Digital measurement

Abstract

Provided herein is a method and apparatus for separating fluid samples. The apparatus comprises a plate comprising microwells. The method comprises depositing the sample on one or both plates when the plates are in an open configuration, wherein the deposition is in the form of a single or multiple droplets of the sample, wherein at least one fifth of the droplets have a volume that occupies more than two microwells, and closing the plates to a closed configuration to separate the sample in the microwells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of prior art to Provisional Application Serial No. 62 / 457,009 filed on February 9, 2017 (ESX-040PRV), Provisional Application Serial No. 62 / 460,076 filed on February 16, 2017 (ESX-040PRV2), Provisional Application Serial No. 62 / 621,475 filed on January 24, 2018 (ESX-040PRV3), Provisional Application Serial No. 62 / 456,603 filed on February 8, 2017 (ESX-033PRV), Provisional Application Serial No. 62 / 457,009 filed on February 9, 2017 (ESX-040PRV3), Provisional Application Serial No. 62 / 460,076 filed on February 16, 2017 (ESX-040PRV2), Provisional Application Serial No. 62 / 621,475 filed on January 24, 2018 (ESX-040PRV3), Provisional Application Serial No. 62 / 456,603 filed on February 8, 2017 (ESX-033PRV), Provisional Application Serial No. 62 / 456,603 filed on February 1 No. 62 / 459,337 filed on February 5, 2017 (ESX-033PRV2), No. 62 / 456,504 filed on February 8, 2017 (ESX-045PRV), No. 62 / 460,062 filed on February 16, 2017 (ESX-045PRV2), and No. 62 / 457,133 filed on February 9, 2017 (ESX-046PRV), all of which are incorporated herein by reference in their entirety. Technical Field

[0003] Among other things, the present invention relates to apparatus and methods for performing biological and chemical analyses. Background Art

[0004] Among other things, the present invention provides devices and methods that allow for more accurate, simpler and faster analysis of analytes in samples than certain prior arts. In certain embodiments, the present invention separates samples into separate or nearly separate microwells of predetermined geometry and volume, and covers that separate or nearly separate the sample in each well from its adjacent wells. The present invention can be used for digital PCR (polymerase chain reaction). Summary of the invention

[0005] An apparatus for performing a digital assay is provided, comprising:

[0006] A first plate, a second plate and a microwell, wherein:

[0007] (a) the first plate and the second plate are movable relative to each other into different configurations and have sample contact areas on their respective surfaces for contacting a fluid sample containing an analyte;

[0008] (b) the second plate has a plurality of microwells in the sample contacting region, wherein each microwell has (i) a predetermined and known geometry, (ii) a pore depth of 200 μm or less, and (iii) a volume substantially smaller than the fluid sample,

[0009] wherein one of the configurations is an open configuration, wherein: the average spacing between the inner surface of the first plate and the edges of the microwells in the second plate is greater than the depth of the wells, and the sample is deposited on one or both plates; and

[0010] Another of the structures is a closed structure, which is a structure after the sample is deposited into the open structure; in the closed structure, at least a portion of the sample is within the microwell, and the average spacing between the inner surface of the first plate and the edge of the microwell in the second plate is less than 1 μm or less than 1 / 10 (one tenth) of the depth of the microwell.

[0011] A method for separating a fluid sample, comprising:

[0012] Obtaining a device or apparatus as claimed in any one of the preceding claims,

[0013] depositing the sample on one or both plates when the plates are in an open configuration, wherein the deposition is in the form of a single or multiple droplets of the sample, wherein at least one of the droplets has a volume that occupies more than two microwells; and

[0014] The plate is closed into a closed configuration to separate the samples in the microwells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present invention in any way. The drawings are not drawn entirely to scale. In the figures where experimental data points are given, the lines connecting the data points are only used to guide the observation of the data and have no other meaning.

[0016] Figure 1 (a) Schematic diagram of two plates: Plate 1 has a flat inner surface, while Plate 2 has a well array on its sample contact surface. (b) Sample liquid is deposited in the center of the well array plate (Plate 2), covered with a flat plate (Plate 1) and the two plates are pressed together. (c) Pressurization separates the liquid into the well array.

[0017] Figure 2 (a) Photograph of a microwell plate fabricated on a 175 μm thick PMMA substrate; (b) Microscope photograph of a hexagonal lattice microwell array with a pore diameter of 30 μm, a pore depth of 8 μm, and a pore center-to-center distance of 34 μm; (c) Microscope photograph of a hexagonal lattice microwell array with a pore diameter of 20 μm, a pore depth of 8 μm, and a pore center-to-center distance of 24 μm.

[0018] Figure 3 . Place two plates (microplate and Figure 1Microscope photographs at (a) 10X and (b) 20X of separation of liquid entering the well array after the plates (described in the closed configuration) are pressed together (with human fingers). In this device, Plate 1 (flat plate) is a flat PET film with a thickness of 50 μm, while Plate 2 (microwell plate) is a PMMA plate with a thickness of 175 μm, and the microarray on the surface has a well diameter of 30 μm, a well depth of 8 μm, and a well center-to-center distance of 34 μm. The liquid is phosphate buffered saline (PBS) at a volume of 2 μL. Note that after depositing the liquid sample and placing the plates in a closed configuration, some microwells are filled and some are empty. Our measurements show that in the closed configuration of the plates, there is a thin residual layer of liquid (-0.5 μm thick or thinner) between the inner surface of Plate 1 and the edges of the wells of the plates.

