickness of the relevant volume of the sample is determined by the spacers, plates and method used and the sample mechanical / fluid properties. For a given sample and a given spacer, plates and plate pressing method, the thickness of the closed phase can be predetermined.

[0283] The terms "the spacing between the inner surfaces of the plates is adjusted by the spacers" or "the sample thickness is adjusted by the plates and the spacers" or the thickness of the sample is adjusted by the spacers and the plates" means that the thickness of the sample in the CROF process is determined by the given plates, spacers, sample and pressing method.

[0284] In some embodiments, the adjusted sample thickness at the closed configuration is the same as the height of the spacer; in this case, in the closed phase, the spacer directly contacts two plates (one of which is the plate that fixes the spacer and the other is the plate that contacts the spacer).

[0285] In certain embodiments, the sample thickness adjusted in the closed phase is greater than the height of the spacer; in this case, in the closed phase, the spacer only directly contacts the plate on whose surface the spacer is fixed or attached, and indirectly contacts another plate (i.e., indirect contact). The term "indirect contact" with the plate means that the spacer and the plate are separated by a thin sample layer called the "residual sample layer", and its thickness is called the "residual thickness". For a given spacer and plate, a given plate pressing method and a given sample, the residual thickness can be predetermined (a predetermined manner before reaching the closed structure), resulting in a predetermined sample thickness in the closed structure. This is because for given conditions (sample, spacer, plate and pressure), the residual layer thickness is the same and can be pre-calibrated and / or calculated. The adjusted sample thickness is approximately equal to the spacer height plus the sample residual thickness.

[0286] In many embodiments, the size and shape of the column is pre-characterized (ie, predetermined) prior to its use, and the predetermined information is used for subsequent analysis, such as determining sample volume (or related volumes), etc.

[0287] In some embodiments, adjustment of sample thickness includes applying a closing (compression) force to the plates to maintain the separation between the plates.

[0288] In some embodiments, adjustment of sample thickness includes establishing spacing between plates with spacers, closing forces applied to the plates, and physical properties of the sample, and optionally wherein the physical properties of the sample include at least one of: viscosity and compressibility.

[0289] 2.2 Board

[0290] In the present invention, generally, the plates of the CROF are made of materials that (i) have a thickness that can be used, together with the spacers, to accommodate a portion or all of the volume of the sample, and (ii) do not have a significant adverse effect on the sample, the assay, or the objectives that the plate is intended to achieve. However, in certain embodiments, specific materials used for the plates (and therefore their properties) are used to achieve certain objectives.

[0291] In some embodiments, the two panels have the same or different parameters for each of the following parameters: panel material, panel thickness, panel shape, panel area, panel flexibility, panel surface properties, and panel optical transparency.

[0292] Sheet material. The sheet is made of single material, composite material, multiple materials, multi-layered materials, alloy or combination thereof. Each material used for the sheet is an inorganic material, an organic material or a mixture, wherein examples of materials are given in the paragraphs of Mat-1 and Mat-2.

[0293] MAT-1. Inorganic materials for plates include, but are not limited to, glass, quartz, oxides, silicon dioxide, silicon nitride, hafnium oxide (HfO), aluminum oxide (AlO), semiconductors: (silicon, GaAs, GaN, etc.), metals (e.g., gold, silver, copper, aluminum, titanium, nickel, etc.), ceramics, or any combination thereof. MAT-2 Organic materials for spacers include, but are not limited to, polymers (e.g., plastics) or amorphous organic materials. The polymer material for the spacer includes, but is not limited to, acrylate polymers, vinyl polymers, olefin polymers, cellulosic polymers, non-cellulosic polymers, polyester polymers, nylon, cyclic olefin copolymer (COC), poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefin polymer (COP), liquid crystal polymer (LCP), polyamide (PA), polyethylene (PE), polyimide (PI), polypropylene (PP), polyphenylene ether (PPE) (POM), polyetheretherketone (PEEK), polyethersulfone (PES), polyethylene phthalate (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polydimethylsiloxane (PDMS), rubber or any combination thereof.

[0294] In some embodiments, the plates are each independently made of at least one of glass, plastic, ceramic, and metal. In some embodiments, each plate independently comprises at least one of glass, plastic, ceramic, and metal.

[0295] In some embodiments, one plate is different from another plate in lateral area, thickness, shape, material or surface treatment. In some embodiments, one plate is the same as another plate in lateral area, thickness, shape, material or surface treatment.

[0296] The material used for the panels can be rigid, flexible or anything in between. Rigidity (ie stiffness) or flexibility is relative to the pressure used to bring the panels into the closed configuration.

[0297] In some embodiments, the choice of a rigid or flexible plate is determined by the need to control sample thickness uniformity in the closed configuration.

[0298] In some embodiments, at least one of the two plates is transparent (to light). In some embodiments, at least a portion or portions of one or both plates are transparent. In some embodiments, the plates are opaque.

[0299] Plate thickness. In some embodiments, at least one of the plates has an average thickness of 2 nm or less, 10 nm or less, 100 nm or less, 500 nm or less, 1000 nm or less, 2 um (micrometer) or less, 5 um or less, 10 micrometers or less, 20 micrometers or less, 50 micrometers or less, 100 micrometers or less, 150 micrometers or less, 200 micrometers or less, 300 micrometers or less, 500 micrometers or less, 800 micrometers or less, 1 mm (millimeter) or less, 2 mm or less, 3 mm or less, or a range between any two values.

[0300] In some embodiments, at least one plate has an average thickness of at most 3 mm (millimeters), at most 5 mm, at most 10 mm, at most 20 mm, at most 50 mm, at most 100 mm, at most 500 mm, or a range between any two values.

[0301] In some embodiments, the thickness of the plate is not uniform across the plate. Using different plate thicknesses at different locations can be used to control plate bending, folding, sample thickness adjustment, etc.

[0302] Plate shape and area. In general, the plate can have any shape as long as the shape allows for compressed open flow of the sample and adjustment of the sample thickness. However, in certain embodiments, a specific shape may be advantageous. The shape of the plate can be circular, oval, rectangular, triangular, polygonal, annular, or any superposition of these shapes.

[0303] In some embodiments, the two plates can have the same size or shape, or different. The area of ​​the plate depends on the application. The area of ​​the plate is at most 1 square millimeter (millimeter square), at most 10 square millimeters, at most 100 square millimeters, at most 1 square centimeter (centimeter square), at most 5 square centimeters, at most 10 square centimeters, at most 100 square centimeters, at most 500 square centimeters, at most 1000 square centimeters, at most 5000 square centimeters, at most 10,000 square centimeters or more than 10,000 square centimeters, or any arrangement between any two values. The shape of the plate can be rectangular, square, circular or other shapes.

[0304] In some embodiments, at least one of the panels is in the form of a belt (or belts) having a width, a thickness, and a length. The width is at most 0.1 centimeters (cm), at most 0.5 cm, at most 1 cm, at most 5 cm, at most 10 cm, at most 50 cm, at most 100 cm, at most 500 cm, at most 1000 cm, or a range between any two values. The length can be as long as it needs to be. The belt can be rolled into a roll.

[0305] Plate surface flatness. In many embodiments, the inner surface of the plate is a flat or very flat plane. In certain embodiments, the two inner surfaces are parallel to each other in the closed configuration. A flat inner surface helps to quantify and / or control sample thickness by simply using a predetermined spacer height in the closed configuration. For a non-flat inner surface of the plate, not only the spacer height needs to be known, but also the topology of the inner surface needs to be known to quantify and / or control sample thickness in the closed configuration. Knowing the surface topology requires additional measurements and / or corrections, which can be complex, time consuming, and costly.

[0306] The flatness of the plate surface is relative to the final sample thickness (the final thickness is the thickness in the closed configuration) and is often characterized by the term "relative surface flatness," which is the variation in plate surface flatness versus final sample thickness.

[0307] In some embodiments, the relative surface is less than 0.01%, 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 30%, less than 50%, less than 70%, less than 80%, less than 100%, or a range between any two of these values.

[0308] Plate parallelism. In some embodiments, the two surfaces of the plate are substantially parallel to each other. In some embodiments, the two surfaces of the plate are not parallel to each other.

[0309] Plate flexibility. In some embodiments, the plate is flexible under the compression of the CROF process. In some embodiments, both plates are flexible under the compression of the CROF process. In some embodiments, a plate is rigid and the other plate is flexible under the compression of the CROF process. In some embodiments, both plates are rigid. In some embodiments, both plates are flexible but have different flexibilities.

[0310] Optical transparency of the plates. In some embodiments, the plates are optically transparent. In some embodiments, both plates are optically transparent. In some embodiments, a plate is optically transparent and the other plate is opaque. In some embodiments, both plates are opaque. In some embodiments, both plates are optically transparent but have different optical transparencies. The optical transparency of a plate refers to a portion or the entire area of ​​the plate.

[0311] Surface wetting properties. In some embodiments, the plate wets (i.e., contact angle is less than 90 degrees) the sample, the transfer liquid, or both. In some embodiments, both plates wet the inner surface of the transfer liquid, or both; have the same or different wettabilities. In some embodiments, a plate wets the inner surface of the transfer liquid, or both; the other plate has a non-wetting inner surface (i.e., contact angle equal to or greater than 90 degrees). Wetting of the inner surface of the plate refers to a portion or the entire area of ​​the plate.

[0312] In some embodiments, the inner surface of the plate has other nano- or micro-structures to control the lateral flow of the sample during CROF. The nano- or micro-structures include, but are not limited to, channels, pumps, etc. Nano- and micro-structures are also used to control the wetting properties of the inner surface.

[0313] 2.3 Spacer

[0314] Function of spacer. In the present invention, the spacer is configured to have one or any combination of the following functions and properties: the spacer is configured to (1) control the thickness of the sample or the relevant volume of the sample together with the plate (preferably, the thickness is precisely controlled, or uniform in a relevant area, or both); (2) allow the sample to have compression regulated open flow (CROF) on the plate surface; (3) do not occupy significant surface area (volume) in a given sample area (volume); (4) reduce or increase the effect of particle or analyte precipitation in the sample; (5) change and / or control the wettability of the inner surface of the plate; (6) identify the location of the plate, the scale of the size and / or information related to the plate, or (7) perform any combination of the above.

[0315] Spacer Architecture and Shape. To achieve the desired sample thickness reduction and control, in certain embodiments, spacers secure their respective plates. In general, spacers can have any shape as long as the spacer is able to adjust sample thickness during the CROF process, but certain shapes are preferred to achieve certain functions, such as better uniformity, less stamping overshoot, etc.

[0316] The spacer(s) are a single spacer or a plurality of spacers (e.g., an array). Some embodiments of a plurality of spacers are arrays of spacers (e.g., pillars) where the inter-spacer distances are periodic or aperiodic, or are periodic or aperiodic in a particular region of the plate, or have different distances in different regions of the plate.

[0317] There are two types of spacers: open spacers and closed spacers. An open isolator is one that allows sample flow through the isolator (i.e. the sample goes around and through the isolator, e.g. a column as an isolator), and a closed isolator is one that stops sample flow (i.e. the sample cannot flow out of the isolator, e.g. a donut-shaped isolator and the sample is inside the ring). Both types of spacers use their height to regularly determine the final sample thickness in a closed configuration.

[0318] In some embodiments, the spacers are only open spacers. In some embodiments, the spacers are only closed spacers. In some embodiments, the spacers are a combination of open spacers and closed spacers.

[0319] The term "columnar spacer" means that the spacer has a columnar shape, and a column refers to an object having a height and lateral shape that allows a sample to flow around it during compressed open flow.

[0320] In some embodiments, the lateral shape of the pillar spacer is a shape selected from the following group: (i) a circle, an ellipse, a rectangle, a triangle, a polygon, a ring, a star, a letter shape (e.g., an L shape, a C shape, letters from A to Z), a number shape (e.g., shapes such as 0, 1, 2, 3, 4, ... to 9); (ii) a shape having at least one rounded corner in group (i); (iii) a shape having a zigzag or rough edge in group (i); and (iv) any overlap of (i), (ii) and (iii). For multiple spacers, different spacers can have different lateral shapes and sizes and different distances from adjacent spacers.

[0321] In some embodiments, the spacers can be and / or can include columns, posts, beads, spheres, and / or other suitable geometries. The lateral shape and dimensions of the spacers (i.e., transverse to the respective plate surfaces) can be anything, except in some embodiments, the following restrictions: (i) the spacer geometry does not cause significant errors in measuring sample thickness and volume; or (ii) the geometry of the spacers does not prevent sample flow out between the plates (i.e., not in a closed form). However, in some embodiments, they require some spacers to be closed spacers to restrict sample flow.

[0322] In some embodiments, the shape of the spacer has rounded corners. For example, a rectangular spacer has one, several, or all of its corners rounded (like a circle, not a 90 degree angle). Rounded corners generally make the manufacturing of the spacer easier and, in some cases, less damaging to the biological material.

[0323] The sidewalls of the pillars can be straight, curved, sloped or of different shapes in different portions of the sidewalls. In some embodiments, the spacers are pillars of various lateral shapes, sidewalls, and pillar height to pillar lateral area ratios.

[0324] In a preferred embodiment, the spacers have a column shape for allowing open flow.

[0325] Material of the spacer. In the present invention, the spacer is generally made of any material that can be used to adjust the thickness of the relevant volume of the sample together with the two plates. In some embodiments, the material of the spacer is different from the material used for the plates. In some embodiments, the material used for the space is at least partially the same as the material used for at least one of the plates.

[0326] The spacer is made of a single material, a composite material, multiple materials, a multi-layer material, an alloy or a combination thereof. Each material used for the spacer is an inorganic material, an organic material or a mixture, wherein examples of materials are given in the paragraphs of Mat-1 and Mat-2. In a preferred embodiment, the spacer is made of the same material as the plate used in the CROF.

[0327] Mechanical strength and flexibility of the spacer. In some embodiments, the mechanical strength of the spacer is strong enough so that during compression of the panels and during the closed phase, the height of the spacer is the same or significantly the same as when the panels are in the open phase. In some embodiments, the difference in the spacer between the open configuration and the closed configuration can be characterized and predetermined.

[0328] The material of the spacer is rigid, flexible or any flexibility in between. Rigidity is relative to the pressure used to bring the plate into the closed configuration: if the space does not deform more than 1% of its height under pressure, the spacer material is considered rigid, otherwise it is flexible. When the spacer is made of a flexible material, the final sample thickness in the closed configuration can still be predetermined by the pressure and mechanical properties of the spacer.

[0329] Spacers in the sample. In order to achieve the desired sample thickness reduction and control, in particular to achieve good sample thickness uniformity, in certain embodiments, spacers are placed within the sample or within the relevant volume of the sample. In some embodiments, one or more spacers are present within the sample or within the relevant volume of the sample, with an appropriate spacer spacing distance. In certain embodiments, at least one of the spacers is located within the sample, at least two spacers within the sample or the relevant volume of the sample, or at least "n" spacers within the sample or the relevant volume of the sample, where "n" can be determined by the sample thickness uniformity or the desired sample flow properties during CROF.

[0330] Spacer height. In some embodiments, all spacers have the same predetermined height. In some embodiments, the spacers have different predetermined heights. In some embodiments, the spacers can be divided into groups or regions, where each group or region has its own spacer height. And in some embodiments, the predetermined height of the spacers is the average height of the spacers. In some embodiments, the spacers have approximately the same height. In some embodiments, a percentage of the number of spacers have the same height.

[0331] The height of the spacer is selected by the final sample thickness and the residual sample thickness that need to be adjusted. The spacer height (predetermined spacer height) and / or the sample thickness is 3 nm or less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1 μm or less, 2 μm or less, 3 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 50 μm or less, 100 μm or less, 150 microns or less, 200 microns or less, 300 microns or less, 500 microns or less, 800 microns or less, 1 mm or less, 2 mm or less, 4 mm or less, or a range between any two values.

[0332] In a preferred embodiment, the spacer height and / or sample thickness is between 1nm and 100nm, in another preferred embodiment, 100nm to 500nm, in a separate preferred embodiment, 500nm to 1000nm, 1um (i.e. 1000nm) to 2, in another preferred embodiment, 2μm to 3μm, in another preferred embodiment, 3μm to 5μm, in another preferred embodiment, 5μm to 10μm, in another preferred embodiment, 10μm to 50μm, 50μm in a separate preferred embodiment, 100μm to 100μm.

[0333] In some embodiments, the spacer height and / or sample thickness is (i) equal to or slightly larger than the smallest dimension of the analyte, or (ii) equal to or slightly larger than the largest dimension of the analyte. "Slightly larger" means it is about 1% to 5% and any number in between.

[0334] In some embodiments, the spacer height and / or sample thickness is larger than the smallest dimension of the analyte (eg, the analyte has an anisotropic shape), but smaller than the largest dimension of the analyte.

[0335] For example, red blood cells have a disc shape with a minimum dimension of 2um (disc thickness) and a maximum dimension of 11um (disc diameter). In one embodiment of the present invention, the spacer is selected so that the inner surface spacing of the plate in the relevant area is 2μm (equal to the minimum dimension) in one embodiment, 2.2μm in another embodiment, or 3 (50% larger than the minimum dimension), but less than the maximum dimension of the red blood cell. Such an embodiment has certain advantages in blood cell counting. In one embodiment, for red blood cell counting, by making the inner surface spacing 2 or 3μm and any numerical value between the two values, the undiluted whole blood sample is evenly confined in the interval, and each red blood cell (RBC) does not overlap with others, so that the red blood cells can be accurately counted visually. (Too much overlap between RBCs will cause serious counting errors).

[0336] In the present invention, in some embodiments, it uses plates and spacers to adjust not only the thickness of the sample, but also the orientation and / or surface density configuration of the analytes / entities in the sample when the plate is closed. When the plate is in the closed phase, the thinner thickness of the sample results in fewer analytes / entities per surface area (i.e., less surface concentration).

[0337] Spacer Transverse Dimension. For open positioners, the transverse dimension can be characterized by the transverse dimensions in two orthogonal directions, x and y (sometimes referred to as width). The transverse dimensions of the spacer in each direction can be the same or different. In some embodiments, the transverse dimension in each direction (x or y) is...

[0338] In some embodiments, the ratio of the lateral dimensions in the x and y directions is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10,000 or any range between two values. In some embodiments, different ratios are used to adjust the sample flow direction; the larger the ratio, the flow is along one direction (the larger dimension direction).

[0339] In some embodiments, different lateral dimensions of the spacers in the x and y directions are used to (a) use the spacers as scale marks to indicate plate orientation, (b) use the spacers to create more sample flow preferred directions, or both.

[0340] In a preferred embodiment, period, width and height.

[0341] In some embodiments, all spacers have the same shape and size. In some embodiments, each spacer has a different lateral dimension.

[0342] For a closed spacer, in some embodiments, the internal lateral shape and size are selected based on the total volume of the sample to be enclosed by the closed spacer, where the volume size has been described in the present disclosure; and in certain embodiments, the external lateral shape and size are selected based on the required strength to support the pressure of the liquid against the spacer and the compression pressure of the clamping plate.

[0343] Aspect ratio of height to average lateral dimension of the column spacers.

[0344] In certain embodiments, the aspect ratio of the height to the average lateral dimension of the pillar spacers is 100,000, 10,000, 1,000, 100, 10, 1, 0.01, 0.001, 0.001, 0.00001 or any range of values ​​therebetween.

[0345] Spacer height accuracy The height of the spacer should be precisely controlled. The relative accuracy of the spacer (i.e., the ratio of deviation from the desired spacer height) is 0.001% or less, 0.01% or less, 0.1% or less; 0.5% or less, 1% or less, 2% or less, 5% or less, 8% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, 99.9% or less, or any range between values.

[0346] The spacer can be a single spacer or multiple spacers on the relevant area of ​​the plate or sample. In some embodiments, the spacers on the plate are configured and / or arranged in an array, and the array is periodic, aperiodic array or periodic at some locations of the plate and aperiodic at other locations.

[0347] In some embodiments, the periodic array of spacers has a lattice of squares, rectangles, triangles, hexagons, polygons, or any combination thereof, where combination means that different positions of the plate have different lattices of spacers.

[0348] In some embodiments, the inter-spacer distance of the spacer array is periodic in at least one direction of the array (ie, uniform inter-spacer distance). In some embodiments, the inter-spacer distance is configured to improve uniformity between plate spacings in a closed configuration.

[0349] The distance between adjacent spacers (i.e., the inter-spacer distance) is 1 micron or less, 5 microns or less, 10 microns or less, 20 microns or less, 30 microns or less, 40 microns or less, 50 microns or less, 60 μm or less, 70 μm or less, 80 μm or less, 90 μm or less, 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, or a range between any two values.

[0350] In some embodiments, the distance between the spacers is 400 or less, 500 or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm or less, 10 mm or less, or any range therebetween. In some embodiments, the distance between the spacers is 10 mm or less, 20 mm or less, 30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, or any range therebetween.

[0351] The distance between adjacent spacers (i.e., the inter-spacer distance) is selected so that for given properties of the plate and the sample, at the closed phase of the plate, the variation in sample thickness between two adjacent spacers is, in some embodiments, at most 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or any range between these values; or in certain embodiments, at most 80%, 100%, 200%, 400%, or a range between any two values.

[0352] Obviously, in order to maintain a given sample thickness variation between two adjacent spacers, a closer inter-spacer distance is required when using a more flexible plate.

[0353] Specifies the precision of the separation distance.

[0354] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars with a height of 2 to 4 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 1 μm to 100 μm.

[0355] In a preferred embodiment, the spacers are a periodic square array wherein the spacers are 2 to 4 μm in height, have an average lateral dimension of 5 to 20 μm, and are spaced 100 to 250 μm apart.

[0356] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 4 to 50 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 1 μm to 100 μm.

[0357] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are 4 to 50 μm in height, have an average lateral dimension of 5 to 20 μm, and are spaced 100 μm to 250 μm apart.

[0358] The period of the spacer array is 1 nm to 100 nm in one preferred embodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to 1000 nm in a separate preferred embodiment, 1 μm (i.e., 1000 nm) to 2 μm in another preferred embodiment, 2 μm to 3 μm in another preferred embodiment, 3 μm to 5 μm in another preferred embodiment, 5 μm to 10 μm in another preferred embodiment, 10 μm to 50 μm in another preferred embodiment, 50 μm to 100 μm in another preferred embodiment, 100 microns to 175 microns in a separate preferred embodiment, and 175 microns to 300 microns in a separate preferred embodiment.

[0359] Spacer density. The spacers are greater than one per square micron, greater than one per 10 square microns, greater than one per 100 square microns, greater than one per 500 square microns, greater than one per 1000 square microns, greater than one per 5000 square microns, greater than one per 0.01 square millimeters, greater than one per 0.1 square millimeters, greater than one per 1 square millimeter, greater than one per 5 square millimeters, greater than one per 10 square millimeters, greater than one per 100 square millimeters, greater than one per 10000 square millimeters, or a range between any two values.

[0360] (3) the spacer is configured to not occupy a significant surface area (volume) in a given sample area (volume);

[0361] Ratio of spacer volume to sample volume. In many embodiments, the ratio of the spacer volume (i.e., the volume of the spacer) to the sample volume (i.e., the volume of the sample) and / or the ratio of the volume of the spacer within the associated volume to the associated volume of the sample to the sample is controlled to obtain certain advantages. Advantages include, but are not limited to, uniformity of sample thickness control, uniformity of analytes, sample flow properties (i.e., flow rate, flow direction, etc.).

[0362] In some embodiments, the ratio of the spacer volume r) to the sample volume, and / or the ratio of the volume of the spacer within the relevant volume of the sample to the relevant volume of the sample is less than 100% at most 99%, at most 70%, at most 50%, at most 30%, at most 10%, at most 5%, at most 3%, at most 1%, at most 0.1%, at most 0.01% at most 0.001%, or any range therebetween.