[0019] Figure 4 It is a pixelated assay QMAX device (Q: quantification; M: amplification; A: addition of reagents; X: acceleration; also known as a compression regulated open flow (CROF) device that can be used for pixelated assays. Figure 4 In the embodiment of the present invention, the QMAX device is an open structure. (a) A device comprising a first plate, a second plate, and microwells on the second plate. (b) Top view of the microwells on the second plate, having (i) square lattice circles, (ii) rectangular lattice with square lattice, (iii) triangles with hexagonal lattice, and (iv) non-periodic circles.

[0020] Figure 5 is an exemplary flow chart showing basic steps in an exemplary process for pixelation measurement using a QMAX device.

[0021] Figure 6 Microscopic examples of isolated hole arrays on a QMAX first plate fabricated on a 0.25 mm thick acrylic substrate are shown, with (a) 20 μm × 20 μm square holes, 100 μm spacing, 30 μm depth; (b) 20 μm × 20 μm square holes, 200 μm spacing, 30 μm depth; (c) 10 μm diameter circular holes, 200 μm spacing, 20 μm depth.

[0022] Figure 7 A schematic diagram showing the preparation of a binding site plate (first plate) and a storage plate (second plate) for performing an exemplary embodiment of the pixelated assay QMAX.

[0023] Figure 8 A schematic diagram showing an exemplary embodiment of a pixelated assay QMAX device in a closed configuration for an incubation process.

[0024] Fig. 9A schematic diagram of an exemplary embodiment of a pixelated assay QMAX device in a closed configuration for use in an amplification process is shown.

[0025] Fig.10 Representative measurements of a pixelated assay with separate wells are shown. (a) The sample volume is estimated by counting the wells loaded with sample during the capture step. (b) The number of molecules in the sample is estimated by counting the number of wells with signal after the amplification step. The final concentration of the analyte in the sample is back-calculated by dividing the number of molecules by the sample volume.

[0026] Fig.11 Schematic diagram showing the preparation of a binding site plate (first plate) and a storage plate (second plate) for an exemplary embodiment of the pixelated assay QMAX. The experimental procedure follows Figure 5 Flowchart of the process.

[0027] Fig.12 Schematic diagram showing an exemplary embodiment of a pixelated QMAX device in a closed configuration for a capture process

[0028] Fig.13 A schematic diagram of an exemplary embodiment of a pixelated assay QMAX device in a closed configuration for use in an amplification process is shown.

[0029] Fig.14 A schematic diagram of an exemplary embodiment of an open-architecture pixelated assay QMAX device for a digital nucleic acid amplification assay is shown.

[0030] Fig.15 Schematic diagram showing an exemplary embodiment of a pixelated assay QMAX device in a closed configuration following sample introduction for a digital nucleic acid amplification assay

[0031] Fig.16 A schematic diagram of an exemplary embodiment of a pixelated assay QMAX device in a closed configuration during a digital nucleic acid amplification process is shown. DETAILED DESCRIPTION

[0032] The following detailed description illustrates some embodiments of the present invention by way of example and not limitation. The section headings and any subheadings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. The table of contents under the section headings and / or subheadings is not limited to the section headings and / or subheadings, but applies to the entire description of the present invention.

[0033] 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 publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0034] A. Principle of microwell array pixelated assay (MAPA).

[0035] GD1 Figure 4 As shown, a pixelated assay device using a microwell array, called MAPA or "microwell array pixelated assay", comprises a first plate, a second plate, and microwells;

[0036] (c) the first plate and the second plate are movable relative to each other into different configurations and have sample contact areas on their respective surfaces for contacting a fluid sample;

[0037] (d) the second plate has a plurality of microwells in the sample contacting region, wherein each microwell has (i) a predetermined and known geometry, (ii) a pore depth of 200 μm or less, and (iii) a volume substantially smaller than the fluid sample,

[0038] wherein one of the configurations is an open configuration, wherein: the average spacing between the inner surface of the first plate and the edge of a microwell in the second plate is greater than the depth of the well, and the sample is deposited on one or both plates; and

[0039] Another of the structures is a closed structure, which is a structure after the sample is deposited in an open structure; in the closed structure, at least a portion of the sample is within the microwell, and the average spacing between the inner surface of the first plate and the edge of the microwell in the second plate is less than 1 μm or less than 1 / 10 (one tenth) of the depth of the microwell.

[0040] GM1 A method for pixelating a fluid sample, comprising:

[0041] i. Get the first board,

[0042] ii. Obtain the second board,

[0043] in

[0044] (a) the first plate and the second plate are movable relative to each other into different configurations and have sample contact areas on their respective surfaces for contacting a fluid sample containing a target analyte;

[0045] (b) the second plate has a plurality of microwells in the sample contact region, wherein each microwell has (i) a pore depth of 200 μm or less, and (ii) a pore volume substantially smaller than a volume of the sample;

[0046] iii. When the plates are in an open configuration, depositing the sample on one or both plates; and

[0047] iv. The plate is made into a closed configuration;

[0048] wherein an open configuration is a configuration in which: the average spacing between the inner surface of the first plate and the edges of the microwells in the second plate is greater than the depth of the wells, and the sample is deposited on one or both plates;

[0049] Among them, the closed structure refers to the structure after the sample is deposited in the open structure; in the closed structure, at least a portion of the sample is in the micropore, and the average distance between the inner surface of the first plate and the edge of the micropore in the second plate is less than 1 μm or less than 1 / 10 (one tenth) of the depth of the micropore.

[0050] In the method of any preceding embodiment, further comprising the step of measuring a signal associated with the analyte when the plate is in the closed configuration.

[0051] In the device or method of any previous embodiment, wherein the additional sealing layer is on the inner surface of one or both plates, wherein the sealing layer is configured to prevent liquid from one hole to its adjacent hole when the plates are in the closed configuration. An embodiment of the sealing layer is a thin adhesive layer.

[0052] In the device or method of any preceding embodiment, wherein the analyte is a molecule. In some embodiments, the analyte is a protein and / or a nucleic acid (e.g., DNA or RNA). In some embodiments, the analyte is a small molecule.

[0053] In the device or method of any preceding embodiment, there is further a binding site on the inner surface of one or both plates, wherein the binding site comprises a capture agent immobilized at the site, and the capture agent is configured to specifically capture the analyte.

[0054] In the device or method of any preceding embodiment, there is further a storage site on the inner surface of one or both plates, wherein the storage site contains a reagent at the site and the reagent is soluble in a liquid.

[0055] In the method of any of the foregoing embodiments, wherein the method further has an amplification step, wherein the amplification makes the analyte more observable than without the amplification, and wherein the analyte signal amplification in the well includes, but is not limited to, a chemical reaction or physical enhancement (e.g., a plasma structure) or both. Examples include, but are not limited to: (a) for nucleic acids, various types of PCR (polymerase chain reaction), LAMP (loop-mediated isothermal amplification), etc., (b) for proteins, ELISA (enzyme-linked immunosorbent assay), light enhancement using a plasma structure (e.g., a plasma metal structure), and (c) for small molecules, a chemical reaction. The chemical reaction includes, but is not limited to, chemiluminescence or other luminescence.

[0056] In the method of any preceding embodiment, there is further a step of subtracting empty wells when determining the actual sample volume, which is performed by (i) identifying the empty wells by imaging the wells in a bright field image and / or by imaging prior to the amplification step, and (ii) subtracting the empty wells in the volume calculation to quantify the analyte concentration.

[0057] Figure 1-3 Some principles of an embodiment of the method are schematically illustrated.

[0058] Spacers

[0059] In certain embodiments of the invention, the device of the previous embodiments further comprises spacers configured to keep the distance between the inner surface of the first plate and the bottom of the well substantially uniform (i.e., substantially the same across the well). In some embodiments, the spacers are fixed within the wells, or on the inner surface of the first plate, or both. Examples of spacers are described in PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, which are incorporated herein in their entirety for all purposes.

[0060] Pixelated assay for samples with low analyte concentrations

[0061] For a given analyte concentration (particularly low concentrations), the volume of each well can be configured so that the well has one analyte or no analyte. In this case, when the plate is in a closed configuration, the signal associated with the analyte in the well (with the analyte) can be amplified without being affected or significantly affected by other wells.

[0062] After analyte signal amplification, the analyte can be detected by examining the presence of wells with observable signals associated with the analyte. The concentration of the analyte in the sample can be determined by counting the number of wells with observable signals associated with the analyte and determining the associated sample volume using the plate.

[0063] When analyzing low analyte concentration samples, each well can be observed at a pixel and the analyte concentration determined by counting the number of pixels with a signal. This assay is also called a digital assay.

[0064] The volume of the sample can be determined by the pore volume and the number of pores and the sample occupancy within the pores.

[0065] B. Pixelated Detection of Nucleic Acids

[0066] In the device or method of any of the foregoing embodiments, the analyte is a nucleic acid and the device or method is configured to perform nucleic acid amplification techniques, including but not limited to different polymerase chain reaction (PCR) methods, for example, as hot-start PCR, nested PCR, touchdown PCR, reverse transcription PCR, RACEPCR, digital PCR, real-time PCR, etc., as well as isothermal amplification methods such as loop-mediated isothermal amplification (LAMP), strand displacement amplification, helicase-dependent amplification, nicking enzyme amplification, rolling circle amplification, recombinase polymerase amplification, etc.

[0067] Digital polymerase chain reaction (digital PCR, digital PCR, dPCR, or dePCR) can be used to directly quantify and clonally amplify nucleic acid chains, including DNA, cDNA, or RNA. The main difference between dPCR and traditional PCR is the method used to determine the amount of nucleic acid, with the former being a more accurate method than PCR but more prone to error in the hands of inexperienced users. [1]:217A "Digital" measurements quantitatively and discretely measure a variable, while "analog" measurements infer certain measurements based on the pattern measured. PCR performs one reaction per single sample. dPCR also performs a single reaction in a sample, however the sample is separated into a large number of parts and the reaction is performed separately in each part. This separation allows for more reliable collection and sensitive measurement of nucleic acid amounts. The method has been shown to be useful for studying variations in genetic sequences, such as copy number variations and point mutations, and is often used for clonal amplification of samples for next-generation sequencing.