[0363] The spacers are fixed to the board. The spacer spacing distance and the orientation of the spacers, which play a key role in the present invention, are preferably maintained during the process of transforming the board from an open phase to a closed phase and / or are preferably predetermined from an open configuration to a closed configuration prior to processing.

[0364] Some embodiments of the present invention are to fix the spacer to one of the panels before the panels are brought to the closed phase. The term "spacer is fixed to its respective panel" means that the spacer is attached to the panel and remains attached when the panel is in use. An example of "spacer is fixed to its respective panel" is that the spacer is made entirely of one piece of material of the panel and the position of the spacer relative to the panel surface does not change. An example of "spacer is not fixed to its corresponding panel" is that the spacer is bonded to the panel by an adhesive, but when the panel is in use, the adhesive cannot keep the spacer in its original position on the panel surface (i.e., the spacer leaves its original position on the panel surface).

[0365] In some embodiments, at least one of the spacers is fixed to its respective plate. In some embodiments, two spacers are fixed to their respective plates. In some embodiments, most of the spacers are fixed to their respective plates. In some embodiments, all of the spacers are fixed to their respective plates.

[0366] In some embodiments, the spacers are integrally secured to the plate.

[0367] In some embodiments, the spacers are secured to their respective plates by one or any combination of the following methods and / or configurations: attached to, bonded to, fused to, embossed to, and etched to.

[0368] The term "embossing" means that the spacers and the board are integrally fixed by embossing (ie, stamping) a piece of material to form the spacers on the board surface. The material can be a single layer of material or multiple layers of material.

[0369] The term "etching" means that the spacers and the board are monolithically attached by etching a piece of material to form the spacers on the board surface. The material can be a single layer of material or multiple layers of material.

[0370] The term "fused with" means that the spacer and the plate are fixed as one by attaching the spacer and the plate together, the original materials of the spacer and the plate are fused to each other, and there is a clear material boundary fusion between the two materials.

[0371] The term "bonded to" means that the spacer and the plate are integrally fixed by bonding the spacer and the plate.

[0372] The term "connected to" means that the spacer and the plate are connected together.

[0373] In some embodiments, the spacers and the plates are made of the same material. In other embodiments, the spacers and the plates are made of different materials. In other embodiments, the spacers and the plates are formed as one piece. In other embodiments, one end of the spacer is fixed to its corresponding plate, while the end is open to accommodate different configurations of the two plates.

[0374] In other embodiments, each spacer is independently attached to, bonded to, fused to, printed on and etched in the corresponding plate. The term "independently" means that one spacer is fixed with its corresponding plate by the same or different method selected from the method of attaching, bonding to, fused to, printed on and etched in the corresponding plate.

[0375] In some embodiments, at least one distance between two spacers is predetermined ("predetermined inter-spacer distance" means that the distance is known when the user uses the panels).

[0376] In some embodiments of all methods and devices described herein, additional spacers are present in addition to the fixed spacers.

[0377] Specific sample thickness. In the present invention, it was observed that a larger plate holding force (i.e., the force holding the two plates together) can be obtained by using a smaller plate spacing (for a given sample area) or a larger sample area (for a given plate spacing) or both.

[0378] In some embodiments, at least one of the plates is transparent in an area surrounding the relevant area, each plate has an inner surface that is configured to contact a sample in a closed phase; in a closed configuration, the inner surfaces of the plates are substantially parallel to each other; the inner surfaces of the plates are substantially planar except for locations with spacers; or any combination thereof.

[0379] 2.4 Final sample thickness and uniformity

[0380] In some embodiments, substantially flat is determined relative to the final sample thickness and, depending on the embodiment and application, has less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, or less than 10%, or a range between any two of these values.

[0381] In some embodiments, the flatness relative to the sample thickness can be less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 50%, or less than 100%, or a range between any two of these values.

[0382] In some embodiments, substantially flat can mean that the surface flatness variation itself (measured from the average thickness) is less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, or less than 10%, or a range between any two of these values. In general, the flatness relative to the plate thickness can be less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 50%, or less than 100%, or a range between any two of these values.

[0383] 2.5 Spacer manufacturing method.

[0384] Spacers can be made on the board in various ways using lithography, etching, imprinting (nanoimprinting), deposition, peeling, fusion or a combination thereof. In some embodiments, the spacers are directly imprinted or imprinted on the board. In some embodiments, the spacers are imprinted in a material (e.g., plastic) deposited on the board. In certain embodiments, the spacers are made by directly imprinting the surface of the CROF board. Nanoimprinting can be done by roll-to-roll technology using a roller imprinter, or wound into a planar nanoimprint. This process has great economic advantages, thus reducing costs.

[0385] In some embodiments, the spacer is deposited on the board. The deposition can be evaporation, pasting or stripping. In pasting, the spacer is first made on a carrier, and then the spacer is transferred from the carrier to the board. In stripping, a removable material is first deposited on the board and a hole is formed in the material; the bottom of the hole exposes the board surface, and then the spacer material is deposited into the hole, and then the removable material is removed, leaving only the spacer on the board surface. In some embodiments, the spacer deposited on the board is fused to the board. In some embodiments, the spacer and the board are made in a single process. The single process includes imprinting (i.e., embossing, molding) or synthesis.

[0386] In some embodiments, at least two spacers are secured to respective plates by different fabrication methods, and optionally, wherein the different fabrication methods include at least one of deposition, bonding, fuse, imprinting, and etching.

[0387] In some embodiments, one or more spacers are secured to the respective plates by a manufacturing method of bonding, fusing, embossing, or etching, or any combination thereof.

[0388] In some embodiments, the fabrication method for forming such a monolithic spacer on a board includes methods of being bonded, fused, stamped, or etched or any combination thereof.

[0389] 2.6 Scale Marking

[0390] The term "ratio marking" refers to a ratio marking that can assist in quantification (i.e., size measurement) or control of an area of ​​interest and / or a relative volume of a sample. In some embodiments, the scale marking is located on the first plate or the second plate, on both plates, on one surface of a plate, on both surfaces of a plate, between plates, near a plate, or any combination thereof. In some embodiments, the scale marking is fixed on the first plate or the second plate, on both plates, on one surface of a plate, on both surfaces of a plate, between plates, near a plate, or them. In some embodiments, the scale marking is deposited on the first plate or the second plate, on both plates, on one surface of a plate, on both surfaces of a plate, between plates, near a plate, or any combination thereof. In some embodiments, some spacers are fixed and some spacers are deposited.

[0391] In some embodiments, the scale markings are etched scale markings, deposited material, or printed material. In some embodiments, the material absorbs light, reflects light, emits light, or any combination thereof.

[0392] In some embodiments, the scale markings are one or more objects of known size and / or known spacing. Examples of objects include, but are not limited to, rectangles, cylinders, or circles.

[0393] In some embodiments, the scale markings have dimensions in nanometers (nm), micrometers (μm), or millimeters (mm), or other size ranges.

[0394] In some embodiments, the scale markings are rulers with scale markings configured to measure the size of an object. In some embodiments, the scale markings are in the range of nanometers (nm), micrometers (μm) or millimeters (mm) or other sizes. In some embodiments, the scale markings are etched scale markings, deposited materials, or printed materials. In some embodiments, the material of the scale markings is a material that absorbs light, reflects light, scatters light, interferes with light, diffracts light, emits light, or any combination thereof.

[0395] In some embodiments, the fabricator is a spacer that serves the dual functions of "adjusting sample thickness" and "providing scale markings and / or sizing". For example, a rectangular spacer with known dimensions or two spacers with a known spacing can be used to measure dimensions associated with the sample around the spacer. From the measured sample dimensions, the volume of the sample-associated volume can be calculated.

[0396] In some embodiments, the scale markings are configured to at least partially define boundaries of an associated volume of the sample.

[0397] In some embodiments, at least one scale marking is configured to have a known dimension parallel to a plane of a lateral region of the associated volume of the sample.

[0398] In some embodiments, at least one pair of scale markings are separated by a known distance parallel to a plane of the side region.

[0399] In some embodiments, the scale markings are configured for optical detection.

[0400] In some embodiments, each scale mark is independently at least one of light absorbing, light reflecting, light scattering, light diffracting, and light emitting.

[0401] In some embodiments, the scale markings are arranged in a regular array with known lateral spacing.

[0402] In some embodiments, each scale mark independently has a lateral profile that is at least one of a square, a rectangle, a polygon, and a circle.

[0403] In some embodiments, at least one scale marking is connected to, bonded to, fused to, embossed into, and etched into one of the plates.

[0404] In some embodiments, at least one scale marking is one of the spacers.

[0405] In some embodiments, some spacers also function as scaling markers to quantify the relative volume of the sample.

[0406] In certain embodiments, binding sites (which immobilize analytes), storage sites, or the like are used as scale marks. In one embodiment, sites with known lateral dimensions interact with light that produces a detectable signal that reflects the known lateral dimensions of the site, thus serving as one or more scale marks.

[0407] In another embodiment, the size of the site is predetermined before the CROF process, and when the plate is in the closed phase, the thickness of the sample portion sitting on the site is significantly less than the average lateral size of the site, and then the incubation time is controlled so that after incubation, (1) most of the analyte / entity bound to the binding site comes from the sample volume located above the binding site, or (2) most of the reagent mixed (diffused) into the sample volume located on top of the binding site comes from the storage site. In these cases, the relevant volume for sample binding or reagent mixing is a volume approximately equal to the area of ​​the predetermined site multiplied by the sample thickness. The key reason for this may be that for a given incubation time, the analyte / entity in the sample volume outside the relevant volume does not have enough time to diffuse to the binding site, or the reagent on the storage site does not have enough time to diffuse into the sample volume outside the relevant volume.

[0408] An example of a method to measure and / or control relevant areas and volumes by using sites with known dimensions and by limiting incubation time is an assay in which a binding site (i.e., area with capture agent) of 1,000 um is placed 1,000 microns on the first plate of a CROF process (whose surface is larger than the binding site); in the closed phase of the plate, a sample with analyte is placed above the binding site, has a thickness of approximately 20 um (in the binding site area) and an area larger than the binding site, and is incubated for a time equal to the target analyte / entity diffusion time through the sample thickness.

[0409] In this case, most of the analyte / entity bound to the binding site comes from the sample volume located above it. Since analyte in the sample portion 20 microns away from the binding site does not have time to diffuse to the binding site (statistically), the binding site is 1,000um×1000um×20um=0.02p.

[0410] In this case, if the signal due to the analyte / entity captured by the binding site is measured after incubation, the concentration of the analyte / entity in the sample in the relevant area and relevant volume can be determined from the relevant area and relevant volume information (provided by the binding site). The analyte concentration is quantified by the amount of analyte captured by the binding site divided by the relevant volume.

[0411] In some embodiments, the relevant volume is approximately equal to the binding site area multiplied by the sample thickness, and the target analyte concentration in the sample is approximately equal to the amount of analyte captured by the binding site divided by the relevant sample volume. The accuracy of the method for quantifying the target analyte volume becomes better as the ratio of the binding site size to the sample thickness becomes larger (assuming that the incubation time is approximately the diffusion time of the target analyte in the sample to the distance of the sample thickness).

[0412] Propagate time in CROF.

[0413] In the present invention, in the method and apparatus for spreading all sections of the sample through two plates, the time to spread the sample to the final thickness in a closed configuration is 0.001 seconds or less, 0.01 seconds, 0.1 seconds, 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 60 seconds, 90 seconds, 100 seconds, 150 seconds, 200 seconds, 300 seconds, 500 seconds, 1000 seconds or a range between any two values.

[0414] In the method and apparatus for spreading all sections of a sample through two plates, in a preferred embodiment, the time to spread the sample to the final thickness in a closed configuration is 0.001 seconds or less, 0.01 seconds, 0.1 seconds, 1 second, 3 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 60 seconds, 90 seconds, 100 seconds, 150 seconds, or a range between any two values.

[0415] In the method and apparatus for spreading all sections of a sample through two plates, in a preferred embodiment, the time to spread the sample to the final thickness in a closed configuration is 0.001 seconds or less, 0.01 seconds, 0.1 seconds, 1 second, 3 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 60 seconds, 90 seconds, or a range between any two values.

[0416] In the method and apparatus for spreading all sections of a sample through two plates, in a preferred embodiment, the time to spread the sample to the final thickness in a closed configuration is 0.001 seconds or less, 0.01 seconds, 0.1 seconds, 1 second, 3 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, or a range between any two values.

[0417] In the method and apparatus for spreading all sections of a sample through two plates, in a preferred embodiment, the time to spread the sample to the final thickness in a closed configuration is 0.001 seconds or less, 0.01 seconds, 0.1 seconds, 1 second, 3 seconds, 5 seconds, 10 seconds, or a range between any two values.

[0418] In the method and apparatus for spreading all sections of a sample through two plates, in a preferred embodiment, the time to spread the sample to the final thickness in a closed configuration is 0.001 seconds or less, 0.01 seconds, 0.1 seconds, 1 second, 3 seconds, or a range between any two values.

[0419] Throughout the description of the present invention, the embodiments described in Section 3 and any combination thereof apply to (ie, are combined with other embodiments).

[0420] In a preferred embodiment, the spacers are integrally manufactured on the X-plate by imprinting (eg nanoimprinting) a plastic film using a mold and are made of the same material.

[0421] In a preferred embodiment, the spacer is integrally fabricated on the X-plate by imprinting (eg, nanoimprinting) a thin plastic film using a mold and is made of the same material, and the thickness of the X-plate is 50um to 500um.

[0422] In a preferred embodiment, the spacers are integrally fabricated on the X-plate by imprinting (eg, nanoimprinting) a thin plastic film using a mold and are made of the same material, and the thickness of the X-plate is 50 um to 250 um.

[0423] In a preferred embodiment, the spacer is integrally fabricated on the X-plate and is made of the same material, and the thickness of the X-plate is from 50 um to 500 um.

[0424] In a preferred embodiment, the spacer is integrally formed as a thin plastic film on the X-plate using a mold and is made of the same material, and the thickness of the X-plate is 50 μm to 250 μm.

[0425] In a preferred embodiment, the spacer is integrally fabricated on the X-plate by imprinting (eg, nanoimprinting) a thin plastic film using a mold, and is made of the same material, wherein the plastic film is PMMA (polymethyl methacrylate) or PS (polystyrene).

[0426] In a preferred embodiment, the spacer is integrally fabricated on the X-plate by imprinting (e.g., nanoimprinting) a thin plastic film using a mold, and is made of the same material, wherein the plastic film is PMMA (polymethyl methacrylate) or PS ((polystyrene), and the thickness of the X-plate is from 50um to 500um.

[0427] In a preferred embodiment, the spacer is integrally fabricated on the X-plate by imprinting (e.g., nanoimprinting) a thin plastic film using a mold, and is made of the same material, wherein the plastic film is PMMA (polymethyl methacrylate) or PS (polystyrene), and the thickness of the X-plate is from 50um to 250um.

[0428] In a preferred embodiment, the spacers are integrally fabricated on the X-plate by imprinting a thin plastic film using a mold (e.g., nanoimprinting), and are made of the same material, wherein the plastic film is PMMA (polymethyl methacrylate) or PS (polystyrene), and the spacers have a square or rectangular shape and have the same spacer height.

[0429] In a preferred embodiment, the spacers have a square or rectangular shape (with or without rounded corners).

[0430] In a preferred embodiment, the spacer has square or rectangular columns with a column width (spacer width in each lateral direction) between 1 μm and 200 μm; a column period (i.e., spacer period) from 2 um to 2000 um and a column height (i.e., spacer height) from 1 um to 100 um.

[0431] In a preferred embodiment, the spacer made of PMMA or PS has square or rectangular columns with a column width (spacer width in each lateral direction) between 1 μm and 200 μm; a pillar period (i.e., spacing period) from 2 um to 2000 um and a pillar height (i.e., spacer height) from 1 um to 100 um.

[0432] In a preferred embodiment, the spacer is integrally fabricated on the X-plate and is made of a plastic material, and the spacer has a square or rectangular column whose column width (spacer width in each lateral direction) is between 1 μm and 200 μm; a column period (i.e., the spacer period) from 2 um to 2000 um and a column height (i.e., the spacer height) from 1 um to 100 um.

[0433] In a preferred embodiment, the spacer is integrally fabricated on the X-plate and made of the same material, and the spacer has a square or rectangular column with a column width (spacer width in each lateral direction) between 1 μm and 200 μm; a pillar period (i.e., spacer period) from 2 um to 2000 um and a pillar height (i.e., spacer height) from 1 um to 10 um.

[0434] In a preferred embodiment, the spacer is integrally manufactured on the X-plate and is made of the same material selected from PS or PMMA or other plastics, and the spacer has a square or rectangular column, wherein the column width (spacer width in each lateral direction) is between 1um and 200um; the pillar period (i.e., the spacer period) is from 2um to 2000um and the pillar height (i.e., the spacer height) is from 10um to 50um.

[0435] In a preferred embodiment of the CROF device, one plate is an X-plate and the other plate is a planar film, wherein the thickness of at least one plate is in the range of 10 μm to 250 μm; wherein the spacer is fixed to the X-plate, and wherein the plate and the spacer can have the same material or different materials and are made of PMMA (polymethyl methacrylate), PS (polystyrene) or a material having similar mechanical properties to PMMA or PS.

[0436] In a preferred embodiment of the CROF device, one plate is an X-plate and the other plate is a planar film, wherein the thickness of at least one plate is in the range of 250 μm to 500 μm; wherein the spacer is fixed to the X-plate, and wherein the plate and the spacer may have the same material or different materials and are made of PMMA (polymethyl methacrylate), PS (polystyrene) or a material having similar mechanical properties to PMMA or PS.

[0437] In a preferred embodiment of the CROF device, one plate is an X-plate and the other plate is a planar film, wherein the thickness of at least one plate is in the range of 10 μm to 250 μm; wherein the spacer is fixed on the X-plate and is an array of square or rectangular columns with a column width (spacer width in each lateral direction) between 1 μm and 200 μm; (i.e., spacing time) is 2um-2000um, and the column height (i.e., spacer height) is 1um-100um, wherein the plate and the spacer can have the same material or different materials and are made of PMMA (polymethyl methacrylate), PS (polystyrene) or a material having similar mechanical properties to PMMA or PS.

[0438] "Similar" in the above paragraph means that the difference in mechanical properties is within 60%.

[0439] Guard ring. Some embodiments have a guard ring to prevent sample from flowing off the plate surface. Some embodiments of the guard ring are a closed wall surrounding the sample area. The height of the wall is equal to the spacer height or different from the spacer height. The wall can be away from the sample measurement area.

[0440] The movable plates in the CROF process may include and / or may be coupled to a hinge, a platform or some other positioning system configured to transition the plates between an open phase and a closed phase. The movable plates may be coupled together with one or more joints in a manner that leaves an opening to access the space between the plates (e.g., to insert and / or remove a sample), provided that at least one joint and / or at least one of the joints of one of the plates is sufficiently flexible to achieve the described open and closed configurations. A membrane pump is not considered a movable plate.

[0441] 3. Uniform plate spacing and sample thickness (U)

[0442] In many applications of the CROF process, it is beneficial to improve the uniformity of the plate spacing and therefore the uniformity of the sample thickness in the closed phase, especially when the spacing is in the micrometer and / or nanometer range. Good uniformity can improve the consistency of the assay. The present invention provides a means to improve uniformity.

[0443] Factors that can reduce the uniformity of plate spacing in CROF include (a) local bending of the plates, (b) non-flatness of the inner surface of the plates, and (c) dust. The effects of these factors become worse as the final plate spacing becomes smaller.

[0444] In order to improve the uniformity of plate spacing (and therefore sample thickness), the present invention utilizes certain design of the plate (mechanical strength, thickness, etc.), spacer size, number of spacers, spacer layout, spacer spacing, accuracy of spacer height, and other factors to overcome factors that cause non-uniformity.

[0445] 3.1 Use spacing distance to achieve uniform sample thickness of flexible board

[0446] In some applications, it is beneficial for one or both of the CROF panels to be flexible. Figure 5 As shown in Fig. 1, for a flexible plate (e.g., a plastic film), if the distance between the spacers is too large, the flexibility of the plate may cause the plate to bend locally (e.g., sag, i.e., bend inward) at a location between two adjacent spacers during the CROF process, resulting in poor sample thickness uniformity. Poor sample thickness uniformity has many disadvantages, such as large errors in determining sample volume and / or analyte concentration, deviations in incubation time, etc.

[0447] One embodiment of the present invention provides a solution to reduce local bowing and thus reduce final sample thickness deviation by using appropriate spacer inter-distance. Figure 5 As shown in FIG. 1 , the CROF device includes a rigid plate with a flat sample surface and a flexible plate. If the distance between the spacers is too large, there is a local bend between two adjacent spacers ( Figure 5 a). To reduce local bending, the distance between spacers is set equal to or less than the critical bending span of the flexible board ( Figure 5 b) When the two sheets are flexible, the inter-spacer distance should be less than the minimum of the critical bending spans of the two sheets.

[0448] U1. A method of uniformly adjusting the thickness of a relevant volume of a sample using two plates, comprising:

[0449] (a) obtaining a sample, wherein the thickness of a relevant volume of the sample needs to be adjusted;

[0450] (b) obtaining two plates that are movable relative to each other into different configurations; one or both of the plates being flexible; and wherein one or both of the plates comprises spacers having a predetermined inter-spacer distance and height, and wherein each of the spacers is secured to a respective plate;

[0451] (c) depositing the sample on one or both of the plates when the two plates are configured in an open configuration; wherein the open configuration is a configuration in which the two plates are either partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0452] (d) after (c), spreading the sample by bringing the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacer and the relevant volume of the sample are between the two plates, and the thickness of the relevant volume of the sample is adjusted by the two plates and the spacer;

[0453] For given two plates, the spacer is configured so that the deviation of the thickness of the relevant volume of the sample over a given area is less than a predetermined value; wherein the relevant volume is a part or the entire volume of the sample.

[0454] In the method of paragraph U1, the configuration of the spacers includes selecting an appropriate distance between the spacers. In some embodiments, the distance between the spacers is selected so that for an allowed sample thickness deviation, for a given two plates and a pressing method, the bending of the two plates under the pressing method is equal to or less than the allowed sample thickness deviation. The adjusted sample thickness in the closed configuration can be thinner than the maximum thickness of the sample when the plates are in the open configuration. U2. A device for adjusting the thickness of a relevant volume of a sample, comprising:

[0455] a first plate and a second plate movable relative to each other into different configurations;

[0456] wherein one or both of the plates are flexible, and wherein one or both of the plates include spacers having a predetermined inter-spacer distance and height, and each of the spacers is secured to its corresponding plate;

[0457] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0458] wherein another of the configurations is a closed phase, which is configured after the sample is deposited in the open phase; and in the closed configuration: the two plates face each other, the spacer and the associated volume of the sample are located between the two plates, and the thickness of the associated volume of the sample is adjusted by the two plates and the spacer;

[0459] Wherein for a given plate, the spacers are configured so that the thickness of a relevant volume of the sample has a thickness deviation over an area less than a predetermined value; wherein the relevant volume is a portion or the entire volume of the sample.

[0460] In the device of paragraph U2, the configuration of the spacers includes selecting an appropriate distance between the spacers. In some embodiments, the distance between the spacers is selected so that for an allowable sample thickness deviation, for a given two plates and a pressing method, the bending of the two plates under the pressing method is equal to or less than the allowable sample thickness deviation. The adjusted sample thickness in the closed configuration can be thinner than the maximum thickness of the sample when the plates are in the open configuration.

[0461] In some embodiments, a small inter-spacer distance also allows the use of flexible films (eg, 100 μm thick plastic films) by making the inter-spacer distance smaller than the bending of the board between two spacers.

[0462] In some embodiments, in order to have uniform sample thickness over a large area in a closed configuration, for a given maximum allowed bending of the flexible sheet, the ratio of the distance between the spacers to the critical bending span of the sheet is at most 0.001%, at most 0.001%, at most 0.001%, at most 0.01%, at most 0.1%, at most 1%, at most 10%, at most 20%, at most 50%, at most 70%, at most 100% and in a range between any two values.