[0068] dPCR improves current PCR practice by dividing the reaction into multiple smaller reactions. The sample is partitioned so that individual nucleic acid molecules in the sample are localized and concentrated in many separate areas. Microplates, capillaries, oil emulsions, and miniaturized chamber arrays with nucleic acid binding surfaces can be used to partition the sample. The PCR solution is prepared similarly to the TaqMan assay, which consists of template DNA (or RNA), fluorescent quencher probes, primers, and PCR premix, which contains DNA polymerase, dNTPs, MgCl2, and reaction buffer at an optimal concentration. The PCR solution is divided into smaller reactions, which are then subjected to PCR separately. After multiple PCR amplification cycles, the fluorescence of the sample is checked with a binary readout of "0" or "1". The fraction of fluorescent droplets is recorded. The partitioning of the sample allows one to estimate the number of different molecules by assuming that the population of molecules follows a Poisson distribution, thereby taking into account the possibility that multiple target molecules reside in a single molecule. Using Poisson's law of small numbers, the distribution of target molecules in the sample can be accurately approximated, allowing for quantification of target chains in PCR products. Based on the fraction of fluorescent droplets (p) represented by the function CPD = -ln(1-p), the model simply predicts that as the number of samples containing at least one target molecule increases, the probability of samples containing more than one target molecule increases. In conventional PCR, the number of PCR amplification cycles is proportional to the starting copy number. However, dPCR does not rely on the number of amplification cycles to determine the initial sample amount, eliminating the reliance on uncertain exponential data to quantify target nucleic acids, and thus providing absolute quantification.

[0069] In the device or method of any previous embodiment, wherein the device is further configured to perform rapid thermal cycling in PCR, wherein the configuration includes, but is not limited to, adding heaters and coolers on or adjacent to the device and other additional devices, materials and / or methods disclosed in U.S. Provisional Application No. 62 / 456,596 filed on February 8, 2017, U.S. Provisional Application No. 62 / 456,504 filed on February 8, 2017, U.S. Provisional Application No. 62 / 459,496 filed on February 15, 2017, U.S. Provisional Application No. 62 / 488,684 filed on April 21, 2017, and U.S. Provisional Application No. 62 / 510,063 filed on May 23, 2017, all of which are incorporated herein in their entirety for all purposes.

[0070] Fig.11 Schematic diagram showing the preparation of a binding site plate (first plate) and a storage plate (second plate) for an exemplary embodiment of the pixelated assay QMAX. The experimental procedure follows Figure 5 Flowchart of the process.

[0071] Specifically, the first plate in this embodiment is a square hole array with a size of 20μm×20μm, a spacing of 100μm, and a depth of 30μm manufactured on a 0.25mm thick acrylic substrate. The substrate was first treated with 1M sodium hydroxide at 45°C for 2 hours and then rinsed with water 3 times. The substrate was then coated with MES buffer (pH4.7) containing 8mg / ml EDC and 11.2mg / ml NHS at room temperature for 2 hours. 20μg / ml streptavidin was then coated on the first plate for 2 hours at room temperature and then rinsed 3 times with PBS. The substrate was then closed with 4% BSA for 1 hour at room temperature and then rinsed 3 times with PBS. At room temperature, a 1μm biotinylated capture probe was coated on the first plate for 2 hours and then washed three times with PBST. Excess liquid was removed and the plate was dried at room temperature.

[0072] The second plate in this example was a 0.175 mm thick flat acrylic film. 200 μL of 1 uM detection probe conjugated with HRP was evenly printed on the second plate and dried at 37°C for 2 hours.

[0073] like Fig.11 As shown, in some embodiments, the first plate includes capture probes that are fully or partially coated on the inner surface of the first plate. In some embodiments, the capture probes are fully or partially located on the bottom or sidewalls or both of the wells on the first plate.

[0074] In some embodiments, the capture probe can be applied to the surface by printing, spraying, dipping or any other method of applying a uniform or partial reagent layer. In certain embodiments, the capture probe is directly applied to the first plate. It should also be noted that in some embodiments, the capture probe is applied to the inner surface of the first plate, but not the inner surface of the second plate; in some embodiments, the capture probe is applied to the inner surface of the second plate instead of the first plate; in some embodiments, the capture probe is applied to the inner surface of both plates. In some embodiments, the concentration range of the coated capture probe is 1fM-1mM.

[0075] In some embodiments, the capture probe is generally 10-50 bp in length, and the 3' end is modified to facilitate coating on the substrate. Common 3' end modifications include, but are not limited to, thiols, dithiols, amines, biotin, etc., and substrates that can be used to fix the capture probe include, but are not limited to, acrylic films, gold surfaces, PS, etc.

[0076] like Fig.11As shown, in some embodiments, the first plate includes a blocking agent coated on the inner surface of the first plate. In some embodiments, the blocking agent blocks any unoccupied sites on the solid surface that may cause unwanted nonspecific binding in the assay. In certain embodiments, the blocking agent reduces nonspecific binding. In certain embodiments, the blocking agent can be applied to the surface by printing, spraying, soaking or any other method of applying a uniform reagent layer. In certain embodiments, the blocking agent is dried on the first plate. It should also be noted that in some embodiments, the blocking agent is coated on the inner surface of the first plate, rather than the inner surface of the second plate; in some embodiments, the blocking agent is coated on the inner surface of the second plate instead of the first plate; in some embodiments, the blocking agent is coated on the inner surface of both plates. In some embodiments, the blocking agent is bovine serum albumin (BSA), casein, or total protein from whole milk, etc. In some embodiments, the blocking agent is a small molecule, such as 6-mercapto-hexanol.

[0077] like Fig.11 As shown, in some embodiments, the first plate includes a stabilizer coated on the inner surface of the first plate. In some embodiments, the stabilizer helps maintain the proper folding of the protein when dry so that the function of the protein is not destroyed during storage. In some embodiments, the stabilizer extends the service life of the reagent, such as, but not limited to, the protein. In some embodiments, the stabilizer can be applied to the surface by printing, spraying, soaking or any other method of applying a uniform reagent layer. In some embodiments, the stabilizer is dried on the first plate. It should also be noted that in some embodiments, the stabilizer is coated on the inner surface of the first plate, but not on the inner surface of the second plate; in some embodiments, the stabilizer is coated on the inner surface of the second plate, but not on the first plate; in some embodiments, the stabilizer is coated on the inner surfaces of both plates. In some embodiments, the stabilizer is a sugar, such as, but not limited to, sucrose and glucose. In some embodiments, the stabilizer is a polymer. In some embodiments, the stabilizer is glycerol.

[0078] like Fig.11 As shown, in some embodiments, the second plate includes a detection probe coated on the inner surface of the second plate. In some embodiments, the detection probe can be applied to the surface by printing, spraying, soaking or any other method of applying a uniform reagent layer. In certain embodiments, the detection probe is dried on the second plate. It should also be noted that in some embodiments, the detection antibody is coated on the inner surface of the second plate instead of the first plate; in some embodiments, the detection antibody is coated on the inner surface of the first plate instead of the inner surface of the second plate; in some embodiments, the detection probe is coated on the inner surface of both plates. In some embodiments, the concentration of the coated detection probe is 1fM-1mm.

[0079] In some embodiments, the detection probe is configured to generate a detectable signal after binding to a nucleic acid target. For example, in some embodiments, the signal can be a colorimetric signal, a luminescent signal, or a fluorescent signal. In some embodiments, for example, the detection probe is labeled with a fluorescent marker that generates a signal after the detection probe binds to a nucleic acid target or a capture probe-target complex. In some embodiments, the fluorescent marker directly labels the detection probe. In some embodiments, the fluorescent marker labels an agent that can bind to the detection probe or the detection probe-target complex. In some embodiments, the detection probe is configured as a chemical substance that can amplify a signal or can amplify a signal from the chemical substance; wherein the amplification method in the amplification step includes, but is not limited to:

[0080] A colorimetric enzymatic reaction, wherein the absorbance signal generated by the substrate is amplified by an enzyme connected to a detection agent; wherein the enzyme includes but is not limited to horseradish peroxidase and alkaline phosphatase; wherein the substrate includes ABTS or TMB;

[0081] A fluorescence-based enzymatic reaction, wherein the fluorescence signal generated by the substrate is amplified by an enzyme connected to a detection agent; wherein the enzyme includes horseradish peroxidase and alkaline phosphatase; wherein the substrate includes but is not limited to Amplex Red;

[0082] Based on the chemiluminescence enzymatic reaction, the chemiluminescent signal generated by the substrate is amplified by an enzyme connected to a detection agent; wherein the enzyme includes horseradish peroxidase and alkaline phosphatase; wherein the substrate includes but is not limited to luminol and isoluminol;

[0083] In some embodiments, examples of commonly used labeling enzymes and chromogenic or fluorescent or chemiluminescent substrates are summarized in Table 1.

[0084] Table 1. Examples of labeling enzymes and substrates

[0085]

[0086]

[0087] Catalytic amplification. The analyte activates the catalyst, which then produces multiple copies of the reporter molecule.

[0088] Catalytic self-amplification. The analyte activates the catalyst, which leads to the production of reporter molecules. These not only generate the signal, but also activate the catalyst.

[0089] Analyte-induced modification of collective properties. Binding of a single analyte molecule to a receptor affects the properties of neighboring units through signal transduction.

[0090] Multivalent surfaces for binding multiple analyte molecules. Recruiting multiple reporter molecules using multivalent scaffolds such as polymers, dendrimers or nanoparticles amplifies the signal.

[0091] The above-mentioned catalysts include Pd(0)-catalyst, apyrase, potassium permanganate, platinum and the like.

[0092] Fig.12 A schematic diagram of an exemplary embodiment of a pixelated QMAX device in a closed configuration for a capture process is shown. In this method,

[0093] 1) Place 1 μL of sample containing a nucleic acid target with a concentration of 1aM to 1mm on the first plate

[0094] 2) Press the second plate on top of the liquid by hand.

[0095] 3) Take a picture of the wells on the first plate. Calculate the total sample volume by counting the wells loaded with sample.

[0096] 4) Incubate for 1 minute.

[0097] 5) Strip second plate / wash first plate 3 times with 5X SSC.