[0463] 3.2 Using flexible boards and spacers to overcome the effects of dust in CROF

[0464] One problem that needs to be overcome in the CROF process is that dust with a thickness greater than the spacer height can disrupt the adjustment of the spacers to achieve the desired final plate spacing (and therefore the final thickness of the sample) (e.g. Figure 6 a). When two rigid plates are used, one such dust particle can destroy the spacer adjustment over the entire area of ​​the plates.

[0465] Certain embodiments of the present invention address this problem by using appropriate distances between the flexible plates and spacers to limit the impact of dust to a small area around the dust, while allowing the area outside the small area to have the final plate spacing and sample thickness set (adjusted) by the spacers.

[0466] For example, Figure 6 b shows that in order to overcome the influence of dust, a flexible plate with appropriate flexibility is used to limit the dust area, and it is used together with a rigid plate with fixed spacers. Figure 6 c shows another embodiment to reduce the dust effect, where the spacer is fixed on the flexible plate. Obviously, another solution is that both plates are flexible.

[0467] The appropriate flexibility of the plate can be selected from the plate thickness and mechanical properties to minimize the impact of dust in the CROF process. Based on the tests shown in the examples, the following are preferred embodiments.

[0468] U3. A method for minimizing the effect of dust on the thickness of a relevant volume of a conditioning sample, comprising:

[0469] (a) obtaining a sample, wherein the thickness of a relevant volume of the sample needs to be adjusted;

[0470] (b) obtaining two plates that are movable relative to each other into different configurations; one or both of the plates being flexible; and wherein one or both of the plates comprises spacers having a predetermined inter-spacer distance and height, and wherein each of the spacers is secured to a respective plate;

[0471] (c) depositing the sample on one or both of the plates when the two plates are configured in an open configuration; wherein the open configuration is a configuration in which the two plates are either partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0472] (d) after (c), spreading the sample by bringing the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacer, the relevant volume of the sample, and one or more dust particles having a thickness greater than the height of the spacer are between the two plates, and the thickness of the relevant volume of the sample is adjusted by the two plates and the spacer;

[0473] wherein the spacer and the plates are configured to minimize the area between the two plates affected by the dust; wherein the area affected by dust is an area where the dust prevents the spacer from adjusting the final spacing between the plates in a closed phase of the plates in the same manner as in the absence of dust; wherein the relevant volume is a portion or the entire volume of the sample.

[0474] In the method of paragraph U3, the configuration of the spacer and the board to minimize the dust affected area includes selecting an appropriate thickness and mechanical properties of the flexible board.

[0475] In some embodiments, the distance between the spacers is selected so that for the allowed sample thickness variation, for a given two plates and compression method, the deflection of the two plates under the compression method is equal to or less than the allowed sample thickness deviation. The conditioned sample thickness in the closed configuration can be thinner than the maximum thickness of the sample when the plates are in the open phase.

[0476] U4. An apparatus for minimizing the effect of dust on the thickness of a relevant volume of a conditioning sample, comprising:

[0477] a first plate and a second plate movable relative to each other into different configurations, and each plate having a sample contacting surface for contacting the sample, wherein one or both of the plates are flexible;

[0478] spacers on the sample contacting surface of one or both plates, the spacers having a predetermined inter-spacer distance and height, and each of the spacers being secured to its corresponding plate;

[0479] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0480] wherein another of the configurations is a closed phase, which is configured after the sample is deposited in the open phase; and in the closed configuration: the two plates face each other, the spacer, the associated volume of the sample, and one or more dust particles having a thickness greater than the height of the spacer are located between the two plates, and the thickness of the associated volume of the sample is adjusted by the two plates and the spacer; and

[0481] wherein the spacer and the plates are configured to minimize the area between the two plates affected by the dust; wherein the area affected by dust is an area where the dust prevents the spacer from adjusting the final spacing between the plates in a closed phase of the plates in the same manner as in the absence of dust; wherein the relevant volume is a portion or the entire volume of the sample.

[0482] 3.3 Use spacers to reduce the impact of surface flatness variations

[0483] In reality, it is impossible for the surface of a plate to be completely flat. Figure 7 As shown in Figure 1, in CROF, the surface flatness variation can be significantly large compared to the desired sample thickness, which can cause large errors in determining the sample thickness. As the final sample thickness in CROF becomes very thin (e.g., in the micrometer or nanometer configuration), the surface flatness variation can increasingly lead to significant errors.

[0484] The surface flatness variation can be characterized by the surface flatness variation distance of the plate, which is the distance from the local maximum to the adjacent local minimum of the surface height (e.g. Figure 7 b).

[0485] The present invention provides a means to make the variation of the final sample thickness in the closed phase of the CROF process smaller than the variation of the surface flatness present on the sample surface of the plate when the plate is in the open phase. The key methods for achieving uniform final sample thickness in the present invention are the use of a flexible plate, appropriate distance between spacers and appropriate pressing force (such as Figure 7 c and d).

[0486] Considering the case where one rigid board and one flexible board are used in the CROF process, under the open configuration of the board, the sample surface of the rigid board has good flatness, but the sample surface of the flexible board has significant surface flatness variation (i.e., significant compared to the expected final sample thickness), as shown in Fig. Figure 7a and b. The present invention corrects the initial flatness change of the sample surface in an open configuration (e.g., to make the flatness change smaller) by using (i) a distance between spacers that is smaller than the distance of the initial surface flatness change; (ii) an appropriate pressing force and / or an appropriate capillary force between the sample and the plate in a closed phase to deform the flexible plate; and (iii) an appropriate flexibility of the flexible plate so that in the final configuration of the plate, the sample surface of the flexible plate deforms to follow the contour of the flat surface of the rigid plate ( Figure 7 c). In addition, to reduce the final sample thickness variation, the distance between spacers should also be smaller than the critical bending span of the flexible board.

[0487] The above method of correcting for surface flatness variation is also applicable to the following cases: (a) the rigid plate has an initial significant sample surface flatness variation, while the flexible plate has a smooth sample surface, (b) both the flexible plate and the rigid plate have significant sample surface flatness variation, and (c) both plates are flexible, and the sample surface of one or both plates has significant surface flatness variation ( Figure 7 d).

[0488] U5. A method for reducing the effect of variations in surface flatness of a plate on the final thickness uniformity of a relevant volume of a sample in a CROF process, comprising:

[0489] (a) obtaining a sample, wherein the thickness of a relevant volume of the sample needs to be adjusted;

[0490] (b) obtaining two plates that are movable relative to each other into different configurations; one or both of the plates being flexible; and wherein one or both of the plates comprises spacers having a predetermined inter-spacer distance and height, and wherein each of the spacers is secured to a respective plate;

[0491] (c) depositing the sample on one or both of the plates when the two plates are configured in an open configuration; wherein the open configuration is a configuration in which the two plates are either partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0492] (d) after (c), spreading the sample by bringing the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacer and the relevant volume of the sample are between the two plates, and the thickness of the relevant volume of the sample is adjusted by the two plates and the spacer;

[0493] wherein the spacer and the plate are configured such that a thickness variation of a relevant volume of the sample in the closed configuration is less than a surface flatness variation of the plate in the open phase; wherein the relevant volume is a portion or the entire volume of the sample.

[0494] U6. An apparatus for reducing the effect of variations in surface flatness of a plate on the uniformity of thickness of a relevant volume of a conditioning sample, comprising:

[0495] a first plate and a second plate movable relative to each other into different configurations, wherein one or both of the plates are flexible and one or both plates have a surface flatness variation;

[0496] a spacer fixed to one or both plates, the spacer having a predetermined height;

[0497] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0498] wherein another of said configurations is a closed phase, which is configured after said sample is deposited in said open phase; and in the closed configuration: the two plates face each other, the spacer and the associated volume of the sample are located between the two plates, and the thickness of the associated volume of the sample is adjusted by the two plates and the spacer; and

[0499] wherein the spacer and the plate are configured such that a thickness variation of a relevant volume of the sample in the closed configuration is less than a surface flatness variation of the plate in the open phase; wherein the relevant volume is a portion or the entire volume of the sample; and an average size of the relevant volume is greater than a surface flatness variation of the plate in the open phase.

[0500] In the method of paragraph U5 and the apparatus of paragraph U6, the configuration of the spacers and plates for reducing the effect of variations in surface flatness of the plates on the uniformity of the final thickness of the relevant volume of the sample includes using an appropriate inter-gap distance (ISD). A preferred embodiment is that the ISD is equal to or less than the initial surface flatness variation distance of the plates in the open configuration.

[0501] In the methods and apparatus of paragraphs U5 and U6, in some embodiments, (1) the spacers are inside the sample in a closed configuration, (2) the spacers are fixed to the corresponding plates, (3) the distance between the spacers is shorter, or (4) any combination thereof.

[0502] In the methods and apparatus of paragraphs U1 to U6, the configuration of the spacers and plates such that the thickness of the relevant volume of the sample is uniform includes the above-mentioned embodiments. In some embodiments, the predetermined spacer distance is configured to limit the local bending of the two plates between the two spacers, wherein the relevant volume is a portion or the entire volume of the sample.

[0503] This includes cases where one or both of the sheets are flexible and various degrees of flexibility (e.g. 100um thick PMMA or PS).

[0504] In a preferred embodiment, one plate is PMMA. In a preferred embodiment, one plate (the first plate) is a glass plate with a thickness of 0.5 to 1.5 mm thick and without any spacers, while the other plate (the second) plate is a 175 μm thick PMMA film with an array of spacers, wherein the spacers are pillars of rectangular shape with rounded corners (40 μm in the x-direction and 30 μm in the y-direction) and have a period of 120 um in the x-direction and 110 um in the y-direction (so the spacing in both the x- and y-directions is 80 μm).

[0505] In the methods and apparatus of Section 3, some embodiments of spacers, spacer materials, and plates within a sample in a closed phase include embodiments of Section 2.

[0506] In the methods and devices of paragraphs U1-6, in some embodiments, the ratio of the column width (or lateral average dimension) to the column height is 0.01 or greater, 0.1 or greater, 1 or greater, 1.5 or greater, 2 or greater, 3 or greater, 5 or greater, 10 or greater, 50 or greater, 100 or greater, or a range between any two values.

[0507] In the methods and apparatus of paragraphs U1-6, in a preferred embodiment, the ratio of the column width (or lateral average dimension) to the column height is 1, 1.5, 2, 10, 20, 30, 50, 100 or a range between any two values.

[0508] In the methods and apparatus of paragraphs U1-6, in some embodiments, the ratio of the column period to the column width (or lateral average dimension) is 1.01, 1.1, 1.2, 1.5, 1.7, 2, 3, 5, 7, 10, 20, 50, 100, 500, 1000, 10,000, or a range between any two of these values.

[0509] In the methods and apparatus of paragraphs U1-6, in a preferred embodiment, the ratio of the column period to the column width (or lateral average dimension) is 1.2, 1.5, 1.7, 2, 3, 5, 7, 10, 20, 30, or a range between any two values.

[0510] In the methods and apparatus of paragraphs U1-6, in a preferred embodiment, the ratio of the column period to the column width (or lateral average dimension) is 1.2, 1.5, 1.7, 2, 3, 5, 7, 10, or a range between any two values.

[0511] For example, in a blood cell counting application, the preferred X-plate column height is between 1 um and 5 um, the column width is between 2 um and 30 um, and the column period is between 4 um and 300 um.

[0512] For example, in immunoassay applications, the preferred X-plate column height is between 5um and 50um, the column width is between 10um and 250um, and the column period is between 20um and 2500um.

[0513] The embodiments described in Section 3 and any combination thereof may be applied to (ie, combined with) other embodiments throughout the specification of the present invention.

[0514] In some embodiments, other factors are also used to control sample thickness uniformity, including but not limited to sample area, mechanical properties of the plate, final sample thickness in the closed configuration, and plate surface wetting properties.

[0515] The following are some preferred embodiments of the methods and apparatus in Section 1 and the remainder of this disclosure.

[0516] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 2 to 4 μm, an average lateral dimension of 5 to 20 um, and a spacer pitch of 1 um to 100.

[0517] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 2 to 4 μm, an average lateral dimension of 5 to 20 um, and a spacer pitch of 100 μm to 250.

[0518] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 4 to 50 um, an average lateral dimension of 5 to 20 um, and a spacer pitch of 1 um to 100.

[0519] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 4 to 50 um, an average lateral dimension of 5 to 20 um, and a spacer pitch of 100 μm to 250.

[0520] The period of the spacer array is 1nm to 100nm in one preferred embodiment, 100nm to 500nm in another preferred embodiment, 500nm to 1000nm in another preferred embodiment, 1um (i.e. 1000nm) to 2um in another preferred embodiment, 3μm to 5μm in another preferred embodiment, 5μm to 10μm in another preferred embodiment, 10μm to 50μm in another preferred embodiment, 10μm to 100μm in another preferred embodiment, 100μm to 175μm in a separate preferred embodiment, and 175μm to 300μm in another preferred embodiment.

[0521] 4. Samples and Deposition

[0522] In the present invention using the method and apparatus of CROF process, the sample is deposited by several methods or a combination of these methods. In one embodiment of deposition, the sample is deposited on only one plate. In certain embodiments, the sample is deposited on two plates (i.e., the first and second plates).

[0523] The sample is deposited while the plate is in an open phase. In some embodiments, the first plate and the second plate are well separated from each other during sample deposition, allowing the sample to be easily deposited on one plate without interfering with the other plate. For example, the first plate and the second plate can be far apart so that the sample drips directly onto the first plate or the second plate as if the other plate does not exist. In certain embodiments of sample deposition, the first plate and the second plate are separated from each other by a certain distance at the open configuration of the plate, and then the sample is deposited on the plate (e.g., by lateral flow or other dripping methods). In certain embodiments, the two plates have one side (e.g., edge) connected together during all operations of the plate ( Fig.30 ). and the opening and closing of the two panels similar to the opening and closing of a book.

[0524] The deposition of the sample can be a single drop or multiple drops. The multiple drops can be at one location or multiple locations on one or both plates. The droplets can be well separated from each other, connected or a combination thereof.

[0525] In some embodiments, the sample includes more than one material, and the materials are deposited together or separately. The materials are deposited separately in parallel or sequentially.

[0526] The sample is deposited onto the plate (i.e., the first plate and the second plate) using a device or directly from the subject to the plate. In some embodiments, a device is used to deposit the sample. The device includes, but is not limited to, a pipette, a needle, a rod, a swab, a tube, an injector, a liquid dispenser, a tip, a rod, an inkjet printer, a printer, a spray device, etc. In certain embodiments, the sample is deposited onto the plate by directly contacting the sample at the sample source and the CROF plate without using any device (i.e., placing the sample and the plate together so that the two are in contact). This is referred to as "direct sample deposition."

[0527] Examples of direct sample deposition onto the plate are (a) direct contact between a pierced finger (or other body part) and the plate, (b) spitting onto the plate, (c) causing tears in a person's eye by direct contact between tears and the plate, (d) direct contact between sweat and the plate, and (e) direct breath onto the plate, depositing breath, etc. This direct deposition method can be used on humans and animals.

[0528] In some embodiments, direct and indirect (through the device) sample deposition are used.

[0529] In the present invention, the volume of the sample deposited on the plate or plate ("sample volume") is at most 0.001 pL (picoliter), at most 0.01 pL, at most 0.1 pL, at most 1 pL, at most 10 pL, at most 100 pL, at most 1 nanoliter (nanoliter), at most 10NL, at most 100NL, at most 1 microliter (microliter), at most 10 microliters, at most 100 microliters, at most 1 milliliter (milliliter), at most 10 milliliters, or a range of any two of these values.

[0530] In some embodiments, the deposition of the sample includes the steps of: (a) placing the sample on one or both plates, and (b) spreading the sample using a method other than second plate compression in the CROF process. Methods of spreading the sample include using other devices (e.g., rods, blades), air blowing, or other devices.

[0531] The sample is deformed.

[0532] During the CROF process, in some embodiments, the sample behaves as an approximately incompressible liquid (which refers to a liquid that maintains a constant volume under shape deformation), so changes in sample thickness will result in changes in sample area. In some embodiments, the sample behaves like a compressible liquid, but its lateral area still expands when its thickness decreases during the CROF process. In certain embodiments, the sample is a liquid, gel, or soft solid, as long as during the CROF process, their lateral area expands when their thickness decreases.

[0533] In the disclosed invention, "facing the first plate and the second plate" is a process of manipulating the position and orientation of the first plate or the second plate or both so that the sample is located between the inner surfaces of the first plate and the second plate. In some embodiments, the action of "facing the first plate and the second plate" is performed by a human hand, a human hand with certain equipment, or an automatic device without a human hand.

[0534] In some embodiments, the thickness is at most 1 millimeter, at most 100 μm, at most 20 μm, at most 10 μm, or at most 2 micrometers. In some embodiments, the thickness is at least 0.1 micrometers. In some embodiments, the thickness is also measured.

[0535] In some embodiments, the thickness of the relevant volume of the sample varies by at most 300%, at most 100%, at most 30%, at most 10%, at most 3%, at most 1%, at most 0.3% or at most 0.1% of the effective diameter of the relevant area.

[0536] In some embodiments, the thickness is determined at least in part by the predetermined height.

[0537] 5. Analytes, entities, binding sites, storage sites, and transport media

[0538] In the present invention, an entity includes, but is not limited to, one of a protein, an amino acid, a nucleic acid, a lipid, a carbohydrate, a metabolite, a cell or a nanoparticle.

[0539] In some embodiments, a binding site includes a binding partner configured to bind a corresponding entity.

[0540] In some embodiments, a binding site includes an entity that binds to the binding site.

[0541] In some embodiments, placing the sample includes placing the sample within a binding site.

[0542] In some embodiments, the agent comprises at least one of a protein, an amino acid, a nucleic acid, a lipid, a carbohydrate, and a metabolite.

[0543] In certain embodiments, the storage site comprises dried reagents.

[0544] In some embodiments, the storage site includes a reagent configured to be released from the storage site upon contact with the sample.

[0545] In some embodiments, the first storage site and the second storage site are located in a common storage site.

[0546] In some embodiments, the transfer medium is a sample. In some embodiments, the transfer medium is a liquid, wherein the reagent or entity can dissolve and diffuse into the liquid.

[0547] In some embodiments, the plate has multiple storage locations. In another embodiment, a storage location has multiple reagents.

[0548] Different release times. In some embodiments, the plate has multiple storage sites at different locations on the plate or one storage site stores multiple reagents, and upon contact with the sample through the storage site, the reagents are released but at different times for the same storage location or reagents at different storage locations.

[0549] In some embodiments, the first reagent is configured to be released from a first storage site upon contact with a sample within a first average release time, and the second reagent is configured to be released with a second storage site average release time after contact with a second storage site, and wherein the first average release time is less than the second average release time.

[0550] In some embodiments, the first reagent is configured to be released from the first storage site upon contact with the sample, and wherein the second reagent is a bound reagent.

[0551] In some embodiments, depositing includes binding at least one reagent to a respective plate.

[0552] In some embodiments, contacting comprises releasing at least one reagent from the respective plate.

[0553] In some embodiments, depositing comprises depositing a first agent and a second agent, and wherein contacting comprises releasing the first agent prior to the second agent.

[0554] In some embodiments, at least one of the plates comprises a storage site including an associated volume of reagent to be added to the sample.

[0555] In some embodiments, the agent comprises at least one of a protein, an amino acid, a nucleic acid, a lipid, a carbohydrate, and a metabolite.

[0556] In some embodiments, the storage site includes dried reagents.

[0557] In some embodiments, the storage site includes a reagent configured to be released from the storage site upon contact with the sample.

[0558] In some embodiments, the storage site is a first storage site and the reagent is a first reagent, wherein the device includes a second storage site comprising a second reagent to be added to a relevant volume of the sample, wherein the second storage site is on a plate.

[0559] In some embodiments, the first storage site and the second storage site are located in a common storage site.

[0560] In some embodiments, the first reagent is configured to be released from a first storage site upon contact with a sample within a first average release time, and the second reagent is configured to be released with a second storage site average release time after contact with a second storage site, and wherein the first average release time is less than the second average release time.

[0561] In some embodiments, at least one reagent is dried on a respective plate.

[0562] In some embodiments of the kit, at least one reagent is bound to a corresponding plate.

[0563] In some embodiments of the kit, at least one reagent is configured to be released from the corresponding plate upon contact with the sample.

[0564] In some embodiments of the kit, a first reagent is located on one or both plates and a second reagent is located on one or both of the plates, wherein the first reagent is configured to be released from the respective plate upon contact with a sample for a first average release time and the second reagent is configured to be released from the respective plate upon contact with the sample for a second average release time, and wherein the first average release time is less than the second average release time.

[0565] In some embodiments of the device, the storage site is a first storage site and the reagent is a first reagent, wherein the device includes a second storage site comprising a second reagent to be added to the associated volume of the sample, wherein the second storage site is located on one of the plates.

[0566] 6. Local combination or mixed sample part (P)

[0567] In some applications, it is desirable to have a binding site to capture (i.e., bind) an analyte in a portion of a sample, rather than in the entire sample. In some cases, it is also desirable to add (i.e., mix) a reagent to a port in a sample, rather than the entire sample. It is generally desirable that there is no fluid separation between that portion of the sample and the rest of the sample. In some multiplexed assays, such a requirement is preferred or necessary.

[0568] The present invention provides a solution to the above requirements by using the CROF method and device to reshape the sample into an ultra-thin film of thickness less than the lateral dimension of the sample portion, where only the portion of the sample with the analyte inside will be captured, or only a portion of the sample will be mixed with the reagent. The working principle of this method is that when the thickness of the sample is less than the lateral dimension of the sample portion, the mixing of the analyte captured by the surface or the reagent placed on the surface can be mainly limited to the diffusion of the analyte and the reagent in the thickness direction, where the diffusion of the lateral diffusion is relatively insignificant. For example, if the sample is reshaped into a 5um thick film, if the lateral dimension of the portion of the sample where the analyte should be captured or the reagent should be mixed is 5mm×5mm, and if the diffusion time of the analyte or reagent on 5 microns is 10 seconds, the lateral diffusion of the analyte or reagent on a distance of 5 millimeters is 1,000,000 seconds (because the diffusion time is proportional to the square of the diffusion distance). This means that by selecting the appropriate ratio of the lateral dimension of the portion of interest of the sample to the thickness of the sample, within a specific time interval, the captured analyte is mainly from the sample portion of interest, or the reagent is mainly mixed to the sample of interest.

[0569] 6.1 Local binding of solids to surfaces in a portion of a sample (P: volume to surface)

[0570] P1. A method for locally binding a target entity in a relevant volume of a sample to a binding site on a surface, comprising:

[0571] (i) performing steps (a) to (d) in the method of paragraph X1, wherein the thickness of the sample in the closed configuration is significantly less than the average linear dimension of the binding site; and wherein the relevant volume is the volume of the sample located on the binding site when the plate is in the closed phase;

[0572] (ii) After (i) and when the panel is in the closed configuration, you may:

[0573] (1) Incubate the sample for a relevant length of time and then stop the incubation; or

[0574] (2) incubating the sample for a time equal to or longer than the minimum of the relevant time length, and then evaluating the binding entity of the target at the binding site for a time period equal to or less than the maximum of the relevant time length;

[0575] The relevant time lengths are:

[0576] i. equal to or greater than the time required for the target entity to pass through the thickness of a uniform thickness layer in a closed configuration; and

[0577] ii. significantly shorter than the time required for the target entity to diffuse laterally across the minimum lateral dimension of the binding site;

[0578] wherein at the end of the incubation in (1) or during the evaluation in (2), a majority of the target entity bound to the binding site is from the relevant volume of the sample;

[0579] wherein said incubating allows said target entity to bind to said binding site, and wherein said relevant volume is the portion of said sample that is above said binding site in said closed configuration.

[0580] The method of paragraph P2, wherein the term "the thickness of the relevant volume of the sample is significantly less than the smallest average dimension of the binding sites" means that the ratio of the smallest average dimension of the binding sites to the thickness of the sample (referred to as the "length to thickness ratio") is at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1,000, at least 10,000, at least 100,000, or any range between values. In preferred embodiments, the length to thickness ratio is at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, or any range between values.

[0581] The method of paragraph P2, wherein the term "significantly shorter than the time for the target entity to diffuse laterally across the smallest lateral dimension of the binding site" means that the time for diffusion across the smallest lateral dimension of the binding site is the time for diffusion through the thickness of the sample (referred to as the "length to thickness diffusion time ratio") is at least 3, at least 10, at least 50, at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,00,000, or any range of values ​​therebetween. In preferred embodiments, the length to thickness diffusion time ratio is at least 3, at least 10, at least 50, at least 10, at least 100, at least 1,000, at least 10,000, or any range of values ​​therebetween.

[0582] P2. A device for locally binding entities in a relevant volume of a sample to binding sites on a surface, comprising:

[0583] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations,

[0584] wherein the first plate has binding sites on its surface, the binding sites having an area smaller than that of the plate and configured to bind a target entity in a sample, wherein the target entity is diffusible in the sample, and wherein each of the two plates comprises spacers and each spacer is affixed to its respective plate and has a predetermined height;

[0585] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates,

[0586] wherein another of said configurations is a closed phase state, which is configured in an open phase state after said sample is deposited; and in the closed phase state: the plates face each other, the spacers, the binding sites and at least a portion of the sample are located between the plates, the sample contacts at least a portion of the binding sites, the volume of the sample of associated thickness is adjusted by the plates and the spacers, and is thinner than the maximum thickness of the sample when the plates are in the open phase state, wherein the associated volume is the volume of the sample located on the binding sites;

[0587] wherein the spacer height is selected to adjust the thickness of the associated volume in the closed phase to be at least 3 times smaller than the average linear dimension of the binding sites.

[0588] The thickness of the volume of interest is adjusted to be 3 times smaller than the average linear dimension of the binding sites, so that the diffusion time of the entities over the sample thickness is 9 times smaller than the diffusion time over a distance equal to the average linear dimension of the sample thickness of the binding sites. This thickness adjustment makes it possible to select an incubation time such that the incubation results in (i) a significant number of target entities in the volume of interest binding to the binding sites, and (ii) a significant number of target entities with binding sites from the volume of interest of the sample, and wherein the incubation is a process that allows the target entities to bind to the binding sites.

[0589] For example, if the incubation time is set equal to the diffusion time of the entity through the thickness of the relevant volume of the sample, then after incubation, most of the entities within the relevant volume have reached and are constrained according to the rate equation, while the entities outside the relevant volume (i.e., before incubation) can initially only diffuse to the periphery of the relevant volume (a relatively small volume), and this volume becomes less important because the ratio of the average linear size of the binding sites to the thickness of the relevant volume becomes larger.

[0590] 6.2 Binding entities stored on a plate surface bind to binding sites on other plate surfaces (surface to surface)

[0591] P. A method for locally binding an entity stored at a storage location on one plate to a binding site on another plate, comprising:

[0592] (a) obtaining a first plate and a second plate movable relative to each other into different configurations, wherein a surface of the first plate has binding sites; and a surface of the second plate has storage sites comprising an entity that binds to the binding sites; wherein the area of ​​the binding sites and the area of ​​the reagent sites are smaller than the area of ​​each plate; and wherein one or both of the plates include spacers, and each of the spacers is fixed to its corresponding plate and has a predetermined height;

[0593] (b) obtaining a transfer medium, wherein the entity is capable of dissolving into and diffusing in the transfer medium;

[0594] (C) a transfer medium deposited on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0595] (d) after (c), unfolding the transfer medium by moving the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacers, the binding sites, the storage locations and at least a portion of the transferred medium are between the plates; at least a portion of the storage locations and a portion of the transfer medium therebetween directly face the binding sites, and the thickness of the associated volume of the transfer medium is adjusted by the plates and the spacers to be greater than the maximum thickness when the plates are in an open phase and significantly less than an average linear dimension of the associated volume in the direction of the plate surface; and

[0596] (e) after (d) and when the plate is in the closed phase, incubating for a period of time and stopping the incubation, wherein the incubation time is selected so that a significant number of entities bound to the binding sites are removed from the storage sites, wherein the relevant volume is the volume of the transfer medium located on the binding sites, and the incubation is the process of allowing the entities to bind to the binding sites.

[0597] The term "at least one port of the storage position directly faces the binding position" means that the shortest distance from a point in the position to the binding position is the same as the thickness of the relevant capacity of the associated configuration in the closed position.

[0598] P4. A device for binding an entity stored at a storage location on one board to a related binding location on another board, comprising:

[0599] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations, wherein a surface of the first plate has binding sites; and a surface of the second plate has storage sites containing entities that bind to the binding sites; wherein an area of ​​the binding sites and an area of ​​the storage sites are smaller than an area of ​​each plate; and wherein one or both of the plates include spacers, and each of the spacers is secured to its corresponding plate and has a predetermined height;

[0600] wherein one of the configurations is an open configuration wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and a transfer medium is deposited on one or both of the plates, wherein the entity at the storage location is able to dissolve into and diffuse into the transfer medium,

[0601] wherein another of the configurations is a closed phase state, which is configured after the transfer medium is deposited in the open phase state; and in the closed configuration: the plates face each other, the spacers, the binding sites, the storage locations and at least a portion of the transfer medium are located between the plates; at least a portion of the storage locations and a portion of the transfer medium therebetween directly face the binding sites, and the thickness of the associated volume of the transfer medium is adjusted by the plates and the spacers and is greater than a maximum thickness of the sample when the plates are in the open phase state;

[0602] wherein the relevant volume is the volume of the transfer medium located at the storage location when the plate is in the closed phase; and

[0603] wherein the spacer height is selected to adjust the thickness of the associated volume in the closed phase to be at least 3 times smaller than the average linear dimension of the binding sites.

[0604] wherein at least one of the spacers is within the sample contact area;

[0605] and a spacer having a predetermined spacing distance and height.

[0606] 6.3 A method for binding entities at multiple storage locations on one plate to multiple corresponding binding sites on another plate

[0607] P5. A method for locally binding entities stored at a plurality of storage sites on one plate to a plurality of corresponding binding sites on another plate, comprising:

[0608] (a) obtaining a first plate and a second plate that are movable relative to each other into different configurations; wherein a surface of the first plate has a plurality of binding sites, and a surface of the second plate has a plurality of corresponding storage locations; wherein each corresponding storage location is located at a position on the second plate corresponding to a position of a binding site, such that when the two plates are placed face to face, each binding site overlaps with only one storage location; and wherein one or both of the plates include spacers, and each of the spacers is fixed to its corresponding plate and has a predetermined height;

[0609] (b) obtaining a transfer medium, wherein the entity at the storage location is capable of dissolving into and diffusing into the transfer medium;

[0610] (C) a transfer medium deposited on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0611] (d) after (c), deploying the transfer medium by moving the plates into a closed configuration, wherein in the closed configuration: the two plates face each other, the spacer, the binding sites, the storage sites and at least a portion of the transfer medium are located between the plates, each binding site directly faces only one corresponding storage site, the transfer medium contacts at least a portion of each binding site and a portion of each storage site, a corresponding volume of transfer medium is regulated by the plates and the spacer, is thinner than the maximum thickness of the transfer medium when the plates are in an open phase, and is significantly smaller than an average linear dimension of the binding sites; and

[0612] (e) After (d) and when the plate is in the closed phase, incubation is carried out for a period of time and the incubation is stopped, wherein the incubation time is selected so that a significant number of entities bind to each binding site from the corresponding reservoir, wherein the relevant volume is the volume of the transfer medium located on the binding site, and the incubation is the process of allowing the entities to bind to the binding sites.

[0613] In some embodiments, the spacing is limited to the binding sample area.

[0614] In some embodiments of method P5, the transfer medium is a sample with a target analyte, the binding site comprises a capture agent, and the entity in the storage site is a detection agent, wherein the target analyte binds the capture agent and the detection agent to form a capture agent-analyte-detection agent sandwich. Method P5 simplifies the analysis steps, and by using a smaller spacer height, the sample thickness can be shortened, and the vertical diffusion time can be shortened, thereby shortening the saturation analysis time, thereby reducing the detection time of the analyte and the detection agent.

[0615] P6. An apparatus for locally binding entities stored at a plurality of storage locations on one board to a plurality of corresponding binding locations on another board, comprising:

[0616] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations;

[0617] wherein the surface of the first plate has a plurality of binding sites, and the surface of the second plate has a plurality of corresponding storage locations; wherein each corresponding storage location is located at a position on the second plate corresponding to the position of the binding site, such that each binding site overlaps with only one storage location when the two plates are placed face to face; and wherein one or both of the plates include spacers, and each of the spacers is secured to its corresponding plate and has a predetermined height;

[0618] wherein one of the configurations is an open configuration wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and a transfer medium is deposited on one or both of the plates, wherein the entity at the storage location is able to dissolve into and diffuse into the transfer medium,

[0619] wherein another of the configurations is a closed phase state, which is configured after the transfer medium is deposited in the open phase state; and in the closed configuration: the two plates face each other, the spacers, the binding sites, the storage locations and at least a portion of the transfer medium are located between the plates, each binding site directly faces only one corresponding storage site, the transfer medium contacts at least a portion of each binding site and a portion of each storage site, and the thickness of the associated volume of the transfer medium is adjusted by the plates and the spacers and is thicker than the maximum thickness of the transfer medium when the plates are in the open phase state;

[0620] wherein the relevant volume is the volume of the transfer medium located at the storage location when the plate is in the closed phase; and

[0621] The predetermined spacer height is selected to adjust the thickness of the relevant volume in the closed configuration to be significantly smaller than the average linear dimension of the binding sites.

[0622] 6.4 Local addition of reagent stored on the surface to a portion of the sample (surface volume)

[0623] P7. A method for locally adding a reagent to a relevant volume of a sample, comprising:

[0624] (a) obtaining a first plate and a second plate movable relative to each other into different configurations, wherein the first plate has storage locations on its surface containing reagents to be added to the associated volume of sample, the reagents being capable of dissolving into and diffusing into the sample, and the storage locations having an area smaller than that of the plates; and wherein one or both of the plates include spacers, and each of the spacers is fixed to its corresponding plate and has a predetermined height;

[0625] (b) Obtain samples;

[0626] (C) placing a sample on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by the spacer;

[0627] (d) after (c), spreading the sample by bringing the plates into a closed state; wherein, in the closed phase: the plates face each other; the spacers, storage locations and at least a portion of the sample are located between the plates; the sample contacts at least a portion of the storage locations and contacts the plates over an area greater than the storage locations; the thickness of the relevant volume of the sample is adjusted by the plates and the spacers, is thinner than the maximum thickness of the sample when the plates are in the open phase, and is significantly smaller than an average linear dimension of the relevant volume in the direction of the plate surface; and

[0628] (e) after (d) and when the plate is in the closed phase, incubating for a period of time and stopping the incubation, wherein the incubation time is selected so that (i) a significant amount of the reagent dissolved in the sample is contained in the relevant volume of the sample and (ii) the reagent is a significant part of the relevant volume, and wherein the relevant volume is the volume of the sample located at the storage site when the plate is in the closed phase, and the incubation is a process that allows the reagent to dissolve and diffuse in the sample.

[0629] P8. A device for locally adding a reagent stored on a plate surface to a relevant volume of a sample, comprising:

[0630] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations,

[0631] wherein the first plate has storage locations on its surface, the storage locations containing reagents to be added to an associated volume of a sample, the reagents being capable of dissolving into and diffusing into the sample; and wherein one or both of the plates include spacers, and each of the spacers is secured to its respective plate and has a predetermined height;

[0632] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0633] wherein another of said configurations is a closed phase state, which is configured in an open phase state after said sample is deposited; and in the closed configuration: the plates face each other, the spacers, the storage locations and at least a portion of the sample are located between the plates, the sample is in contact with at least a portion of the storage locations, and when a surface of at least one of the plates is outside the storage locations, the thickness of a relevant volume of the sample is adjusted by the plates and the spacers, and when the plates are in the open phase state, its thickness is thinner than the maximum thickness of the sample, and wherein the relevant volume is the volume of the sample located at the storage locations when the plates are in the closed configuration;

[0634] The spacer height is selected to adjust the thickness of the relevant volume in the closed phase of the plate to be at least less than 3 times the average linear dimension of the relevant volume in the direction of the plate surface.

[0635] 7. Formation of a capture-analyte-detection sandwich at the binding site (W)

[0636] One aspect of the invention is to form a capture-analyte-detection sandwich on binding sites on a solid surface in a single step by using CROF processing and placing binding sites on one plate onto corresponding storage sites storing detection reagents on another plate.

[0637] 7.1 Capture-analyte-detection sandwich at the binding site in one incubation step (general) (W)

[0638] W1. A method for forming a capture-analyte-detection sandwich at a binding site of a plate, comprising:

[0639] (a) obtaining a sample containing a target analyte, wherein the target analyte is capable of diffusing into the sample;

[0640] (b) obtaining a capture agent and obtaining a detection agent, wherein the capture agent and the detection agent are (capable of) binding to the target analyte to form a capture agent-target analyte-detection agent sandwich;

[0641] (C) obtaining a first plate and a second plate that are movable relative to each other into different configurations; wherein the first plate has binding sites where a capture agent is immobilized on the sites, and the second plate has storage sites where the detection agent is stored; wherein when the storage sites are in contact with the sample, the detection agent is capable of dissolving into the sample and diffusing into the sample; and wherein one or both of the plates include spacers, and each of the spacers is fixed to its corresponding plate and has a predetermined height;

[0642] (d) placing a sample on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0643] (e) after (d), unfolding the sample by moving the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacers and the associated volume of the sample are between the plates, the volume of the sample of associated thickness is adjusted by the plates and the spacers and is thinner than the sample thickness when the plates are in an open phase, and the sample is in contact with the binding sites and the storage sites; and

[0644] (f) after (e), while the plate is in a closed phase, incubating for a period of time to allow formation of a capture agent-target analyte-detection agent sandwich;

[0645] The relevant volume is at least a portion or the entire volume of the sample.

[0646] W2. An apparatus for forming a capture-analyte detection sandwich on a binding site of a plate, comprising:

[0647] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations;

[0648] wherein the first plate has a binding site having a capture agent immobilized thereon, and the second plate has a storage site for storing a detection agent; wherein the capture agent and the detection agent are (capable of) binding to a target analyte in a sample to form a capture agent-target analyte-detection agent sandwich; wherein the detection agent is capable of dissolving into and diffusing into the sample when the storage site is in contact with the sample; and wherein one or both of the plates include spacers, and each of the spacers is affixed to its corresponding plate and has a predetermined height;

[0649] wherein one of the configurations is an open phase, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0650] wherein another of said configurations is a closed phase, which is deposited when said sample is in an open phase; in the closed configuration: the plates face each other, the spacer and the associated volume of the sample are located between the plates, the thickness of the associated volume of the sample is adjusted by the plates and the spacer and is thinner than the thickness of the sample when the plates are in an open phase and the sample is in contact with the binding sites and the storage sites;

[0651] The relevant volume is at least a portion or the entire volume of the sample.

[0652] 7.2 Using analytes from a portion of the sample (ie, localized), a capture-analyte detection sandwich is formed at the binding site in a one-step incubation.

[0653] W3. A method of forming a capture-analyte-detection sandwich on a binding site of a plate using an analyte from a portion of a sample, comprising:

[0654] (a) obtaining a sample containing a target analyte, wherein the target analyte is capable of diffusing into the sample;

[0655] (b) obtaining a capture agent and obtaining a detection agent, wherein the capture agent and the detection agent are capable of binding to the target analyte to form a capture agent-target analyte-detection agent sandwich;

[0656] (c) obtaining a first plate and a second plate movable relative to each other into different configurations; wherein the first plate has binding sites where a capture agent is immobilized on the sites, and the second plate has storage sites where the detection agent is stored, the agent being able to dissolve into the sample and diffuse into the sample when the storage sites are in contact with the sample; and wherein one or both of the plates include spacers, and each of the spacers is fixed to its corresponding plate and has a predetermined height;

[0657] (d) placing a sample on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0658] (e) after (d), unfolding the sample by moving the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacers, the binding sites and the storage locations are between the plates, the binding sites and the storage locations are in contact with a relevant volume of the sample, and the thickness of the relevant volume of the sample is regulated by the plates and the spacers and is thinner than the sample thickness when the plates are in an open phase; and is significantly smaller than an average linear dimension of the binding sites; and

[0659] (f) after (e) and when the plate is in a closed phase, incubating for a period of time and stopping the incubation, wherein the incubation time is selected to allow a large number of capture-analyte-detection sandwich structures to be formed at the binding site, including analytes from the relevant volume sample, wherein the relevant volume is the volume of the sample located at the binding site, and the incubation is a process that allows the capture-analyte detection sandwich to be formed.

[0660] In some embodiments, the ratio of pitch to site size may be less than 1 / 5.

[0661] W4. A device for forming a capture-analyte-detection sandwich at a binding site on a plate, the capture-analyte-detection sandwich having an analyte from a portion of a sample, the device comprising:

[0662] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations;

[0663] wherein the first plate has a binding site having a capture agent immobilized thereon, and the second plate has a storage site for storing a detection agent; wherein the capture agent and the detection agent are (capable of) binding to a target analyte in a sample to form a capture agent-target analyte-detection agent sandwich; wherein the detection agent is capable of dissolving into and diffusing into the sample when the storage site is in contact with the sample; and wherein one or both of the plates include spacers, and each of the spacers is affixed to its corresponding plate and has a predetermined height;

[0664] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0665] wherein another of said configurations is a closed phase state, which is configured in an open phase state after said sample is deposited; and in the closed phase state: the plates face each other, the spacers, the binding sites and the storage sites are between the plates, the binding sites and the storage sites are in contact with a relevant volume of the sample, and the relevant volume of the sample having a thickness adjusted by the plates and the spacers and being thinner than the sample thickness when the plates are in the open phase state; and wherein the relevant volume is the volume of the sample located on the binding sites; and

[0666] wherein the spacer height is selected to adjust the thickness of the associated volume in the closed configuration to be significantly smaller than the average linear dimension of the binding site. 7.3 Method for reducing the time to form a capture-analyte detection sandwich at a binding site by reducing the diffusion distance (W, X).

[0667] W5. A method of reducing the time to form a capture-analyte-detection sandwich at a binding site of a plate, comprising:

[0668] (a) obtaining a sample containing a target analyte, wherein the target analyte is capable of diffusing into the sample;

[0669] (b) obtaining a capture agent and obtaining a detection agent, wherein the capture agent and the detection agent are capable of binding to the target analyte to form a capture agent-target analyte-detection agent sandwich;

[0670] (c) obtaining a first plate and a second plate movable relative to each other into different configurations; wherein the first plate has binding sites where a capture agent is immobilized on the sites, and the second plate has storage sites where the detection agent is stored, the agent being able to dissolve into the sample and diffuse into the sample when the storage sites are in contact with the sample; and wherein one or both of the plates include spacers, and each of the spacers is fixed to its corresponding plate and has a predetermined height;

[0671] (d) placing a sample on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0672] (e) after (d), spreading the sample by moving the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacers, the binding sites and the storage sites are between the plates, the binding sites overlap with the storage sites, the binding sites and the storage sites are in contact with a relevant volume of the sample, and the thickness of the relevant volume of the sample is adjusted by the plates and the spacers, and the plates are in an open state when the sample thickness is less than the sample thickness; and the reduction in sample thickness thereby reduces the time for analytes and detection reagents to diffuse vertically across the sample thickness, wherein the relevant volume is at least a portion of the entire volume of the sample.

[0673] The time period during which the target entity in the relevant volume is allowed to bind to the binding site is shorter than the time period during which the structure is not closed.

[0674] The method may further include a washing step to remove the sample between the plates, and the washing step is performed when the plates are in the closed phase or the open phase.

[0675] The method further comprises a reading step of reading the signal from the capture-analyte-detection sandwich immobilized on the binding sites. The reading is performed after washing or without any washing.

[0676] The method may further be multiplexed as described above or below.

[0677] W6. A device for reducing the time to form a capture-analyte detection sandwich on a binding site of a plate, comprising:

[0678] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations;

[0679] wherein the first plate has a binding site having a capture agent immobilized thereon, and the second plate has a storage site for storing a detection agent; wherein the capture agent and the detection agent are (capable of) binding to a target analyte in a sample to form a capture agent-target analyte-detection agent sandwich; wherein the detection agent is capable of dissolving into and diffusing into the sample when the storage site is in contact with the sample; and wherein one or both of the plates include spacers, and each of the spacers is affixed to its corresponding plate and has a predetermined height;

[0680] wherein one of the configurations is an open phase, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0681] Another of the configurations is a closed phase in which the sample is deposited in an open phase; in the closed phase: the plates face each other, the spacers, the binding sites and the storage sites are between the plates, the binding sites overlap with the storage sites, the binding sites and the storage sites are associated with a volume of the sample, and the thickness of the associated volume of the sample is adjusted by the plates and the spacers and is thinner than the thickness of the sample when the plates are in an open phase; and the reduced thickness of the sample thereby reduces the time for the analyte and the detection reagent to diffuse vertically through the thickness of the sample, wherein the associated volume is at least a portion of the entire volume of the sample.

[0682] In these embodiments, the method may include a plate to which a capture agent is attached, wherein the attachment is accomplished by a chemical reaction of the capture agent with a reactive group on a flat plate. Another plate may include a dry detection agent patch at a position so that after the plate is closed, the attached capture agent and the detection agent patch face each other. Then, as described above, the method may include a sample-analyte and device containing a target contact and closing the plate. The detection agent dissolves and diffuses into the sample. Since the target analyte is in a solution, the target analyte will be bound to the capture agent and fixed to the surface of one of the plates. The detection agent may be bound to the target analyte before or after binding to the capture agent. In some cases, the method may include removing any analyte that is not bound to the capture agent target, or any unbound detection agent (e.g., by washing the surface of the plate in a binding buffer); the detection agent may be conjugated with an optically detectable marker, thereby providing a means for detecting the target analyte. After optionally removing the detection agent that is not bound to the target analyte, the system can read, for example, using a readout system to read out the light signal (e.g., the wavelength of light is between 300nm and 1200nm), which is bound to the plate. In addition, as described above, the detection agent can be directly labeled (in this case, the detection agent can be strongly linked to the luminescent label before being deposited on one of the plates), or indirectly labeled (i.e., by a second capture agent that binds to the detection agent, for example, a labeled second antibody or a labeled nucleic acid, specifically binds to the detection agent and is linked to the luminescent label). In some embodiments, the method can include a capping agent to prevent non-specific binding of the capture agent to non-target analytes. Binding conditions for specific target analytes include suitable temperature, time, solution pH level, ambient light level, humidity, chemical reagent concentration, antigen-antibody ratio, etc., which are all well known or can be easily derived from the present disclosure. General methods for methods of molecular interaction between capture agents and their binding partners (including analytes) are well known in the art (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, 1st edition (1988) Cold spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, Wiley & Sons, 1995). The methods described above and below are exemplary; the methods herein are not the only way to conduct an assay.

[0683] In certain embodiments, nucleic acid capture agents can be used to capture protein analytes (e.g., DNA or RNA binding proteins). In certain embodiments, protein capture agents (e.g., DNA or RNA binding proteins) can be used to capture nucleic acid analytes.

[0684] The sample may be a sample of cells, tissues or body fluids from a clinical setting. Body fluids of interest include, but are not limited to, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (ear wax), chyle, campanulate, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomitus, urine and exhaled condensate.