[0098] As used herein, "sample" can be any nucleic acid containing or not containing sample, including but not limited to human body fluids, such as whole blood, plasma, serum, urine, saliva and sweat, and cell cultures (mammals, plants, bacteria, fungi). Samples can be freshly obtained or stored or processed in any desired or convenient manner (e.g., by dilution or addition of buffer, or other solutions or solvents). Cellular structures can be present in the sample, for example, human cells, animal cells, plant cells, bacterial cells, fungal cells and viral particles.

[0099] The term "nucleic acid" as used herein refers to any DNA or RNA molecule, or a DNA / RNA hybrid, or a mixture of DNA and / or RNA. Therefore, the term "nucleic acid" includes but is not limited to genomic or chromosomal DNA, plasmid DNA, amplified DNA, cDNA, total RNA, mRNA and small RNA. The term "nucleic acid" is intended to include natural DNA and / or RNA molecules, or synthetic DNA and / or RNA molecules. In some embodiments, there is cell-free nucleic acid in the sample, as used herein, "cell-free" means that nucleic acid is not included in any cell structure. In some other embodiments, nucleic acid is included in the cell structure, which includes but is not limited to human cells, animal cells, plant cells, bacterial cells, fungal cells and / or viral particles. There can be nucleic acid in the form of cell-free nucleic acid or in the cell structure or in its combined form in the sample. In some further embodiments, nucleic acid is purified before being introduced into the inner surface of the first plate. In other embodiments, nucleic acid can be in a complex combined with other molecules (such as proteins and lipids).

[0100] The method of the present invention is applicable to samples of a certain volume range. Samples with different volumes can be introduced into plates with different sizes.

[0101] As used herein, the terms "nucleic acid" and "nucleotide" are intended to be consistent with their use in the art, and include naturally occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids can hybridize with nucleic acids in a sequence-specific manner or can be used as templates for replication of specific nucleotide sequences. Naturally occurring nucleic acids generally have a backbone containing a phosphodiester bond. Analog structures can have alternative backbone connections, including backbone connections of any kind known in the art. Naturally occurring nucleic acids generally have deoxyribose (e.g., found in deoxyribonucleic acid (DNA)) or ribose (e.g., found in ribonucleic acid (RNA)). Nucleic acids can contain nucleotides with any analogs of these sugar moieties known in the art. Nucleic acids can include natural or non-natural nucleotides. In this regard, natural deoxyribonucleic acids can have one or more bases selected from adenine, thymine, cytosine or guanine, and ribonucleic acids can have one or more bases selected from uracil, adenine, cytosine or guanine. Useful non-natural bases that can be included in nucleic acids or nucleotides are known in the art. When the term "probe" or "target" is used to refer to a nucleic acid, it is used as a semantic identifier of the nucleic acid in the context of the methods or compositions herein, and does not necessarily limit the structure or function of the nucleic acid beyond the scope of otherwise explicitly indicated. The terms "probe" and "target" can be similarly applied to other analytes, such as proteins, small molecules, cells, etc.

[0102] As used herein, the term "capture probe" refers to a nucleic acid that hybridizes to a nucleic acid having a complementary sequence.

[0103] The term "complementary" as used herein refers to a nucleotide sequence that base pairs with a target nucleic acid of interest through hydrogen bonds. In typical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), and guanine (G) forms a base pair with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). Therefore, A is complementary to T and G is complementary to C. Generally, "complementary" refers to a nucleotide sequence that is completely complementary to the target of interest, so that each nucleotide in the sequence is complementary to each nucleotide at the corresponding position in the target nucleic acid. When a nucleotide sequence is not completely complementary to a non-target sequence (100% complementary), but due to the complementarity of certain fragments of the nucleotide sequence to the non-target sequence, it is still possible to base pair with the non-target sequence, the complementary percentage can be calculated to assess the possibility of non-specific (off-target) binding. Generally, 50% or less complementarity does not result in non-specific binding. In addition, 70% or less complementary sequences do not result in non-specific binding under stringent hybridization conditions.

[0104] In some embodiments, hybridization agents promote hybridization between two complementary sequences of nucleic acids, including but not limited to sodium chloride, sodium acetate, polysucrose, dextran, polyvinyl pyrrolidone, bovine serum albumin, and the like.

[0105] In certain embodiments, the predetermined period of time is equal to or longer than the time required for the target nucleic acid to diffuse through the layer of uniform thickness into the sample.

[0106] In certain embodiments, the predetermined period of time is equal to or longer than the time required for target nucleic acid.

[0107] Fig.13 A schematic diagram of an exemplary embodiment of a pixelated assay QMAX device in a closed configuration for use in an amplification process is shown. In this method,

[0108] 1) Add 3 μL (excess) TMB amplification substrate to the first plate;

[0109] 2) Press the second amplification plate onto the top of the liquid by hand;

[0110] 3) Incubate for 1 minute. During this process, only well-captured targets are amplified and show signals (color or fluorescence);

[0111] 4) Take a picture of the wells on the first plate and count the number of wells using the signal

[0112] Fig.14 A schematic diagram of an exemplary embodiment of an open-architecture pixelated assay QMAX device for a digital nucleic acid amplification assay is shown.