[0685] In one embodiment of the assay, the plate is contacted with a sample containing a target analyte (e.g., a target protein) and the plate is closed. The sample contains or is modified to contain all necessary reagents (e.g., salts, etc.) suitable for specific binding conditions. The capture agent (e.g., an antibody) and the detection agent specifically bind to the target analyte in the sample, resulting in a detectable patch of labeled analyte.

[0686] As in any embodiment, the amount of the target analyte in the sample can be measured to provide a qualitative or quantitative measurement of the amount of the target analyte in the sample. In some embodiments, the magnitude of the signal provides a quantitative determination of the amount of the target analyte in the sample. In some cases, the assessment can be compared with a standard curve (e.g., a standard curve of a second analyte or a spiked analyte) that may be in a known concentration in some cases. The comparison can be facilitated by depositing capture agents of different densities (e.g., different concentrations) and reading signals from each capture agent block.

[0687] 8 Combine and add samples and reagents using small volumes (V)

[0688] In many applications, it is highly desirable to use the smallest possible volume of sample or reagent. However, in microfluidic channel devices (the most common method for using small samples today), a large amount of sample is wasted flowing from the inlet of the device to the test (detection) area, resulting in the need for a larger sample volume than the volume of the test location. One aspect of the present invention is to significantly reduce the volume of sample or reagent used in the test by depositing a micro volume of sample or reagent on a plate and then reshaping the volume into a thin film with a smaller thickness, but a larger area than before. This reshaping can also speed up the reaction rate.

[0689] 8-1 Bind the target entity in a small volume of sample on the surface binding site by diffusing the sample.

[0690] V1. A method for binding a target entity in a sample to a binding site, comprising:

[0691] (a) obtaining a first plate and a second plate movable relative to each other into different configurations, wherein the first plate has binding sites on its surface, and wherein one or both of the plates include spacers and each spacer is secured to its respective plate and has a predetermined height;

[0692] (b) obtaining a sample containing a target entity to be bound to the binding site;

[0693] (c) placing a sample on one or both plates when the plates are configured in an open phase; wherein in the open phase: the two plates are partially or completely separated, the spacing between the plates is not limited by a spacer, and the sample does not cover an area or a portion of an area of ​​the binding site when deposited;

[0694] (d) after (c), spreading the sample by moving the plates into a closed state; wherein in the closed phase: the plates face each other, the spacer and the associated volume of the sample are between the plates, the sample contacts more area of ​​the binding site than when the plates are in an open phase, and the thickness of the associated volume of the sample at the binding site is adjusted by the plates and the spacer, wherein the associated volume is a portion or all of the volume of the sample.

[0695] V2. A device for binding a target entity in a sample to a surface binding site, comprising:

[0696] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations;

[0697] wherein the first plate has binding sites on its surface that bind to target entities in the sample, and wherein the area of ​​the binding sites is greater than the contact area of ​​the sample when the sample is deposited on only one of the plates, contacting the other plate;

[0698] wherein one or both of the plates include spacers, and each of the spacers is secured to its corresponding plate and has a predetermined height;

[0699] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates and the cover, as preserved, whether in the area of ​​the binding site or a portion thereof;

[0700] Another of the configurations is a closed phase, which is configured in an open phase after the sample is deposited; and in the closed phase: the plates face each other, the spacer and the sample are between the plates, the sample contacts more areas of the binding site than when the plates are in the open phase, and the thickness of the sample at the binding site is regulated by the plates and the spacer.

[0701] 8-2 Adding reagents to small sample volumes by diffusing the sample

[0702] V3. A method for binding a target entity in a sample to a binding site, comprising:

[0703] (a) obtaining a first plate and a second plate movable relative to each other into different configurations, wherein the first plate has storage locations on its surface containing reagents to be added to the sample, and wherein the plates include spacers and each spacer is affixed to its respective plate and has a predetermined height;

[0704] (b) placing a sample on one or both plates when the plates are configured in an open phase; wherein in the open phase: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample does not contact an area or a portion of an area of ​​a storage location when deposited;

[0705] (c) after (b), spreading the sample by moving the plates into a closed state; wherein in the closed phase: the plates face each other, the spacer and the associated volume of the sample are between the plates, the sample contacts more area of ​​the storage location than when the plates are in an open phase, and the thickness of the associated volume of the sample is adjusted by the spacer; and wherein the associated volume is a portion of the sample at the storage location.

[0706] V4. A device for binding a target entity in a sample to a binding site, comprising:

[0707] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different phases,

[0708] wherein the first plate has storage locations on its surface containing reagents and the reagents are to be added to the sample, and wherein one or both of the plates include spacers and each of the spacers is secured to its corresponding plate and has a predetermined height;

[0709] wherein one or both of the plates include spacers, and each of the spacers is secured to its corresponding plate and has a predetermined height;

[0710] wherein one of the phases is an open configuration wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates,

[0711] Another of the configurations is a closed phase, which is configured in an open phase after the sample is deposited; and in the closed configuration: the plates face each other, the spacer and the associated volume of the sample are located between the plates, the sample contacts more area of ​​the storage location than when the plates are in the open phase, and the amount of associated volume of the thickness sample is adjusted by the spacer; and wherein the associated volume is a portion of the sample at the storage location.

[0712] In the methods of V1 and V2 and the devices of V3 and V4, due to the small volume of the sample and the wettability of the surface, in some cases even if the sample is deposited in the binding site area or the storage area, the contact area of ​​the deposited sample with the plate will be smaller than the area of ​​the binding site or the storage site. Therefore, spreading, especially precise spreading, is required.

[0713] The sample drop may be multiple drops, and in the closed configuration, the drops merge into a film with a thickness less than the maximum thickness.

[0714] In the present invention, in the methods of paragraphs V1 to V7 and the devices of paragraphs V2 to V8, the volume of the sample deposited on the plate or plate ("sample volume") is at most 0.001pL (picoliter), at most 0.01pL, at most 0.1pL, at most 1pL, at most 10pL, at most 100pL, at most 1nL (nanoliter), at most 10nL, at most 100nL, at most 1uL (microliter), at most 10uL, at most 100uL, at most 1mL (milliliter), at most 10mL or a range of any two of these values.

[0715] 9Use uniform sample thickness for uniform binding or uniform addition of reagents (UAB)

[0716] For analytical and chemical reactions, it is advantageous to have a thin sample with uniform thickness over a relatively large area. Examples of this include physically binding a sample to a surface binding site, adding reagents to a sample, quantifying the relevant volume of a sample, quantifying an analyte, etc.

[0717] For a method that uses two plates to reduce and adjust the thickness of a relevant volume (partial or full volume) of a sample, precision, uniformity and ease of use are crucial.

[0718] One aspect of the present invention is to improve the accuracy, uniformity or ease of use of a method and / or apparatus for adjusting the thickness of a relevant volume of a sample by compressing the sample with two plates.

[0719] 9.1 Methods for uniformly binding entities in the sample to binding sites on the plate

[0720] UAB1. A method for uniformly binding entities in a sample to binding sites on a plate, comprising:

[0721] (a) obtaining a sample containing a target entity capable of diffusing in the sample;

[0722] (b) obtaining a first plate and a second plate movable relative to each other into different configurations, wherein the first plate has binding sites on its surface configured to bind the target entity, wherein one or both of the plates include spacers and each spacer is fixed to its corresponding plate and has a predetermined height;

[0723] (c) placing a sample on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0724] (d) after (c), spreading the sample by bringing the plates into a closed phase, wherein in the closed phase: the plates face each other, the spacer and the relevant volume of the sample are between the plates, the binding site is in contact with the relevant volume, the thickness of the relevant volume of the sample is adjusted by the plates and the spacer, and is thinner than the maximum thickness of the sample and more uniform across the binding site than when the plates are in an open phase;

[0725] wherein the spacer and the plate are configured such that the adjusted thickness of the relevant volume in the plate closed configuration is more uniform than the adjusted thickness of the corresponding volume in the plate open configuration; and wherein the relevant volume is a portion or the entire volume of the sample. It also has a storage location on the plate opposite the binding site for forming a uniform sandwich.

[0726] UAB2. A device for uniformly binding entities in a sample to binding sites on a plate, comprising:

[0727] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations;

[0728] wherein the first plate has binding sites on its surface configured to bind the target entity, wherein one or both of the plates include spacers, and each of the spacers is fixed to its corresponding plate and has a predetermined height;

[0729] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0730] wherein another of said configurations is a closed phase, which is configured in an open phase after said sample is deposited; and in a closed state: the plates face each other, the spacer and the relevant volume of the sample are between the plates, the binding site is in contact with the relevant volume, the thickness of the relevant volume of the sample is determined by the plates and the spacer, and is thinner than the maximum thickness of the sample and more uniform over the binding site than the plates in the open phase;

[0731] wherein the spacer and the plate are configured such that the adjusted thickness of the relevant volume in the plate closed configuration is more uniform than the adjusted thickness of the relevant volume in the plate open configuration; and wherein the relevant volume is a portion or the entire volume of the sample.

[0732] 9.2 Method for adding reagents on the plate evenly to the sample

[0733] UAB3. A method for uniformly adding a reagent to a sample of a relevant volume, comprising:

[0734] (a) obtaining a first plate and a second plate movable relative to each other into different configurations, wherein the first plate has storage locations on its surface containing a reagent to be added to an associated volume of a sample, the reagent being capable of dissolving into and diffusing into the sample; and wherein one or both of the plates include spacers, and each of the spacers is secured to its respective plate and has a predetermined height;

[0735] (b) obtaining samples;

[0736] (c) placing a sample on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0737] (d) after (c), unfolding the sample by bringing the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacer and the associated volume of the sample are between the plates, the storage position is in contact with the associated volume, and the thickness of the associated volume of the sample is adjusted by the plates and the spacer and is thinner than the maximum thickness of the sample when the plates are in an open phase;

[0738] wherein the spacer and plate are configured such that the thickness of the relevant volume of the sample is more uniform over an area of ​​the relevant volume in the plate closed configuration than over an area of ​​the relevant volume in the plate open phase; and wherein the relevant volume is a portion or the entire volume of the sample.

[0739] UAB4. A device for uniformly adding a reagent to a sample of a relevant volume, comprising:

[0740] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations;

[0741] wherein the first plate has storage locations on its surface, the storage locations containing reagents to be added to an associated volume of a sample, the reagents being capable of dissolving into and diffusing into the sample; and wherein one or both of the plates include spacers, and each of the spacers is secured to its respective plate and has a predetermined height;

[0742] One of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0743] wherein another of the configurations is a closed phase, which is configured in an open phase after the sample is deposited; and in a closed state: the plates face each other, the spacer and the associated volume of the sample are located between the plates, the storage location is in contact with the associated volume, and the thickness of the associated volume of the sample is adjusted when the plates are in the open phase, the thickness of the plates and the spacer is less than the maximum thickness of the sample;

[0744] wherein the spacer and plate are configured such that the thickness of the relevant volume of the sample is more uniform over an area of ​​the relevant volume in the plate closed configuration than over an area of ​​the relevant volume in the plate open phase; and wherein the relevant volume is a portion or the entire volume of the sample.

[0745] 9.3 A method for uniformly forming a capture-analyte-detection sandwich at a binding site

[0746] UAB5. A method for uniformly capturing an analyte detection sandwich at binding sites on a plate, comprising:

[0747] (a) obtaining a sample containing a target analyte;

[0748] (b) obtaining a capture agent and obtaining a detection agent, wherein the capture agent and the detection agent are (capable of) binding to the target analyte to form a capture agent-target analyte-detection agent sandwich;

[0749] (c) obtaining a first plate and a second plate movable relative to each other into different configurations; wherein the first plate has binding sites where a capture agent is immobilized on the sites, and the second plate has storage sites where the detection agent is stored, and when the storage sites are in contact with the sample, the detection agent is capable of being dissolved into the sample and diffusing into the sample; and wherein one or both of the plates include spacers, and each of the spacers is fixed to its corresponding plate and has a predetermined height;

[0750] (d) placing a sample on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0751] (e) after (d), unfolding the sample by bringing the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacer and the associated volume of the sample are between the plates, the volume of the associated thickness of the sample is adjusted by the plates and the spacer and is thinner than the thickness of the sample when the plates are in an open phase, and the sample is in contact with the binding site and the storage site;

[0752] wherein the spacer and plate are configured such that the thickness of the relevant volume of the sample is more uniform over an area of ​​the relevant volume in the plate closed configuration than over an area of ​​the relevant volume in the plate open phase; and wherein the relevant volume is a portion or the entire volume of the sample.

[0753] UAB6. A device for uniformly capturing an analyte detection sandwich at binding sites on a plate, comprising:

[0754] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations;

[0755] wherein the first plate has a binding site, the binding site has a capture agent immobilized on the site, and the capture agent is capable of binding to a target analyte in the sample;

[0756] wherein the second plate has a storage location for storing the detection agent, wherein the storage location is capable of (a) dissolving the detection agent into the sample and diffusing into the sample when the storage location is in contact with the sample; and (b) binding to the target analyte and forming a capture agent-target analyte-detection agent sandwich;

[0757] wherein one or both of the plates include spacers, and each of the spacers is secured to its corresponding plate and has a predetermined height;

[0758] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0759] wherein another of said configurations is a closed phase, which is configured in an open phase after said sample is deposited; and in the closed configuration: the plates face each other, the spacer and the associated volume of the sample are located between the plates, the thickness of the associated volume of the sample is adjusted by the plates and the spacer and is thinner than the thickness of the sample when the plates are in the open phase and the sample is in contact with the binding sites and the storage sites;

[0760] wherein the spacers and plates are configured such that a thickness of a relevant volume of the sample is more uniform over an area of ​​the relevant volume in the plate closed configuration than in the plate open phase; and wherein the relevant volume is a portion or the entire volume of the sample.

[0761] 9.4 Uniformly adjust the thickness of the sample-related volume between the two plates.

[0762] UAB7. A method for adjusting the thickness of a relevant volume of a sample, comprising:

[0763] (a) obtaining a sample, wherein the thickness of a relevant volume of the sample is to be adjusted;

[0764] (b) obtaining two plates movable relative to each other into different configurations, wherein one or both of the plates include spacers having a predetermined spacer distance and height, and each of the spacers is secured to its respective plate;

[0765] (c) placing a sample on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not regulated by the spacer;

[0766] (d) after (c), unfolding the sample by bringing the plates into a closed configuration, wherein in the closed configuration: the plates face each other, the spacer and the relevant volume of the sample are located between the plates, the volume of the sample of relevant thickness is adjusted by the plates and the spacer, and is thinner than the maximum thickness of the sample when the plates are in an open phase;

[0767] wherein the spacer and plate are configured such that the thickness of the relevant volume of the sample is more uniform over an area of ​​the relevant volume in the plate closed configuration than over an area of ​​the relevant volume in the plate open phase; and wherein the relevant volume is a portion or the entire volume of the sample.

[0768] UAB8. An apparatus for adjusting the thickness of a relevant volume of a sample, comprising:

[0769] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations;

[0770] wherein one or both of said plates include spacers having a predetermined spacer distance and height, and each of said spacers is secured to its respective plate;

[0771] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates;

[0772] wherein another of said configurations is a closed phase, which is configured in an open phase after said sample is deposited; and in the closed configuration: the plates face each other, the partitions and the associated volume of the sample are located between the plates, the thickness of the associated volume of the sample is adjusted by the plates and the partitions and is greater than a maximum thickness of the sample when the plates are in the open phase;

[0773] wherein the spacer and the plate are configured such that the thickness of the relevant volume of the sample is more uniform over an area of ​​the relevant volume when the plate is in a closed configuration than over an area of ​​the relevant volume when the plate is in an open phase; and wherein the relevant volume is a portion or all of the volume of the sample in the methods and apparatus in paragraphs U1 to U8, the construction of the spacer and the plate that makes the thickness of the relevant volume of the sample uniform has embodiments described in the present disclosure.

[0774] Uniformity of sample thickness. In the methods and apparatus of paragraphs U1 to U8, the uniformity of the thickness of the relevant volume of the sample is such that the sample thickness in the closed phase has a relative variation of at most 0.001%, at most 0.01%, at most 0.05%, at most 0.1%, at most 0.5%, at most 1%, at most 2%, at most 5%, at most 10%, at most 20%, at most 30%, at most 50%, at most 75%, at most 90%, less than 100%, or a range between any two of these values.

[0775] In a preferred embodiment of the methods and devices in paragraphs U1 to U8, the uniformity of the thickness of the relevant volume of the sample is such that the sample thickness in the closed phase has a relative variation of at most 0.1%, at most 0.5%, at most 1%, at most 2%, at most 5%, at most 10%, at most 20%, at most 30%, at most 50% or a range between any two of these values.

[0776] Another parameter that may be important in reducing the saturation incubation time is the uniformity of the sample thickness. If the thickness has large variations across the binding site, the saturation incubation time may vary from position to position in the binding site, forcing longer saturation incubation times to ensure that all positions in the binding site are saturated.

[0777] 10 Enhanced Surface

[0778] One of the current major obstacles for PoC diagnostics and any analysis using small sample amounts is poor sensitivity. It is desirable to enhance the signal of the assay. One aspect of the present invention relates to devices and methods for placing binding sites on a signal amplification surface (SAS) to amplify the signal to achieve higher sensitivity. The signal amplification surface may also be referred to as a signal amplification layer (SAL).

[0779] The general structure of a SAL includes a nanoscale metal-dielectric / semiconductor-metal structure that amplifies local surface electric fields as well as gradients and optical signals. The amplification is high at sharp (i.e., large curvature) edges of metal structures and at locations between small gaps of two metal structures. The highest enhancement regions are those with sharp edges and small gaps. In addition, the size of all metal and non-metal micro / nano structures is typically smaller than the wavelength (i.e., sub-wavelength) of the light amplified by the SAL.

[0780] In some embodiments, the SAL layer has as many sharp metal edges and small gaps as possible. This requires a dense group of metal nanostructures and small gaps between the nanostructures. The SAL structure may contain several different layers. In addition, the SAL layer itself can be further improved by a method that can further cover the metal material portion that does not have sharp edges and small gaps, such as U.S. Provisional Application Serial No. 61 / 801,424 filed on March 15, 2013 and PCT Application WO2014197096 filed on March 15, 2014 (incorporated herein by reference for all purposes) and PCT / US2014 / 028417 (Chou et al., "Analyte Detection Enhancement By TargetedImmobilization, Surface Amplification, and Pixelated Reading And Analysis"), which are incorporated herein by reference for all purposes.

[0781] A specific embodiment of a signal amplification surface is a D2PA array (disk coupled dot-on-pole antenna array), which may also include a molecular adhesion layer covering at least a portion of the metal dot structure, the metal disk and / or the metal backing plate and optionally a capture agent that specifically binds to the analyte, wherein the capture agent is attached to the molecular adhesion layer of the D2PA array. When the analyte binds to the capture agent, the nanosensor can amplify the optical signal from the analyte. In some embodiments, the size of one, several or all of the key metal and dielectric components of the SAL is smaller than the wavelength of the light being sensed. Details of the physical structure of disk-coupled point-to-post antenna arrays, their fabrication methods, methods of linking capture agents to disk-coupled point-to-post antenna arrays, and using disk-coupled point-to-post antenna arrays for detecting analytes are described in WO2012024006, WO2013154770, Li et al. (Optics Express 2011 19, 3925-3936), Zhang et al. (Nanotechnology 2012 23: 225-301); and Zhou et al. (Anal. Chem. 2012 84: 4489), which are incorporated herein by reference for all purposes.

[0782] 10.1 Amplification of the signal of the target entity in the volume of the sample

[0783] M1. A method for amplifying a signal of a target entity in a volume of interest of a sample, comprising:

[0784] (a) Obtain a sample containing the target entity;

[0785] (b) obtaining two plates movable relative to each other into different configurations, wherein one of the plates comprises a binding site on a surface thereof, the binding site comprising a signal amplification surface, the signal amplification surface being configured to bind the target entity and amplify the optical signal, on or near the signal amplification surface; and wherein one or both of the plates comprises spacers, and each of the spacers is on its respective plate and has a predetermined height;

[0786] (c) placing a sample on one or both plates when the plates are configured in an open phase; wherein the open phase is a configuration wherein the two plates are separated and the spacing between the plates is not adjusted by the spacer;

[0787] (d) after (c), unfolding the sample by moving the plates into a closed configuration, wherein in the closed phase: the plates face each other, the spacers and the associated volume of the sample are located between the plates, the volume of the associated thickness sample is adjusted by the plates and the spacers and is thinner than the sample when the plates are in the open phase, and the associated volume of the sample is in contact with the binding sites; and

[0788] (e) after (d), incubating while the plate is in the closed phase for a period of time to allow the target entity in the relevant volume of the sample to bind to the binding site;

[0789] wherein the volume of interest is the portion of the sample that is in contact with the binding sites when the plate is in the closed phase.

[0790] M2. A device for amplifying a signal when analyzing a target entity in a relevant volume of a sample, comprising:

[0791] a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations,

[0792] wherein the first plate comprises a binding site on a surface thereof, and the binding site comprises a signal amplification surface, the signal amplification surface being configured to (i) bind a target entity in a sample and (ii) amplify a signal at or near the signal amplification surface;

[0793] wherein one or both of the plates include spacers, and each of the spacers is on its respective plate and has a predetermined height;

[0794] wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacer, and the sample is deposited on one or both of the plates,

[0795] wherein another of said configurations is a closed phase, which is configured in an open phase after said sample is deposited; and in the closed configuration: the plates face each other, the spacer and the associated volume of the sample are located between the plates, the thickness of the associated volume of the sample is adjusted by the plates and the spacer, and is thicker than when the plates are in the open state;

[0796] wherein the volume of interest is the portion of the sample that is in contact with the binding sites when the plate is in the closed phase.

[0797] In some embodiments, the signal amplifying surface includes at least one of a metal-dielectric nanostructure, a metal-semiconductor nanostructure, and a disk-coupled point-and-post antenna array.

[0798] In some embodiments, the signal amplification surface includes a metal layer.

[0799] 11 Saving reagent volume in rapid binding tests (S)

[0800] In the case of binding entities in a reagent to binding sites on a surface (e.g., coating a plate with a capture agent or staining a biological sample surface), it is desirable to have a short incubation time. One approach to short incubations is to significantly increase the concentration of the entity in the reagent. However, this approach is wasteful of the entity and therefore expensive, because only a small portion of the entity in the reagent that is close to the binding site can reach the binding site during a very short incubation time, and the rest of the position is too far to diffuse to the binding site for binding and is useless and wasted. For typical diffusion constants of common reagents in ordinary solutions, the typical diffusion lengths are about 10 microns, 33 microns, and 100 microns for incubation times of 1 second (seconds), 10 seconds, and 100 seconds, respectively. The typical thickness of a droplet on a typical surface is 2.5 mm, which is at least 25 times thicker than the above diffusion length, resulting in significant waste (expensive) if the incubation time is 100 seconds or less. One aspect of the present invention is to drop the reagent over a large area but at a very thin thickness (thinner than natural dripping) to save reagents and thus reduce costs.

[0801] 11.1 A method for storing a reagent containing a target entity bound to a surface binding site by a spreading reagent. (A volume having a smaller natural contact area than the binding site)

[0802] S1. A method for storing a reagent containing a target entity bound to a surface binding site, comprising:

[0803] (a) obtaining a first plate and a second plate movable relative to each other into different configurations, wherein the first plate has binding sites on its surface, and wherein one or both of the plates include spacers and each spacer is secured to its respective plate and has a predetermined height;

[0804] (b) wherein (i) comprises a reagent capable of binding to a target entity of a binding site, and (ii) has a volume and wetting properties such that the contact area of ​​the reagent deposited on the binding site that does not contact the other plate is larger than the area of ​​the binding site;

[0805] (c) placing a sample on one or both plates when the plates are configured in an open phase; wherein in the open phase: the two plates are partially or completely separated and the spacing between the plates is not limited by the spacer;

[0806] (d) after (c), spreading the sample by moving the plates into a closed state; wherein in the closed configuration: the plates face each other, the spacer and the sample are between the plates, the sample contacts more area of ​​the binding site than the thickness of the sample when the plates are in an open phase, and the thickness of the sample is regulated by the plates and the spacer at the binding site and is thinner than when the plates are in an open structure.

[0807] In the method of paragraph S1, it further includes incubating for a period of time and stopping the incubation after (d) and when the plate is in a closed phase, wherein the incubation time is approximately equal to the time for the target entity to diffuse across the maximum sample thickness when the plate is in a closed phase, and wherein the incubation is a process that allows the entity to bind to the binding site.

[0808] S2. A device for storing a reagent containing a target entity bound to a surface binding sit...

Claims

1. A kit for staining and analyzing tissues, comprising: Device and staining solution, wherein the device comprises a first plate, a second plate and a spacer, wherein: (i) the first and second plates are movable relative to each other into different configurations, including an open configuration and a closed configuration; (ii) the first plate and / or the second plate are flexible plates; (iii) each plate has a contact area on its respective surface for a staining solution to contact and / or stain the tissue; (iv) one or both plates include spacers fixed to the respective plates, wherein the spacers are columnar, the predetermined height is uniform and is less than or equal to 250 μm, and wherein a plurality of the spacers are within the contact region; and The thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-um; The fourth power of the distance between the spacers ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE)≤5x10 6 um 3 / GPa; wherein, in an open configuration, the two plates are separated, the spacing between the two plates is not regulated by a spacer, and the tissue is deposited on one or both plates; and wherein the tissue is deposited in the open configuration to form the closed configuration, wherein the tissue and at least a portion of the staining solution are sandwiched between two plates to form a layer of uniform thickness, wherein the uniform thickness of the layer is defined by the contact area of ​​the two plates and adjusted by the plates and the spacer.

2. The kit according to claim 1, wherein The first plate and the second plate are connected by a hinge so that the two plates rotate relative to each other about the hinge.

3. A method for staining a tissue, comprising: (i) obtaining the kit according to claim 1 or 2; (ii) placing tissue and staining solution on one or both plates in a developed configuration of the plates; (iii) Compressing the panels into a closed configuration.

4. The method according to claim 3, further comprising: (a) imaging the tissue by acquiring one or more images after the compression into the closed configuration in step (iii); and (b) analyzing the image and obtaining analysis results.

5. An apparatus for staining a tissue sample containing or suspected of containing an analyte, the apparatus comprising: (i) the kit according to claim 1 or 2, and (ii) an imager for imaging the tissue sample and the analyte.

6. A system for staining and analyzing tissue, comprising: (i) the apparatus of claim 5; (ii) computing unit; and (iii) Wireless mobile communication unit.

7. The system of claim 6, further comprising a light source from the mobile communication unit or an external source.

8. The method according to claim 4, further comprising the steps of: The image or the analysis result is transmitted to a mobile communication device far away from the staining apparatus using a wireless mobile communication unit.

9. The kit according to claim 1, wherein the staining solution is used for immunohistochemical staining.

10. The kit according to claim 1, wherein the staining solution is used for nucleic acid detection.

11. The kit according to claim 1, wherein the tissue contains or is suspected of containing an analyte, and the analyte is a protein, DNA, RNA or a cell.

12. The kit according to claim 1, further comprising a dry staining reagent coated on one or both plates, wherein the staining solution is a transfer solution for transferring the dry staining reagent onto the tissue.

13. The kit according to claim 1, wherein The uniform thickness has a value of 50 μm or less.

14. The kit according to claim 1, wherein The uniform thickness has a value in the range of 0.5 um to 10 um.

15. The kit according to claim 1, wherein The material of the plate and the spacer is selected from PMMA, PC, COC, COP and another plastic.

16. The kit according to claim 1, wherein The distance between the spacers is in the range of 1 um to 200 um.

17. The kit according to claim 1, wherein The distance between the spacers is in the range of 200um to 1000um.

18. The kit according to claim 1, wherein for a spacer for adjusting a layer of uniform thickness, the Young's modulus of the spacer multiplied by the filling factor of the spacer is equal to or greater than 20 MPa, wherein, The fill factor is the ratio of the spacer contact area to the total plate area.

19. The kit according to claim 1, wherein: The distance between the spacers is in the range of 1um to 200um; For each spacer, a ratio of a lateral dimension of the spacer to its height is at least 1; At least some of the spacers are arranged periodically.

20. The kit of claim 1, wherein for the flexible board, the Young's modulus of the spacer multiplied by the filling factor of the spacer is equal to or greater than 2 MPa, wherein, Fill factor is the ratio of the spacer contact area to the total board area.

21. The kit according to claim 1, wherein One or both plates include position markings on the surface or inside the plate, which provide position information of the plate.

22. The kit according to claim 1, wherein One or both plates include a scale on the surface or inside the plate which provides information on the lateral dimensions of the structure of the sample and / or the plate.

23. The kit according to claim 1, wherein One or both plates include imaging markers on the surface or within the plate that are used to assist in imaging the sample to be tested.

24. The kit according to claim 1, wherein The spacers are used as position marks, scale marks, imaging marks or any combination thereof.

25. The kit according to claim 1, wherein The spacers are posts having a cross-sectional shape selected from polygonal, circular, elliptical, or any combination thereof.

26. The kit of claim 1, wherein the tissue is from a nasal swab, mucus, earwax, glandular secretions, throat swab, skin, biopsy tissue, sputum, pus, microbiota, breast milk, stool sample, connective tissue, muscle tissue, neural tissue, epithelial tissue, and cartilage.

27. The kit of claim 1, wherein the dye used for the staining solution is selected from the group consisting of: acid fuchsin, alcian blue 8GX, alizarin red S, aniline blue WS, auramine O, azo carmine B, azo carmine G, azure A, azure B, azure C, basic fuchsin, berberine brown Y, brilliant cresyl blue, brilliant green, carmine, chlorconazole black E, Congo red, CI cresyl violet, crystal violet, light red, eosin B, eosin Y, erythrosine, ethyl eosin, ethyl green, fast green FCF, fluorescein isothiocyanate, Giemsa stain, hematoxylin, hematoxylin-eosin, indigo carmine Lipored, Janus Green B, Jenner Stain 1899, Light Green SF, Malachite Green, Martius Yellow, Methyl Orange, Methyl Violet 2B, Methylene Blue, Methylene Blue, Methylene Violet, Bernsen, Neutral Red, Aniline Black, Nile Blue A, Nuclear Fast Red, Oil Red, Orange G, Orange II, Lichen Red, Methyl Violet, Fluorescent Pink B, Protein Silver S, Pyrro Red B, Pyrro Red, Resazurin, Bengal Red, Safranin O, Sudan Black B, Sudan III, Sudan IV, MacNeal Tetrachrome Stain, Thionine, Toluidine Blue, Iron Hematoxylin, Swiss Stain, and any combination thereof.

28. The kit according to claim 1, wherein the staining solution is a cell viability dye selected from the group consisting of propidium iodide, 7-AAD, trypan blue, calcein AM, fixable viability dyes, SYTO9 and other nucleic acid dyes, resazurin and formazan. and other mitochondrial dyes, and any combination thereof.

29. The kit according to claim 1, wherein The staining solution contains antibodies configured to specifically bind to protein analytes in the sample to be tested.

30. The kit according to claim 1, wherein The staining solution includes oligonucleotide probes, and the oligonucleotide probes are configured to specifically bind to DNA and / or RNA in the sample to be tested.

31. The kit of claim 1, wherein each of the spacers serves as a position marker, a ratio marker, an imaging marker, or any combination thereof.

32. The kit according to claim 1, wherein The spacer has a columnar shape and has a flat top surface.

33. The kit according to claim 32, wherein For each spacer, a ratio of a lateral dimension of the spacer to its height is at least 2.

34. The kit according to claim 18, wherein A ratio of the width to the height of the spacer is equal to or greater than 2.

35. The kit according to claim 34, wherein The product of the fill factor and the Young's modulus of the spacer is 20 MPa or greater, wherein the fill factor is a ratio of a gasket contact area to a total board area.

36. The kit according to claim 1, wherein The spacers are periodic.

37. The kit according to claim 1, wherein The plate has a plurality of staining reagent sites, wherein, in a closed configuration, a ratio of a distance between two adjacent staining reagent sites to a sample thickness is 5 or greater.

38. The kit according to claim 1, wherein The device also includes a dry staining reagent or a release time control material or both on one or both plates.

39. The kit according to claim 18, wherein The spacers regulating the uniform thickness layer are periodic and have a fill factor of at least 2%, wherein the fill factor is the ratio of the spacer contact area to the total plate area.

40. The kit according to claim 1, wherein One or both plates include scale markings.

41. The kit according to claim 1, wherein The thickness of the flexible board is in the range of 10 μm to 200 μm.

42. The kit according to claim 1, wherein The device also includes electrodes on one or both of the first plate and the second plate, the electrodes being used to measure the charge, current, capacitance, impedance or resistance or any combination thereof of the sample to be tested.

43. The kit according to claim 40, wherein The device also includes an amplifying surface, which is a surface that enhances the fluorescence produced by the detection agent.

44. The kit according to claim 43, wherein The amplifying surface may also be a surface that enhances the luminescence produced by the detection agent.

45. The kit according to claim 1, wherein The device also includes a dry binding site on one of the two plates, the dry binding site comprising a capture agent, an antibody, a nucleic acid, a labeling reagent, or a cell stain.

46. ​​The kit according to claim 1, wherein The spacer is fixed on the plate by directly stamping the plate or injection molding the plate, and wherein the material of the plate and the spacer is selected from PMMA, PC, COC, COP or other plastics.

47. A method for analyzing a liquid sample, comprising: (a) obtaining a sample comprising an analyte; (b) obtaining a first plate and a second plate that are movable relative to each other into different phases, wherein each plate has a sample contacting surface that is planar, one or both plates are flexible, one or both plates include spacers affixed to the respective sample contacting surfaces, and wherein the spacers have a predetermined uniform height and a predetermined constant inter-spacer distance that is at least 2 times greater than the size of the analyte, up to 200 microns; The thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-um; The fourth power of the distance between the spacers ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE)≤5x10 6 um 3 / GPa; (c) depositing a sample on one or both plates when the first plate and the second plate are in an open phase, wherein the open phase is a phase in which the two plates are partially or completely separated and the interval between the plates is not adjusted by a spacer; (d) after completing step c), using two plates to compress at least a portion of the sample into a thin layer of sample of uniform thickness confined by sample contacting surfaces of the plates, wherein the uniform thickness of the layer is adjusted by spacers of the plates, wherein the compressing comprises: Bring the two boards together; Conformally pressing, in parallel or sequentially, one region of at least one of the plates to press the plates together into a closed phase, wherein the conformal pressing produces uniform pressure on the plates over at least a portion of the sample and the pressing causes at least a portion of the sample to spread laterally between sample contacting surfaces of the first and second plates, wherein the closed phase is a phase in which the spacing between the uniformly thick thin layers of one plate is adjusted by a spacer; and (e) analyzing the analyte in a thin layer of uniform thickness while the plate is in a closed phase; Wherein, conformal pressing is a method of making the pressure in a region constant regardless of how the outer surfaces of the first plate and the second plate are deformed; Among them, parallel pressing is to apply pressure on the intended area at the same time, while sequential pressing is to gradually transfer pressure to other intended areas after applying pressure on the intended area.

48. The method of claim 47, wherein after step d and before step e, the method further comprises, after the first plate and the second plate are in a closed phase, removing the conformal pressing pressure, wherein Thickness of uniform thin layer after removing the conformal pressing pressure: (i) the same thickness as the uniform thin layer before the pressure of the conformal pressing is removed; (ii) Deviation from the spacer height is less than 10%.

49. The method of claim 47, wherein the sample deposition of step c is directly from the subject to the plate without the use of any transfer device.

50. The method of claim 47, wherein during the deposition process of step c, the amount of sample deposited on the plate is unknown.

51. The method of claim 47, wherein the analyzing step e comprises measuring a lateral area of ​​the sample-associated volume and calculating a volume of the sample-associated volume by the lateral area and a height of a predetermined spacer.

52. The method of claim 47, wherein the analyzing step e comprises measuring: (i) luminescence of any of photoluminescence, electroluminescence, and electrochemiluminescence; (ii) surface Raman scattering; (iii) an electrical impedance selected from resistance, capacitance and inductance; or (iv) Any combination of i-iii.

53. The method of claim 47, wherein the analyzing step e comprises reading, image analysis, or counting of the analytes, or a combination thereof.

54. The method of claim 47, wherein the sample contains one or more analytes and the sample contacting surface of one or both plates comprises one or more binding sites, wherein each binding site binds and immobilizes a corresponding analyte.

55. The method of claim 47, wherein the sample contact surface of one or both plates comprises one or more storage points, each of which stores one or more reagents, wherein the reagents are dissolved and diffused in the sample during or after step c.

56. The method of claim 47, wherein one or both of the plate sample contact surfaces comprises one or more enhancement surfaces, each of which enhancement surfaces is capable of enhancing a signal from an analyte or a marker of the analyte when the analyte or marker is within 500 nanometers of an enhancement site.

57. The method of claim 47, wherein: (i) the sample contacting surface of one or both plates comprises one or more binding sites, each of which binds and immobilizes a corresponding analyte; or (ii) the sample contacting surface of one or both plates comprises one or more storage sites, each of which stores one or more reagents, wherein the reagents are dissolved and diffused in the sample during or after step c, wherein the sample comprises one or more analytes; (iii) one or both of the plate sample contacting surfaces include one or more enhancement surfaces, each of which is capable of enhancing a signal from the analyte or a marker for the analyte when the analyte or marker is within 500 nanometers of an enhancement site; (iv) Any combination of i to iii.

58. The method of claim 47, wherein the conformal compression of step d is provided by a pressurized liquid, a pressurized gas, or a conformable material.

59. The method of claim 47, wherein the analyzing comprises counting cells in a thin layer of uniform thickness.

60. The method of claim 47, wherein said analyzing comprises performing an assay on a thin layer of uniform thickness.

61. The method of claim 47, wherein said analysis is a binding assay or a biochemical assay.

62. The method of claim 47, wherein the deposited sample has a total volume of less than 0.5 microliters.

63. The method of claim 47, wherein multiple droplets of the sample are deposited onto one or both plates.

64. A device for analyzing a liquid sample, comprising: A first plate and a second plate, wherein: i. The first plate and the second plate are movable relative to each other to enter different phases; ii. One or both boards are flexible boards; iii. Each plate has a sample contact area on its respective surface for contacting the sample including the analyte; iv. one or both plates include spacers affixed to respective sample contact areas, wherein the spacers have a predetermined uniform height and a predetermined constant inter-spacer distance that is at least 2 times greater than the size of the analyte, up to 200 microns, and wherein at least one of the spacers is within the sample contact area; The thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-um; v. The fourth power of the distance between the spacers ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE)≤5x10 6 um 3 / GPa; wherein one of the phases is an open phase, in which: the first plate and the second plate are separated from each other, the interval between the plates is not adjusted by a spacer, and the sample is deposited on one or both plates; Among them, another phase state is a closed phase state, which is a phase state formed after the sample is deposited in the open phase state; in the closed phase state: at least a portion of the sample is pressed by two plates into a thin layer with uniform thickness, wherein the uniform thickness of the thin layer is defined by the sample contact surfaces of the first plate and the second plate, and is adjusted by the plates and the spacer.

65. The device of claim 64, wherein the inter-spacer distance is in the range of 1 micron to 120 microns.

66. The device of claim 64, wherein the flexible sheet has a thickness between 20 microns and 250 microns.

67. The device of claim 66, wherein the Young's modulus of the flexible plate is in the range of 0.1 to 5 GPA.

68. The device according to claim 64, wherein the sample layer of uniform thickness is at least 1 mm 2 is uniform in the plane area.

69. The apparatus of claim 64, wherein the thickness uniform sample layer has a thickness uniformity of at most + / - 5%.

70. The device of claim 64, wherein the spacers are posts having cross-sectional shapes comprising: Circular, polygonal, oval, elliptical, or any combination thereof.

71. The device of claim 64, wherein the spacer has a pillar shape with a flat top surface and has a uniform cross-section, wherein: For each spacer, a ratio of a lateral dimension to a height of the spacer is at least 1.

72. The device of claim 64, wherein the inter-spacer distances are periodic.

73. The device of claim 64, wherein the spacer has a fill factor of 1% or greater, wherein the fill factor is the ratio of the spacer contact area to the total plate area.

74. The device of claim 64, wherein the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 20 MPa, wherein the fill factor is the ratio of the spacer contact area to the total plate area.

75. The device of claim 64, wherein the spacing between the two plates in the closed phase is less than 200 microns.

76. The apparatus of claim 64, wherein the spacing between the two plates in the closed phase is a value selected between 1.8 microns and 3.5 microns.

77. The device of claim 64, wherein the spacer is fixed to the plate by direct stamping or injection molding.

78. The device according to claim 64, wherein the material of the plate and the spacer is selected from the following group of materials: PMMA, PC, COC, COP, or other plastics.

79. The device of claim 64, wherein the spacer has a pillar shape, and a sidewall corner of the spacer has a rounded shape with a radius of curvature of at least 1 micron.

80. The device of claim 64, wherein the spacers have at least 1000 / mm 2 density.

81. The device of claim 64, wherein at least one of the plates is transparent.

82. The apparatus of claim 64, wherein the mold used to make the spacers contains a pattern directly obtained by reactive ion etching or ion beam etching, or a pattern obtained by single or multiple replication of reactive ion etching or ion beam etching.

83. A method for analyzing a blood sample, comprising: (a) obtaining a blood sample; (b) obtaining a first plate and a second plate, the first plate and the second plate being movable relative to each other into different phases, wherein each plate has a sample contacting surface that is flat, one or both plates are flexible, and one or both plates include a spacer fixed to the respective sample contacting surface, and wherein the spacer has: i. A predetermined consistent height; ii. The shape of the pillars having a uniform cross-section and a flat top surface; iii. a ratio of width to height equal to or greater than one; iv. The predetermined constant distance between the spacers is in the range of 10 microns to 200 microns; v. Fill factor equal to 1% or greater; vi. The product of the filling factor and Young's modulus of the spacer is 2 GPa or more, wherein the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-um; The fourth power of the distance between the spacers ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE)≤5x10 6 um 3 / GPa; (c) depositing a blood sample on one or both plates when the two plates are in an open phase, wherein the open phase refers to a phase in which the two plates are partially or completely separated from each other and the interval between the plates is not regulated by a spacer; (d) after completing step (c), using two plates to compress at least a portion of the blood sample into a thin layer of sample with uniform thickness confined by the sample contacting surfaces of the plates, wherein the uniform thickness of the layer is adjusted by the spacers of the plates and has an average value with a deviation of less than 10% within the range of 1.8 microns to 3 microns, wherein the compressing comprises: Bring the two boards together; Conformally pressing, in parallel or sequentially, one region of at least one of the plates to press the plates together into a closed phase, wherein the conformal pressing produces uniform pressure on the plates over at least a portion of the sample and the pressing causes at least a portion of the sample to spread laterally between sample contacting surfaces of the first and second plates, wherein the closed phase is a phase in which the spacing between the uniformly thin regions of thickness of one plate is adjusted by a spacer; and (e) When the two plates are in a closed phase, a thin layer of blood of uniform thickness is analyzed. where the fill factor is the ratio of the contact area of ​​the spacer to the total plate area; Among them, conformal pressing is a method that makes the pressure in a region constant regardless of how the outer surfaces of the first plate and the second plate are deformed.

84. The method of claim 83, wherein the method comprises removing the external force after the first plate and the second plate are in a closed phase; imaging a thin layer of blood cells of uniform thickness while the two plates are in a closed phase; and (f) counting blood cells in an area of ​​the image.

85. The method of claim 83, wherein the inter-spacer distance is in the range of 20 microns to 200 microns.

86. The method of claim 83, wherein the inter-spacer distance is in the range of 5 microns to 20 microns.

87. The method of claim 83, wherein the surface has an error of less than 30 nanometers.

88. The method of claim 83, wherein the blood sample of step (b) is undiluted whole blood to which no anticoagulant is added.

89. The method of claim 83, wherein the depositing step (b) is accomplished by: i. puncturing human skin to release blood on the skin; ii. Without using a blood transfer tool, touch the blood to one or both plates.

90. The method of claim 83, wherein the counting step (f) comprises counting the number of red blood cells.

91. The method of claim 83, wherein the counting step (f) comprises counting the number of white blood cells.

92. The method of claim 83, wherein the counting step (f) comprises staining cells in the sample and counting neutrophils, lymphocytes, monocytes, eosinophils and basophils.

93. The method of claim 83, wherein the imaging and the amounting are accomplished by: i. Illuminate blood cells in a thin layer of uniform thickness; ii. Taking one or more images of blood cells using a CCD or CMOS sensor; iii. Use computers to identify cells in the images; iv. Count blood cells in one area of ​​the image.

94. The method of claim 83, wherein the external force of step (c) is provided by human hands.

95. The method of claim 83, further comprising measuring the levels of sodium, potassium, chloride, bicarbonate, urea, nitrogen, magnesium, creatinine, glucose, calcium, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol and / or the levels of triglycerides in the thin layer of uniform thickness.

96. The method of claim 83, further comprising a dry reagent coated on one or both plates.

97. The method of claim 83, wherein the sample of thin layers of uniform thickness has a thickness uniformity of up to ±5%.

98. The method of claim 83, wherein the spacers are struts comprising cross-sectional shapes that are circular, polygonal, oval, elliptical, or any combination thereof.

99. The method of claim 83, wherein the inter-spacer distance is the average thickness of a red blood cell.

100. The method of claim 83, wherein the analyzing step comprises imaging cells in the blood.

101. The method of claim 83, wherein the cells comprise red blood cells, white blood cells, or platelets.

102. The method of claim 83, wherein said analyzing said blood comprises imaging cancer cells, viruses or bacteria in the blood.

103. The method of claim 83, wherein said analyzing said blood comprises detection of proteins or nucleic acids.

104. The method of claim 83, wherein said analyzing said blood comprises measurement of blood cells, comprising determining sample thickness using said spacer, determining said lateral area by imaging, and calculating the area of ​​red blood cells using a two-dimensional image.

105. The method of claim 83, wherein said analyzing said blood comprises measuring a concentration of red blood cells in the blood.

106. The method of claim 83, wherein said analyzing said blood comprises measuring the concentration of white blood cells in the blood.

107. The method of claim 83, wherein said analyzing said blood comprises measuring a concentration of platelets in the blood.

108. A device for analyzing a liquid sample, comprising: A first plate and a second plate, wherein: i. The first plate and the second plate can be moved relative to each other into different phases; ii. One or both boards are flexible boards; iii. Each plate has a sample contact area on its respective surface for contacting a blood sample; iv. one or both plates include spacers affixed to respective sample contact regions, wherein the spacers have a predetermined uniform height and a predetermined constant inter-spacer distance in the range of between 7 microns and 200 microns, and wherein at least one of the spacers is within the sample contact region; wherein one of the phases is an open phase, in which: the first plate and the second plate are separated from each other, the interval between the plates is not adjusted by a spacer, and the sample is deposited on one or both plates; Among them, another phase state is a closed phase state, which is a phase state formed after the sample is deposited in the open phase state; in the closed phase state: at least a portion of the sample is pressed by two plates into a thin layer of uniform thickness, wherein the uniform thickness of the thin layer is defined by the sample contact surfaces of the first plate and the second plate, adjusted by the plates and spacers, and has an average value with a slight error in the range of 1.8 microns to 3 microns.

109. The device of claim 108, wherein the spacers are posts having cross-sectional shapes comprising: Round, polygonal, oval, elliptical.