[0113] In some embodiments, the dried reagent includes a cell lysis reagent, which includes but is not limited to salts, detergents, enzymes and other additives. The term "salt" herein includes but is not limited to lithium salts (e.g., lithium chloride), sodium salts (e.g., sodium chloride), potassium (e.g., potassium chloride). The term "detergent" herein can be ionic, including anionic and cationic, nonionic or zwitterionic. The term "ionic detergent" used herein includes any detergent that is partially or completely in ionic form when dissolved in water. Suitable anionic detergents include but are not limited to sodium dodecyl sulfate (SDS) or other alkali metal alkyl sulfates or similar detergents, sarkosyl or a combination thereof. The term "enzyme" herein includes but is not limited to lysozyme, cellulase and protease. In addition, chelating agents (including but not limited to EDTA, EGTA and other polyaminocarboxylic acids) and some reducing agents (e.g., dithiothreitol (dTT)) may also be included in the cell lysis reagent. The composition of the necessary reagents herein varies according to the rational design of different amplification reactions.

[0114] In some embodiments, "dry reagents" include PCR reagents, including but not limited to primers, deoxynucleotides (dNTPs), divalent cations (e.g., Mg2+), monovalent cations (e.g., K+), buffers, enzymes, and reporters. As used herein, in some embodiments, "primers" may refer to a pair of forward and reverse primers. In some embodiments, primers may refer to multiple primers or primer sets. As used herein, enzymes suitable for nucleic acid amplification include but are not limited to DNA-dependent polymerases, or RNA-dependent DNA polymerases, or DNA-dependent RNA polymerases.

[0115] As used herein, the term "reporter molecule" refers to any marker, label or dye that can bind to or intercalate into a nucleic acid molecule or be activated by a byproduct of an amplification process to visualize the nucleic acid molecule or the amplification process. Suitable reporter molecules include, but are not limited to, fluorescent markers or labels or dyes, intercalators, molecular beacon labels, or bioluminescent molecules, or combinations thereof.

[0116] In some embodiments, "dry reagents" include stabilizers, which include but are not limited to protein stabilizers, examples of which include but are not limited to polyols, sugars, amino acids, amines and salting-out salts; polymers and proteins, examples of which include but are not limited to PEG, polysaccharides, dextran, hydroxyethyl starch (HETA), PEG-4000 and gelatin; examples of surfactants include but are not limited to Tween 20, Tween 80, Triton X-100, Brij 35, Pluronic F127 and SDS; examples of amino acids include but are not limited to histidine, arginine and glycine; examples of preservatives include but are not limited to benzyl alcohol, m-cresol and phenol.

[0117] Fig.15A schematic diagram of an exemplary embodiment of a pixelated assay QMAX device in a closed configuration after sample introduction for a digital nucleic acid amplification assay is shown. In the method,

[0118] 1) Drop the sample containing the nucleic acid target on the first plate

[0119] 2) Press the second plate on top of the liquid by hand.

[0120] 3) Take a picture of the wells on the first plate. Calculate the total sample volume by counting the wells loaded with sample.

[0121] In some embodiments, a "sample" can be any nucleic acid containing or not containing sample, including but not limited to human body fluids, such as whole blood, plasma, serum, urine, saliva and sweat, and cell culture (mammals, plants, bacteria, fungi). The sample can be freshly obtained or stored or processed in any desired or convenient manner (e.g., by dilution or addition of buffer, or other solution or solvent). Cellular structures can be present in the sample, such as human cells, animal cells, plant cells, bacterial cells, fungal cells and viral particles.

[0122] The term "nucleic acid" as used herein refers to any DNA or RNA molecule, or a DNA / RNA hybrid, or a mixture of DNA and / or RNA. Therefore, the term "nucleic acid" is intended to include, but is not limited to, genomic or chromosomal DNA, plasmid DNA, amplified DNA, cDNA, total RNA, mRNA, miRNA and microRNA. The term "nucleic acid" also includes natural DNA and / or RNA molecules, or synthetic DNA and / or RNA molecules. In some embodiments, there is cell-free nucleic acid in the sample, as used herein, "cell-free" means that nucleic acid is not included in any cell structure. In some other embodiments, nucleic acid is included in the cell structure, which includes but is not limited to human cells, animal cells, plant cells, bacterial cells, fungal cells and / or viral particles. There may be nucleic acid in the form of cell-free nucleic acid or in the cell structure or in its combined form in the sample. In some further embodiments, nucleic acid is purified before being introduced into the inner surface of the first plate. In other embodiments, nucleic acid can be in a complex combined with other molecules such as protein and lipid.

[0123] The method of the present invention is applicable to samples of a certain volume range. Samples with different volumes can be introduced into plates with different sizes.

[0124] In some embodiments, after the sample is introduced, Fig.14 The dried reagent in is dissolved in the sample.

[0125] Fig.16A schematic diagram of an exemplary embodiment of a pixelated assay QMAX device in a closed configuration during a digital nucleic acid amplification process is shown.

[0126] As used herein, "amplicon" refers to various nucleic acids produced by nucleic acid amplification techniques. The types of nucleic acid amplification products herein include, but are not limited to, single-stranded DNA, single-stranded RNA, double-stranded DNA, linear DNA, or circular DNA, etc. In some embodiments, the nucleic acid amplification products can be identical nucleic acids of the same length and structure. In some other embodiments, the nucleic acid amplification products can be multiple nucleic acids of different lengths and structures.