110. The device of claim 108, wherein the inter-spacer distance is in the range of 14 microns to 200 microns.

111. The device of claim 108, wherein the distance between the spacers is in the range of 7 microns to 20 microns.

112. The device of claim 109, wherein the shape of the spacer further comprises: Any combination of circles, polygons, ovals, and ellipses.

113. The device of claim 108, wherein the spacer has a pillar shape with a flat top surface and a uniform cross-section, wherein: For each spacer, a ratio of a lateral dimension to a height of the spacer is at least 1.

114. The device of claim 108, wherein the spacer has a pillar shape, and a sidewall corner of the spacer has a circular shape with a radius of curvature of at least 1 micron.

115. The device of claim 108, wherein the spacers have at least 1000 / mm 2 density.

116. An apparatus according to claim 108, wherein at least one of the plates is transparent.

117. The device of claim 108, wherein at least one of the plates is made of a flexible polymer.

118. The device of claim 108, wherein the spacer is incompressible and / or independent and only one of the plates is flexible.

119. The device of claim 108, wherein the thickness of the flexible sheet is in the range of 50 microns to 200 microns.

120. The device of claim 108, further comprising a dry reagent coated on one or both plates.

121. The apparatus of claim 108, wherein the error is less than 10%.

122. A method for locally binding a target entity in a portion of a liquid sample: (a) obtaining a sample comprising a target entity capable of diffusing in the sample; (b) obtaining a first plate and a second plate, the first plate and the second plate being movable relative to each other into different phases, one or both of the first plate and the second plate being a flexible plate, wherein one or both of the plates include a spacer fixed to the respective plate, wherein The spacers have a predetermined uniform predetermined height, and wherein the first plate includes, on its surface, a bonding point having a predetermined area and for bonding and fixing the target entity; Wherein the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-um; the fourth power of the distance between the spacers ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE)≤5x10 6 um 3 / GPa; (c) depositing a sample on one or both plates when the first plate and the second plate are in an open phase, wherein the open phase refers to a phase in which the two plates are partially or completely separated from each other and the spacing between the plates is not regulated by a spacer; (d) after completing c, compressing the sample by causing the first plate and the second plate to enter a closed phase, wherein the closed phase is a phase in which at least a portion of the sample is pressed into a uniform thickness layer in contact with the contact joint confined by the inner surfaces of the two plates, wherein the uniform thickness of the layer is adjusted by the plates and the spacers, is less than 250 microns and is substantially less than the linear dimension of the predetermined area of ​​the joint; (e) After completing step d, when the first plate and the second plate are in a closed phase: (1) incubating the sample for a relevant length of time and then stopping the incubation; or (2) incubating the sample for a time period equal to or greater than a minimum of the relevant time period, and assessing binding of the target entity within the binding site for a time period equal to or less than a maximum of the relevant time period; The relevant time length is: i. equal to or greater than the length of time required for the target entity to diffuse across the thickness of a thin layer of uniform thickness in the closed phase; ii. significantly less than the length of time required for the target entity to diffuse laterally across the minimum lateral dimension of the binding site; Thus, a reaction is generated wherein, at the end of the incubation in step (1) or at the time of the evaluation in step ), the majority of the target entity bound to the binding site is derived from the relevant volume of the sample; wherein the incubation allows the target entity to bind to the binding site, wherein the relevant volume refers to a portion of the sample located above the binding site in the closed phase.

123. The method of claim 122, further comprising a step of evaluating a target entity bound to a binding site, wherein: The evaluation is performed i after completing step e, or ii while performing step e, but the evaluation occurs after the sample has been incubated for a period of time equal to or longer than the time required for the analyte to diffuse across the thickness of the uniformly thin layer.

124. The method of claim 123, further comprising a step of calculating the concentration of the analyte in the sample-related volume, wherein: The calculation is based on the volume of the associated sample volume defined by the predetermined area of ​​the binding site, the thickness of a thin layer of sample of uniform thickness in a closed phase, and the resulting estimated amount of target entity bound to the binding site.

125. The method of claim 124, wherein the target entity is a capture agent, an analyte, or a probe.

126. The method of claim 125, wherein the volume of sample at the analyte assay site is equal to the predetermined area of ​​the binding site multiplied by the predetermined height of the spacer.

127. The method of claim 126, wherein the first plate includes a first predetermined measurement point and a second predetermined measurement point on a surface thereof, wherein: The distance between the edges of the assay points is substantially greater than the thickness of the uniform thickness layer when the two plates are in a closed phase, wherein at least a portion of the uniform thickness layer is located above the predetermined assay points, and wherein the sample has one or more analytes that are diffusible in the sample.

128. The method of claim 127, wherein the first plate comprises a first measurement point and a second measurement point, wherein the method comprises: The first assay point is evaluated for a first analyte and a second analyte is determined at a second predetermined assay point.

129. The method of claim 128, wherein the method comprises assaying the same analyte in the first and second predetermined assay points.

130. A method according to claim 129, wherein the first plate also has at least three analyte measurement points on its surface, the distance between the edges of any two adjacent measurement points is significantly greater than the thickness of the uniform thin layer when the first plate and the second plate are in a closed phase, wherein at least a portion of the uniform thin layer is located above the measurement points, and wherein the sample has one or more analytes capable of diffusing in the sample.

131. The method of claim 130, wherein the method comprises measuring a different analyte at each measuring point.

132. A method according to claim 131, wherein each assay point further includes a sub-analyte assay point, wherein at least two adjacent sub-analyte assay points are separated by a distance less than the thickness of a uniform thin layer of thickness when the first plate and the second plate are in a closed phase, wherein at least a portion of the uniform thin layer of thickness is located above the assay point, and wherein the sample has one or more analytes capable of diffusing in the sample.

133. The method of claim 132, wherein the analyte determination site is between a pair of electrodes.

134. The method of claim 133, wherein the assay site is defined by a sheet of dried reagent.

135. The method of claim 134, wherein the assay site binds and immobilizes an analyte.

136. The method of claim 135, wherein the assay site enhances the signal from the analyte.

137. The method of claim 136, wherein the assay site is defined by a piece of binding reagent which, upon contact with the sample, dissolves into the sample, diffuses in the sample, and binds to the analyte.

138. The method of claim 137, wherein the sample thickness is less than 200 microns.

139. The method of claim 138, wherein the first plate and the second plate include storage points located at positions opposite to each other when the first plate and the second plate are in a closed phase.

140. The method of claim 139, wherein a ratio of a linear dimension of the bond points to the uniform thickness is greater than 5.

141. The method of claim 140, wherein the reaction is saturated in less than 60 seconds.

142. A method according to claim 141, wherein the relevant time length is in the range of 60 seconds to 30 minutes.

143. The method of claim 142, wherein step (e)(2) comprises, after stopping the incubation, evaluating the target entity bound to the binding site.

144. The method of claim 143, wherein the method comprises one or more elutions.

145. An apparatus for locally binding a target entity in a portion of a liquid sample, comprising: A first plate and a second plate, wherein: i. The first plate and the second plate are movable relative to each other into different phases; ii. One or both plates are flexible; iii. Each plate has a sample contact area on its respective surface that contacts an entity that can diffuse in the sample; iv. one of the first plate and the second plate comprises a binding point having a predetermined area and binding and fixing the target entity in its sample contact area; The one or both plates include spacers affixed to the respective plate, wherein the spacers have a predetermined uniform height and a predetermined constant inter-spacer distance, wherein at least one of the feature spacers is within a sample contact region; Wherein the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-um; the fourth power of the distance between the spacers ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE)≤5x10 6 um 3 / GPa; wherein one of the phases is an open phase, in which: the first plate and the second plate are separated from each other, the interval between the plates is not adjusted by a spacer, and the sample is deposited on one or both plates; Among them, another of the phases is a closed phase, which is a phase formed after the sample is deposited in the open phase; in the closed phase: at least a portion of the sample is pressed by two plates into a thin layer with uniform thickness, wherein at least a portion of the thin layer with uniform thickness is located above the bonding point, wherein the uniform thickness of the thin layer is defined by the sample contact surfaces of the first plate and the second plate, adjusted by the plates and spacers, less than 250 microns, and significantly smaller than the average linear dimension of the predetermined area of ​​the bonding point.

146. The apparatus of claim 145, wherein the first plate further comprises, on its surface, a first predetermined measurement point and a second predetermined measurement point, wherein, The distance between the edges of the assay points is substantially greater than the thickness of the uniform thickness layer when the two plates are in a closed phase, wherein at least a portion of the uniform thickness layer is located above the predetermined assay points, and wherein the sample has one or more analytes that are diffusible in the sample.

147. A device according to claim 146, wherein the first plate has at least three analyte measurement points on its surface, and the distance between the edges of any two adjacent measurement points is significantly greater than the thickness of the uniform thin layer when the first plate and the second plate are in a closed phase, wherein at least a portion of the uniform thin layer is located above the measurement points, and wherein the sample has one or more analytes capable of diffusing in the sample.

148. A device according to claim 147, wherein the first plate has at least two adjacent measurement points on its surface, and the two measurement points are not separated by a distance that is significantly greater than the thickness of the uniform thin layer when the first plate and the second plate are in a closed phase, wherein at least a portion of the uniform thin layer is located above the measurement points, and wherein the sample has one or more analytes capable of diffusing in the sample.

149. The device of claim 148, wherein the analyte determination site is between a pair of electrodes.

150. The device of claim 149, wherein the assay site is defined by a sheet of dried reagent.

151. The device of claim 150, wherein the assay site binds and immobilizes an analyte.

152. The device of claim 151, wherein the assay site is defined by a piece of binding reagent which, upon contact with the sample, dissolves into the sample, diffuses in the sample, and binds to the analyte.

153. The device of claim 152, wherein the inter-spacer distance is in the range of 14 microns to 200 microns.

154. The device of claim 153, wherein the inter-spacer distance is in the range of 7 microns to 20 microns.

155. The device of claim 154, wherein the spacer is a post having a cross-sectional shape that is circular, polygonal, oval, elliptical, or any combination thereof.

156. The device of claim 155, wherein the spacer has a pillar shape with a flat top surface and has a uniform cross-section, wherein: For each spacer, a ratio of a lateral dimension to a height of the spacer is at least 1.

157. The device of claim 156, wherein the spacer has a pillar shape and a sidewall corner of the spacer has a circular shape with a radius of curvature of at least 1 micron.

158. The device of claim 157, wherein the spacers have at least 1000 / mm 2 density.

159. An apparatus according to claim 158, wherein at least one of the plates is transparent.

160. The device of claim 159, wherein at least one of the plates is made of a flexible polymer.

161. A device according to claim 160, wherein at least one of the first plate and the second plate is flexible.

162. A method for locally releasing an agent into a portion of a liquid sample, comprising: (a) obtaining a sample; (b) obtaining a first plate and a second plate, wherein the first plate and the second plate are movable relative to each other into different phases, wherein: (i) one or both panels are flexible panels and include spacers secured to the respective panels; (ii) the spacers have a predetermined uniform height; (iii) the first plate includes, on its surface, a storage point having a predetermined area and including a reagent capable of dissolving into and diffusing in the sample upon contact with the sample; The thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-um; the fourth power of the distance between the spacers ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE)≤5x10 6 um 3 / GPa; (c) depositing a sample on one or both plates when the first plate and the second plate are in an open phase, wherein the open phase refers to a phase in which the first plate and the second plate are partially or completely separated from each other and the spacing between the plates is not adjusted by a spacer; (d) after completing step (c), compressing the sample by causing the two plates to enter a closed phase, wherein the closed phase is a phase in which at least a portion of the sample is compressed into a thin layer of uniform thickness at the contact joint bounded by the inner surfaces of the two plates, wherein the uniform thickness of the thin layer is adjusted by the plates and the spacers, is less than 250 microns and is substantially smaller than the linear dimension of the predetermined area of ​​the storage point; (e) after completing step (d), while the first plate and the second plate are in a closed phase, incubating the sample for a relevant length of time, and then stopping the incubation, The relevant time length is: i. equal to or greater than the length of time required for the target entity to diffuse across the thickness of a thin layer of uniform thickness in the closed phase; ii. significantly less than the length of time required for the target entity to diffuse laterally across the minimum lateral dimension of the binding site; Thus, from a reaction, after incubation, most of the reagents originally present at the storage point are present in the relevant volume of the sample, The incubation is a process to allow the reagent to bind or mix with the sample, wherein the relevant volume is the portion of the sample that is above the binding site when in the closed phase.

163. The method of claim 162, wherein the reagent is a capture agent, an analyte, or a probe agent.

164. The method according to claim 162, wherein the surface of the second plate of step (b) further comprises binding points having corresponding predetermined areas and corresponding positions and for binding and immobilizing reagents.

165. A method according to claim 162, wherein in step (d) the binding point and the storage point are aligned in the closed phase.

166. The method of claim 162, wherein at the end of the incubation in step (e), most of the reagent initially at the storage point has bound to the binding site and the binding of the binding site to the reagent in the relevant volume has reached equilibrium.

167. The method of claim 162, further comprising a step of calculating the concentration of the analyte in the sample-related volume, wherein: The calculation is based on the volume of the associated sample volume defined by the predetermined area of ​​the binding site, the thickness of a thin layer of sample of uniform thickness in the closed phase, and the resulting estimated amount of target entity bound to the binding site.

168. The method of claim 162, wherein the target entity is a capture agent, an analyte, or a probe.

169. The method of claim 162, wherein the volume of the sample at the predetermined area is equal to the predetermined area multiplied by the predetermined height of the spacer.

170. The method of claim 162, wherein the first plate comprises, on a surface thereof, a first storage point and a second storage point, wherein: The distance between the edges of the storage spots is substantially greater than the thickness of the uniform thickness layer when the two plates are in a closed phase, wherein at least a portion of the uniform thickness layer is located above the storage spots, and wherein the sample has one or more analytes that are capable of diffusing in the sample.

171. The method of claim 162, wherein the method comprises evaluating a first analyte at the first storage point and determining a second analyte at a second predetermined storage point.

172. The method of claim 162, wherein the method comprises measuring the same analyte at the first and second storage points.

173. A method according to claim 162, wherein the first plate further has at least three storage points on its surface, the distance between the edges of any two adjacent storage points is significantly greater than the thickness of the uniform thin layer of thickness when the first plate and the second plate are in a closed phase, wherein at least a portion of the uniform thin layer of thickness is located above the predetermined area, and wherein the sample has one or more analytes capable of diffusing in the sample.

174. The method of claim 162, wherein the method comprises measuring different analytes at each storage point.

175. The method of claim 162, wherein the storage point is defined by a piece of dried reagent.

176. The method of claim 162, wherein the storage site is defined by a piece of binding reagent which, upon contact with the sample, dissolves into the sample, diffuses in the sample, and binds to the analyte.

177. The method of claim 162, wherein the sample thickness is less than 200 microns.

178. The method of claim 162, wherein when the first plate and the second plate are in a closed phase, positions of the first and second storage points are relative to each other.

179. The method of claim 162, wherein the ratio of the linear dimension of the bond points to the uniform thickness is greater than 5.

180. The method of claim 162, wherein the reaction is saturated in less than 60 seconds.

181. A method according to claim 162, wherein the relevant time length is in the range of 60 seconds to 30 minutes.

182. The method of claim 162, wherein the method comprises one or more elutions.

183. A device for locally releasing a reagent to a portion of a liquid sample, comprising: A first plate and a second plate, wherein: i. The first plate and the second plate are movable relative to each other into different phases; ii. One or both plates are flexible; iii. Each plate has a sample contact area on its respective surface for contacting the sample; iv. one of the first plate and the second plate comprises a storage point having a predetermined area and comprising a reagent capable of dissolving in the sample, diffusing in the sample and binding to a target entity after contacting the sample in its sample contact area; v. one or both plates include spacers affixed to the respective plates, wherein the spacers have (a) a predetermined uniform height of no greater than 250 microns and substantially less than an average linear dimension of a predetermined area of ​​the storage sites, and (b) a predetermined constant inter-spacer distance, wherein at least one of the spacers is within a sample contact region; Wherein the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-um; the fourth power of the distance between the spacers ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE)≤5x10 6 um 3 / GPa; wherein one of the phases is an open phase, in which: the first plate and the second plate are separated from each other, the interval between the plates is not adjusted by a spacer, and the sample is deposited on one or both plates; Among them, another phase state is a closed phase state, which is a phase state formed after the sample is deposited in the open phase state; in the closed phase state: at least a portion of the sample is pressed by two plates into a thin layer with uniform thickness, wherein at least a portion of the thin layer with uniform thickness is located above the bonding point, wherein the uniform thickness of the thin layer is defined by the sample contact surfaces of the first plate and the second plate, and adjusted by the plates and the spacer.

184. An apparatus according to claim 183, wherein the first plate further comprises, on its surface, a second storage point having a predetermined area, wherein a distance between edges of the first and second storage points is substantially greater than a thickness of a thin layer of uniform thickness when the first plate and the second plate are in a closed phase, wherein at least a portion of the thin layer of uniform thickness is located above the storage point, wherein the sample has one or more analytes capable of diffusing in the sample.

185. A device according to claim 183, wherein the first plate has at least three storage points on its surface, the distance between the edges of any two adjacent storage points is significantly greater than the thickness of the uniform thin layer when the first plate and the second plate are in a closed phase, wherein at least a portion of the uniform thin layer is located above the storage points, and wherein the sample has one or more analytes capable of diffusing in the sample.

186. A device according to claim 183, wherein the first plate has at least two adjacent storage points on its surface, and the two storage points are not separated by a distance that is significantly greater than the thickness of the uniform thin layer when the first plate and the second plate are in a closed phase, wherein at least a portion of the uniform thin layer is located above the storage points, and wherein the sample has one or more analytes capable of diffusing in the sample.

187. A device according to claim 183, wherein the storage point is defined by a piece of dried reagent.

188. A device according to claim 183, wherein the storage point is defined by a piece of binding reagent, which, after contacting the sample, dissolves into the sample, diffuses in the sample, and binds to the analyte.

189. The device of claim 183, wherein the inter-spacer distance is in the range of 14 microns to 200 microns.

190. The device of claim 183, wherein the inter-spacer distance is in the range of 7 microns to 20 microns.

191. The device of claim 183, wherein the spacer is a post having a cross-sectional shape that is circular, polygonal, oval, elliptical, or any combination thereof.

192. The device of claim 183, wherein the spacer has a pillar shape with a flat top surface and a uniform cross-section, wherein: For each spacer, a ratio of a lateral dimension to a height of the spacer is at least 1.

193. The device of claim 183, wherein the spacer has a pillar shape, and a sidewall corner of the spacer has a circular shape with a radius of curvature of at least 1 micron.

194. The device of claim 183, wherein the spacers have at least 1000 / mm 2 density.

195. An apparatus according to claim 183, wherein at least one of the plates is transparent.

196. The device of claim 183, wherein at least one of the plates is made of a flexible polymer.

197. A device according to claim 183, wherein at least one of the first plate and the second plate is flexible.

198. A method for reducing the time required to bind a target entity in a sample-related volume in a binding site on a plate, comprising: (a) obtaining a sample comprising a target entity capable of diffusing in the sample; (b) obtaining a first plate and a second plate, the first plate and the second plate being movable relative to each other into different phases, wherein one or both plates include spacers fixed to the respective plates and one or both plates are flexible plates; wherein the spacers have a predetermined uniform predetermined height and a predetermined constant distance between the spacers, and wherein the first plate includes on its surface a bonding point having a predetermined area and bonding and fixing the target entity; wherein the thickness of the flexible plate multiplied by the Young's modulus of the flexible plate is in the range of 60 to 750 GPa-um; the fourth power of the distance between the spacers ISD divided by the thickness h of the flexible plate and the Young's modulus E of the flexible plate, ISD 4 / (hE)≤5x10 6 um 3 / GPa; (c) depositing a sample on one or both plates when the first plate and the second plate are in an open phase, wherein the open phase refers to a phase in which the two plates are partially or completely separated from each other and the spacing between the plates is not regulated by a spacer; (d) after completing step (c), compressing the sample by causing the two plates to enter a closed phase, wherein the closed phase is a phase in which the thickness of the relevant volume of the sample is reduced compared to the open phase to form a lateral area of ​​at least 1 mm 2 a thin layer of uniform thickness, bounded by the inner surfaces of the two plates and covering the junction, wherein the uniform thickness of the thin layer is adjusted by the plates and the spacers, is less than 250 microns and is substantially smaller than the linear dimension of the predetermined area of ​​the junction, wherein the relevant volume is part or all of the volume of the sample; Thus, a reaction is created wherein decreasing the thickness of the relevant volume of the sample decreases the time required for the binding between the binding site and the target entity in the relevant volume to reach equilibrium.

199. The method of claim 198, comprising reducing at least a portion of the sample thickness.

200. The method of claim 198, wherein the predetermined area is at least 1 mm 2 .

201. The method of claim 198, wherein the predetermined area is less than 1 mm 2 .

202. The method according to claim 198 also includes a step of evaluating the target entity bound to the binding site, wherein the evaluation is performed after the sample is incubated for a certain period of time, which is equal to or longer than the time required for the target entity to diffuse across the thickness of the uniform thin layer.

203. The method of claim 198, wherein the time is reduced to less than 60 seconds.

204. The method of claim 198, further comprising a step of calculating the concentration of the analyte in the sample-related volume, wherein: The calculation is based on the volume of the associated sample volume defined by the predetermined area of ​​the binding site, the thickness of a thin layer of sample of uniform thickness in the closed phase, and the resulting estimated amount of target entity bound to the binding site.

205. The method of claim 198, wherein the target entity is a capture agent, an analyte, or a probe.

206. A method according to claim 198, wherein the volume of the sample at the predetermined area is equal to the predetermined area multiplied by the predetermined height of the spacer.

207. The method of claim 198, wherein the first plate includes a first bonding point and a second bonding point on a surface thereof, wherein: The distance between the edges of the binding sites is substantially greater than the thickness of the uniform thickness layer when the two plates are in a closed phase, wherein at least a portion of the uniform thickness layer is located above the binding sites, and wherein the sample has one or more analytes that are capable of diffusing in the sample.

208. The method of claim 198, wherein the method comprises: The first analyte is assessed in the first binding spot and the second analyte is measured in the second binding spot.

209. The method of claim 198, wherein the method comprises measuring the same analyte in the first and second binding sites.

210. A method according to claim 198, wherein the first plate has at least three binding points on its surface, the distance between the edges of any two adjacent binding points is significantly greater than the thickness of a uniform thin layer of thickness when the first plate and the second plate are in a closed phase, wherein at least a portion of the uniform thin layer of thickness is located above a predetermined area, and wherein the sample has one or more analytes capable of diffusing in the sample.

211. The method of claim 198, wherein the method comprises measuring different analytes in each binding site.

212. A method according to claim 198, wherein each binding point further includes a sub-analyte binding point, wherein at least two adjacent sub-analyte binding points are separated by a distance less than the thickness of a uniform thin layer when the first plate and the second plate are in a closed phase, wherein at least a portion of the uniform thin layer is located above the assay point, and wherein the sample has one or more analytes capable of diffusing in the sample.

213. The method of claim 198, wherein the junction is between a pair of electrodes.

214. The method of claim 198, wherein the binding site is defined by a piece of dried reagent.

215. The method of claim 198, wherein the binding site binds and immobilizes an analyte.

216. The method of claim 198, wherein the binding site enhances the signal from the analyte.

217. The method of claim 198, wherein the binding site is defined by a piece of binding reagent which, upon contact with the sample, dissolves into the sample, diffuses in the sample, and binds to the analyte.

218. The method of claim 198, wherein the sample thickness is less than 200 microns.

219. The method of claim 198, wherein the first and second plates include storage points located relative to each other when the first and second plates are in a closed phase.

220. The method of claim 198, wherein the ratio of the linear dimension of the bond points to the uniform thickness is greater than 5.

221. The method of claim 198, wherein the reaction is saturated in less than 60 seconds.

222. A method according to claim 198, wherein the relevant time length is in the range of 60 seconds to 30 minutes.

223. An apparatus for reducing the time required to bind a target entity in a sample-related volume in a binding point on a binding plate, comprising: A first plate and a second plate, wherein: i. The first plate and the second plate are movable relative to each other into different phases; ii. One or both plates are flexible; iii. Each plate has a sample contact area on its respective surface that contacts an entity that can diffuse in the sample; iv. one of the first plate and the second plate comprises a binding point having a predetermined area and binding and fixing the target entity in its sample contact area; The one or both plates include spacers fixed to the respective plates, wherein the spacers have a predetermined uniform height and a predetermined constant inter-spacer distance, wherein at least one of the spacers is within the sample contact area; wherein the thickness of the flexible plate multiplied by the Young's modulus of the flexible plate is in the range of 60 to 750 GPa-um; the fourth power of the inter-spacer distance ISD divided by the thickness h of the flexible plate and the Young's modulus E of the flexible plate, ISD 4 / (hE)≤5x10 6 um 3 / GPa; wherein one of the phases is an open phase, in which: the first plate and the second plate are separated from each other, the interval between the plates is not adjusted by a spacer, and the sample is deposited on one or both plates; wherein another of the phases is a closed phase, which is a phase formed after the sample is deposited in the open phase; in the closed phase: at least a portion of the sample is pressed by the two plates into a thin layer of uniform thickness, wherein at least a portion of the thin layer of uniform thickness is located above the bonding point, wherein the uniform thickness of the thin layer is defined by the sample contact surfaces of the first plate and the second plate, adjusted by the plates and the spacer, less than 250 microns, and substantially less than the average linear dimension of the predetermined area of ​​the bonding point; Lowering the thickness of the associated volume of the sample reduces the time required for the binding between the binding site and the target entity in the associated volume to reach equilibrium.

224. An apparatus according to claim 223, wherein the first plate comprises a first binding point and a second binding point on its surface, the distance between the edges of the binding points is significantly greater than the thickness of the uniform thin layer when the two plates are in a closed phase, and at least a portion of the uniform thin layer is located above the binding points, and the sample has one or more analytes capable of diffusing in the sample.

225. A device according to claim 223, wherein the first plate has at least three analyte binding sites on its surface, the distance between the edges of any two adjacent binding sites is significantly greater than the thickness of the uniform thin layer when the first plate and the second plate are in a closed phase, and at least a portion of the uniform thin layer is located above the binding sites, and the sample has one or more analytes capable of diffusing in the sample.

226. A device according to claim 223, wherein the first plate has at least two adjacent analyte binding sites on its surface, the two binding sites are not separated by a distance significantly greater than the thickness of the uniform thin layer when the first plate and the second plate are in a closed phase, and at least a portion of the uniform thin layer is located above the binding sites, and the sample has one or more analytes capable of diffusing in the sample.

227. A device according to claim 223, wherein the binding site is defined by a piece of dried reagent.

228. The device of claim 223, wherein the binding sites bind and immobilize an analyte.

229. A device according to claim 223, wherein the binding point is defined by a piece of binding reagent, which, after contacting the sample, dissolves into the sample, diffuses in the sample, and binds to the analyte.

230. The device of claim 224, wherein the inter-spacer distance is in the range of 14 microns to 200 microns.

231. A device according to claim 223, wherein the distance between the spacers is in the range of 7 microns to 20 microns.

232. The device of claim 223, wherein the spacer is a post having a cross-sectional shape that is polygonal, circular, oval, elliptical, or any combination thereof.

233. The device of claim 223, wherein the spacer has a pillar shape with a flat top surface and a uniform cross-section, wherein: For each spacer, a ratio of a lateral dimension to a height of the spacer is at least 1.

234. A device according to claim 223, wherein the spacer has a shape of a pillar, and the side wall corners of the spacer have a circular shape with a curvature radius of at least 1 micron.

235. The device of claim 223, wherein the spacers have at least 1000 / mm 2 density.

236. An apparatus according to claim 223, wherein at least one of the plates is transparent.

237. A device according to claim 223, wherein at least one of the plates is made of a flexible polymer.

238. A device according to claim 223, wherein at least one of the first plate and the second plate is flexible.

239. A method for measuring liquid samples in parallel, multiplexed, and without fluid isolation, comprising: (a) obtaining a sample comprising one or more target analytes that are capable of diffusing in the sample; (b) obtaining a first plate and a second plate, wherein the first plate and the second plate are movable relative to each other into different phases, wherein: i. One or both of the plates include a spacer fixed to the respective plates, and one or both of the plates are flexible; ii. The spacers have a predetermined uniform height and a predetermined constant distance between the spacers; iii. The first plate comprises, on its surface, one or more binding sites, each binding site comprising a predetermined area, which comprises a capture agent capable of binding and fixing the target analyte of step (a); iv. the second plate includes one or more corresponding storage points on its surface, each storage point includes a predetermined area, which includes a reagent of a certain concentration, which dissolves into the sample and diffuses in the sample after contacting the sample, Wherein, each capture agent, target analyte and corresponding probe agent can form a sandwich structure of capture agent-target analyte-probe agent at the binding point of the first plate; Wherein the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-um; the fourth power of the distance between the spacers ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE)≤5x10 6 um 3 / GPa; (c) depositing a sample on one or both plates when the first plate and the second plate are in an open phase, wherein the open phase refers to a phase in which the first plate and the second plate are partially or completely separated from each other and the spacing between the plates is not adjusted by a spacer; (d) after completing (c), compressing the sample by causing the two plates to enter a closed phase, wherein the closed phase is a phase in which: i. at least a portion of the sample is compressed into a uniform layer of thickness that contacts and is confined by the inner surfaces of the two plates, contacts one or more bonding points and one or more storage points, ii. the one or more corresponding storage points are above the one or more binding points; iii. The uniform thickness of the thin layer is adjusted by the plate and the spacer, less than 250 microns, and substantially smaller than the linear dimension of the predetermined area of ​​each storage point; (e) After completing step (d), when the first plate and the second plate are in a closed phase: (1) incubating the sample for a relevant length of time and then stopping the incubation; or (2) incubating the sample for a time period equal to or greater than a minimum value of the relevant time period, and evaluating the binding of each target analyte to the binding site for a time period equal to or less than a maximum value of the relevant time period; The relevant time length is: i. equal to or greater than the length of time required for the target analyte in step (a) to diffuse across the thickness of the uniform thin layer in the closed phase; ii. significantly less than the time required for the target analyte to diffuse laterally across the minimum lateral dimension of the predetermined area of ​​the storage point or binding point in step (a); Thus, a reaction is generated wherein, at the end of the incubation in step (1) or at the time of the evaluation in step (2), a majority of the target analyte bound to the capture agent-target analyte-probe agent sandwich structure at the binding site is derived from the corresponding relevant volume of the sample; wherein the incubation allows each target analyte to bind to a binding site and a probe, wherein the corresponding relevant volume refers to a portion of the sample located above a corresponding storage site in a closed phase, wherein the spacing between adjacent storage sites and the spacing between adjacent binding sites in the closed phase are both greater than the distance that the target analyte or probe can diffuse within a relevant time, and wherein there is no fluid isolation between the binding sites and / or storage sites.

240. The method of claim 239, wherein the first plate comprises a plurality of said bonding points on its surface.

241. The method of claim 239, wherein each of the plurality of binding sites binds a different target analyte.

242. A method according to claim 239, wherein the second plate includes a plurality of said corresponding storage points on its surface.

243. A method according to claim 239, wherein each of the plurality of corresponding storage sites binds a different target analyte.

244. A method according to claim 239, wherein the first plate includes a plurality of the binding points on its surface, and the second plate includes a plurality of the corresponding storage points on its surface, wherein each binding point faces a corresponding storage point when the first plate and the second plate are in a closed phase.

245. A method according to claim 239, wherein the first plate includes a plurality of said binding points on its surface, and the second plate includes a storage point on its surface, wherein at least some of the binding points face an area of ​​the storage point when the first plate and the second plate are in a closed phase.

246. A method according to claim 239, wherein the first plate includes a bonding point on its surface, and the second plate includes a plurality of storage points on its surface, wherein at least some of the storage points face an area of ​​the bonding point when the first plate and the second plate are in a closed phase.

247. The method of claim 239, wherein the first plate comprises a plurality of binding sites on its surface, wherein the binding sites comprise different capture agents capable of binding to and immobilizing different target analytes.

248. The method of claim 239, wherein the first plate comprises a plurality of binding sites on a surface thereof, wherein the binding sites comprise the same capture agent.

249. The method of claim 239, wherein the capture agents have different densities at different binding sites.

250. A method according to claim 239, wherein there is a gap between two adjacent binding points or two adjacent storage points, and the ratio of the gap to the thickness of the sample in the closed phase is at least 3.

251. The method according to claim 239 also includes a step of calculating the concentration of one or more analytes in the sample-related volume, wherein the calculation is based on the volume of the related sample volume defined by the predetermined area of ​​the binding point, the thickness of a thin layer of sample with uniform thickness in a closed phase, and the amount of the target entity detected.

252. The method of claim 239, wherein the target entity is a capture agent, an analyte, or a probe.

253. A method according to claim 239, wherein the volume of the sample in a predetermined area is equal to the area of ​​the predetermined area multiplied by the predetermined height of the spacer.

254. The method of claim 239, wherein the method comprises measuring the same analyte in related amounts located at at least two binding sites.

255. The method of claim 239, wherein the method comprises measuring different analytes in related amounts located at at least two binding sites.

256. The method of claim 239, wherein the storage locations are defined by multiple pieces of dried reagents.

257. A method according to claim 239, wherein the storage points are defined by multiple pieces of binding reagents, which, after contacting the sample, dissolve into the sample, diffuse in the sample and bind to the analyte.

258. The method of claim 239, wherein the sample has a thickness of less than 200 microns.

259. A method according to claim 239, wherein the ratio of the linear dimension of the bonding point to the uniform thickness is greater than 5.

260. The method of claim 239, wherein the method comprises one or more elutions.

261. A device for parallel, multiplex, and fluid-free isolation measurement of liquid samples, comprising a first plate and a second plate, wherein: i. The first plate and the second plate are movable relative to each other into different phases; One or both boards are flexible boards; ii. One or both of the plates include a spacer fixed to the corresponding plate; the spacer has a predetermined uniform height and a predetermined constant distance between the spacers; wherein the thickness of the flexible plate multiplied by the Young's modulus of the flexible plate is in the range of 60 to 750 GPa-um; the fourth power of the distance between the spacers ISD divided by the thickness h of the flexible plate and the Young's modulus E of the flexible plate, ISD 4 / (hE)≤5x10 6 um 3 / GPa; iii. Each plate has a sample contact area on its respective surface that contacts one or more target analytes that can diffuse in the sample; iv. the first plate has one or more binding sites on its surface, wherein each binding site has a predetermined area and comprises a capture agent capable of binding and fixing a target analyte corresponding to the sample; v. the second plate has one or more corresponding storage spots on its surface, wherein each storage spot has a predetermined area and includes a certain concentration of a probe, which, after contacting the sample, dissolves into the sample and diffuses in the sample, wherein each capture agent, target analyte and corresponding probe agent are capable of forming a capture agent-target analyte-probe agent sandwich structure in the binding site of the first plate; wherein one of the phases is an open phase, in which: the first plate and the second plate are separated from each other, the interval between the plates is not adjusted by a spacer, and the sample is deposited on one or both plates; Among them, another of the phases is a closed phase, which is a phase formed after the sample is deposited in the open phase; in the closed phase: i. At least a portion of the sample is pressed by two plates into a thin layer of uniform thickness, wherein the uniform thickness of the thin layer is defined by the inner surfaces of the first plate and the second plate and covers one or more binding points and one or more storage points; ii. the one or more corresponding storage sites are located above the one or more binding sites; iii. The uniform thickness of the thin layer is adjusted by the spacer and the first and second plates, less than 250 microns, and substantially smaller than the linear dimension of the predetermined area of ​​each storage point; iv. There is no fluid isolation between the binding points and / or the storage points; Wherein the spacing between the edges of adjacent storage spots and the spacing between the edges of adjacent binding spots is greater than the distance that the target analyte or probe can diffuse within the relevant time, and there is no fluid isolation between the binding spots and / or the storage spots.

262. A device according to claim 261, wherein the first plate includes a plurality of said bonding points on its surface.

263. A device according to claim 261, wherein each of the plurality of binding sites binds a different target analyte.

264. An apparatus according to claim 261, wherein the second plate includes a plurality of said corresponding storage points on its surface.

265. A device according to claim 261, wherein each of the plurality of corresponding storage sites binds a different target analyte.

266. The device of claim 261, wherein the first plate comprises a plurality of the binding points on its surface, and the second plate comprises a plurality of the corresponding storage points on its surface, wherein: When the first plate and the second plate are in a closed phase, each binding point faces a corresponding storage point.

267. The device according to claim 261, wherein the first plate includes a plurality of said binding points on its surface, and the second plate includes one said storage point on its surface, wherein, When the first plate and the second plate are in the closed phase, at least some of the binding sites face an area of ​​the storage sites.

268. The device according to claim 261, wherein the first plate includes one said binding point on its surface, and the second plate includes a plurality of said storage points on its surface, wherein, When the first plate and the second plate are in the closed phase, at least some of the storage points face a region of the binding points.

269. The device of claim 261, wherein the first plate comprises a plurality of said binding sites on its surface, wherein said binding sites comprise different capture agents capable of binding and fixing different target analytes.

270. The device of claim 261, wherein said first plate comprises a plurality of said binding sites on its surface, wherein said binding sites comprise the same capture agent.

271. A device according to claim 270, wherein the capture agent has different density distributions at different binding sites.

272. An apparatus according to claim 268, wherein there is a gap between two adjacent binding points or between two adjacent storage points.

273. A device according to claim 272, wherein the distance between the spacers is in the range of 1 micron to 120 microns.

274. The device of claim 272, wherein the ratio of the spacing to the thickness of the sample in the closed phase is at least 3.

275. A device according to claim 261, wherein the ratio of the spacing to the thickness of the sample in the closed phase is at least 3.

276. The apparatus of claim 261, wherein the sample layer has a uniform thickness of at least 1 mm. 2 is uniform in the plane area.

277. The apparatus of claim 261, wherein the thickness uniform sample layer has a thickness uniformity of at most + / - 5%.

278. The device of claim 261, wherein the spacers are posts having cross-sectional shapes comprising: Circular, polygonal, oval, elliptical, or any combination thereof.

279. The device of claim 261, wherein the spacer has a pillar shape with a flat top surface and a uniform cross-section, wherein For each spacer, a ratio of a lateral dimension to a height of the spacer is at least 1.

280. A device according to claim 261, wherein the distance between the spacers is periodic.

281. A device according to claim 261, wherein the spacer has a fill factor of 1% or higher, wherein the fill factor is the ratio of the spacer contact area to the total plate area.

282. A device according to claim 261, wherein the Young's modulus of the spacer multiplied by the filling factor of the spacer is equal to or greater than 20 MPa, wherein the filling factor is the ratio of the spacer contact area to the total plate area.

283. The apparatus of claim 261, wherein: The separation between the two plates in the closed phase is less than 200 microns.

284. The apparatus of claim 261, wherein: In the closed phase the spacing between the two plates is a value between 1.8 microns and 3.5 microns.

285. The device of claim 261, wherein the spacer is fixed to the plate by direct stamping or injection molding.

286. The device according to claim 261, wherein the material of the plate and the spacer is selected from the following group of materials: polystyrene, PMMA, PC, COC, COP, or other plastics.

287. A device according to claim 261, wherein the spacer has a pillar shape, and the side wall corners of the spacer have a circular shape with a curvature radius of at least 1 micron.

288. The device of claim 261, wherein the spacers have at least 1000 / mm 2 density.

289. An apparatus according to claim 261, wherein at least one of the plates is transparent.

290. A system for rapid sample analysis using a mobile phone, comprising: (a) An apparatus as claimed in any one of claims 261 to 289, wherein the two plates are movable relative to each other into different phases; wherein: i. One of the phases is an open phase, in which: the first plate and the second plate are separated from each other, the interval between the plates is not adjusted by a spacer, and the sample is deposited on one or both plates; ii. Another of the phases is a closed phase, which is a phase formed after the sample is deposited in the open phase; in the closed phase: at least a portion of the sample is pressed by two plates into a thin layer of uniform thickness, wherein the uniform thickness of the thin layer is defined by the sample contact surfaces of the first plate and the second plate, and is adjusted by the plates and the spacer; (b) a mobile communications device comprising: i. one or more cameras for detecting and / or imaging the sample; ii. Electronics, signal processors, and hardware and software for receiving and / or processing detected signals and / or images of said samples and for remote communication; (c) A light source belonging to the mobile communication device or an external light source.

291. A system according to claim 290, wherein one of the first plate and the second plate includes a binding site for a bound analyte, wherein at least a portion of the thin layer of uniform thickness is located above the binding site and is significantly smaller than an average lateral linear dimension of the binding site.

292. The system of claim 290, further comprising: A housing configured to hold a sample and mountable on a mobile communication device.

293. A system according to claim 290, wherein the housing includes an optical system for facilitating imaging and / or signal processing of the sample by the mobile communication device, and a mounting member configured to hold the sample on the mobile communication device.

294. The system of claim 290, wherein the mobile communication device is configured as a device that can communicate the test results to a medical professional, a medical facility, or an insurance company.

295. The system of claim 290, wherein the mobile communication device is further configured to communicate subject information with a medical professional, medical facility, or insurance company.

296. The system of claim 290, wherein the mobile communication device is configured to receive a prescription, diagnosis or recommendation from a medical professional.

297. A system according to claim 290, wherein the mobile communication device is configured with hardware and software to: (a) acquire a sample image; (b) analyze a test area and a control area in the image; and (c) compare the value obtained from the analysis of the test area with a threshold value characterizing a rapid diagnostic test.

298. A system according to claim 290, wherein at least one of the first plate and the second plate includes a storage point for storing assay reagents.

299. A system according to claim 290, wherein at least one of the cameras reads a signal from the CROF device.

300. The system of claim 290, wherein the mobile communication device communicates with a remote location via a wifi or mobile phone network.

301. The system of claim 290, wherein the mobile communication device is a mobile phone.

302. A method for quickly analyzing a sample using a mobile phone, comprising: (a) depositing a sample on a device in a system as claimed in any one of claims 290 to 301; (b) analyzing a sample deposited on the device to obtain a result; (c) communicating the results from the mobile communication device to a location remote from the mobile communication device.

303. The method according to claim 302, comprising: analyzing the results at the remote location to provide an analysis result; And transmit the analysis result from the remote place to the mobile communication device.

304. The method of claim 302, wherein the analysis is performed remotely by a medical professional.

305. The method of claim 302, wherein the mobile communication device receives a prescription, diagnosis, or advice from a remote medical professional.

306. The method of claim 302, wherein the sample is a body fluid.

307. The method of claim 302, wherein the body fluid is blood, saliva, or urine.

308. The method of claim 302, wherein the analyzing step comprises detecting an analyte in a sample.

309. The method of claim 302, wherein the analyte is a biomarker.

310. The method of claim 302, wherein the analyte is a protein, a nucleic acid, a cell, or a metabolite.

311. The method of claim 302, comprising counting red blood cells.

312. The method of claim 302, comprising counting white blood cells.

313. The method of claim 302, comprising staining cells in the sample and counting neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

314. A method for analyzing a liquid sample, comprising: (a) obtaining a sample comprising an analyte capable of diffusing in the sample; (b) The apparatus of claim 290, wherein the first plate and the second plate are movable relative to each other into different phases, wherein one or both plates include spacers affixed to the respective sample contacting surfaces, and wherein the spacers have a predetermined uniform height, and wherein the first plate includes analyte assay sites having predetermined areas on its surface; (c) depositing a sample on one or both plates when the plates are in an open phase, wherein the open phase is a phase in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by a spacer; (d) after completing (c), compressing at least a portion of the sample using two plates into a thin layer of the sample of uniform thickness confined by sample contacting surfaces of the plates, wherein the uniform thickness of the layer is adjusted by spacers of the plates, wherein the compressing comprises: Bring the two boards together; Applying an external force to the outer surfaces of the first plate and the second plate to press the two plates together into a closed phase, wherein the external force generates pressure on the plates above at least a portion of the sample and presses so that at least a portion of the sample is laterally spread between the inner surfaces of the first plate and the second plate, wherein the closed phase is a phase in which the spacing between the uniform thickness thin layer regions of the plates is adjusted by the spacer; (e) incubating the sample while the plate is in the closed phase for a time that is (i) greater than, equal to, or longer than the time required for the analyte to diffuse across the thickness of the uniformly thick layer; and (ii) substantially shorter than the time required for the analyte to diffuse across the area of ​​the analyte assay site; (f) immediately stopping the incubation after step (e) and measuring the analyte at the assay site, or continuing the incubation while the first plate and the second plate are in a closed phase and measuring the analyte at the assay site at a time point that is significantly shorter than the time required for the analyte to diffuse across the area of ​​the analyte assay site.

315. The method of claim 314, further comprising: (g) Calculate the concentration of the analyte in the relevant volume of the sample layer at the measurement point.

316. A method according to claim 314, wherein the sample volume of the sample thin layer at the measurement point is equal to the predetermined area of ​​the measurement point multiplied by the predetermined height of the spacer.

317. The method of claim 314, wherein the first plate includes a first measurement point and a second measurement point, the method comprising: The same analyte is analyzed at the first assay point and the second assay point.

318. The method of claim 314, wherein the analyte determination site is between a pair of electrodes.

319. The method of claim 314, wherein the assay site is defined by a sheet of dried reagent.

320. The method of claim 314, wherein the assay site binds and immobilizes an analyte.

321. The method of claim 314, wherein the assay site is defined by a piece of binding reagent which, upon contact with the sample, dissolves into the sample, diffuses in the sample, and binds to the analyte.

322. A method according to claim 314, wherein the first plate further comprises a plurality of analyte determination points on its surface, and the distance between the edges of two adjacent determination points is significantly greater than the thickness of a thin layer of uniform thickness when the first plate and the second plate are in a closed phase, wherein the sample comprises one or more analytes capable of diffusing in the sample.

323. The method of claim 314, wherein at least two of the adjacent assay sites are not isolated from one another to prevent diffusion of analytes between the assay sites.

324. The method of claim 314, wherein the sample thickness is less than 200 microns.

325. The method of claim 314, wherein the first and second plates include storage points located relative to each other when the first and second plates are in a closed phase.

326. A method according to claim 314, wherein the ratio of the linear dimension of the bonding point to the uniform thickness is greater than 5.

327. The method of claim 314, wherein the reaction is saturated in less than 60 seconds.

328. A method according to claim 314, wherein the relevant time length is in the range of 60 seconds to 30 minutes.

329. The method of claim 314, wherein step e(i) comprises, after stopping the incubation, evaluating the target entity bound to the binding site.

330. The method of claim 314, wherein the method comprises one or more elutions.

331. The method of claim 314, wherein the inter-spacer distance is in the range of 14 microns to 200 microns.

332. The method of claim 314, wherein the inter-spacer distance is in the range of 7 microns to 20 microns.

333. The method of claim 314, wherein the spacers are posts having cross-sectional shapes comprising: Circular, polygonal, oval, elliptical, or any combination thereof.

334. The method of claim 314, wherein the spacer has a pillar shape, has a flat top surface, and has a uniform cross-section, wherein: For each spacer, a ratio of a lateral dimension to a height of the spacer is at least 1.

335. The method of claim 314, wherein the spacer has a pillar shape, and a sidewall corner of the spacer has a circular shape with a radius of curvature of at least 1 micron.

336. The method of claim 314, wherein the spacers have at least 1000 / mm 2 density.

337. A method according to claim 314, wherein at least one of the plates is transparent.

338. A method according to claim 314, wherein at least one of the first plate and the second plate is made of flexible plastic.

339. A method according to claim 314, wherein only one of the first plate and the second plate is flexible.

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