[0127] As used herein, "nucleic acid amplification" includes any technique for detecting nucleic acids by amplifying (producing a large number of copies of) a target molecule in a sample, where "target" refers to a sequence or partial sequence of a nucleic acid of interest. Suitable nucleic acid amplification techniques include, but are not limited to, different polymerase chain reaction (PCR) methods, such as hot start PCR, nested PCR, touchdown PCR, reverse transcription PCR, RACEPCR, digital PCR, etc., and isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), strand displacement amplification, helicase-dependent amplification, nicking enzyme amplification, rolling circle amplification. Recombinase polymerase amplification, etc.

[0128] As used herein, the term "reporter molecule" refers to any marker, label or dye that can bind to or intercalate into a nucleic acid molecule or be activated by a byproduct of an amplification process to visualize the nucleic acid molecule or the amplification process. Suitable reporter molecules include, but are not limited to, fluorescent markers or labels or dyes, intercalators, molecular beacon labels, or bioluminescent molecules, or combinations thereof.

[0129] In some embodiments, a reporter molecule is used to quantify the accumulated nucleic acid after nucleic acid amplification.As defined and used above, a reporter molecule has a quantifiable characteristic that is related to the presence or absence or amount of nucleic acid amplicons accumulated in a closed chamber.

[0130] C. Another Example of a QMAX Device for Nucleic Acid Capture in Hybridization Assays

[0131] Figure 4 is a schematic diagram of an exemplary embodiment of a QMAX (Q: quantification; M: amplification; A: addition of reagents; X: acceleration); also known as a compression regulated open flow (CROF) device, which can be used, for example, to capture nucleic acids for hybridization assays. Figure 4 In the example, the QMAX device is an open structure.

[0132] DD1. A device for pixelating a fluid sample, comprising:

[0133] a first plate, a second plate and a microwell, wherein

[0134] (a) the first plate and the second plate are movable relative to each other into different configurations and have sample contact areas on their respective surfaces for contacting a fluid sample containing a target analyte;

[0135] (b) the second plate has a plurality of microwells in the sample contact area, wherein each microwell has (i) a pore depth of 200 μm or less, (ii) a pore volume significantly smaller than the sample volume, and (iii) a binding site comprising a capture agent immobilized at the site, and the capture agent is configured to capture a target analyte;

[0136] wherein one of the configurations is an open configuration, wherein: the average spacing between the inner surface of the first plate and the edge of the microwells in the second plate is at least 250 μm, and the sample is deposited on one or both plates;

[0137] Another of the structures is a closed structure, which is the structure after the sample is deposited in an open structure; in the closed structure, at least a portion of the sample is within the microwell, and the average spacing between the inner surface of the first plate and the edge of the microwell in the second plate is less than 1 / 10 (one tenth) of the depth of the microwell.

[0138] Figure 4 (b) shows a top view of the microwells on the second plate, which has (i) circles in a square array, (ii) rectangles in a square array, (iii) triangles in a hexagonal array, and (iv) non-spaced circles.

[0139] Figure 6 Microscope embodiments of isolated hole arrays on a QMAX first plate fabricated on a 0.25 mm thick acrylic substrate are shown, with (a) 20 μm × 20 μm square holes, 100 μm spacing, 30 μm depth; (b) 20 μm × 20 μm square holes, 200 μm spacing, 30 μm depth; (c) 10 μm circular hole diameter, 200 μm spacing, 20 μm depth.

[0140] DD2 A kit for pixelation assay, comprising:

[0141] The device of embodiment DD1, and

[0142] Imager for imaging the sample contact area.

[0143] DD3 A kit for pixelation assay, comprising:

[0144] Implement the device in DD1,

[0145] Reagents to be added to the QMX card with microwells, and

[0146] Imager for imaging the sample contact area.

[0147] The kit according to any of the preceding embodiments, wherein the reagent is a wash solution.

[0148] The kit as described in any of the preceding embodiments, wherein the reagent is a detection agent.

[0149] The kit according to any of the preceding embodiments, wherein the reagent is an enzyme solution capable of generating light in a substrate.

[0150] M1. A method for pixelating a fluid sample, comprising:

[0151] iii. Get the first board,

[0152] iv. Obtain the second board,

[0153] in

[0154] (a) the first plate and the second plate are movable relative to each other into different configurations and have sample contact areas on their respective surfaces for contacting a fluid sample containing a target analyte;

[0155] (b) the second plate has a plurality of microwells in the sample contact area, wherein each microwell has (i) a pore depth of 200 μm or less, (ii) a pore volume significantly smaller than the sample volume, and (iii) a binding site comprising a capture agent immobilized at the site, and the capture agent is configured to capture a target analyte;

[0156] iii. depositing the sample on one or two plates; and

[0157] v. Make the board into a closed configuration;

[0158] wherein one of the configurations is an open configuration, wherein: the average spacing between the inner surface of the first plate and the edge of the microwells in the second plate is at least 250 μm, and the sample is deposited on one or both plates;

[0159] Another of the structures is a closed structure, which is the structure after the sample is deposited in an open structure; in the closed structure, at least a portion of the sample is within the microwell, and the average spacing between the inner surface of the first plate and the edge of the microwell in the second plate is less than 1 / 10 (one tenth) of the depth of the microwell.

[0160] In the method of embodiment M1, the method further comprises, after step (iv), a step of partially or completely separating the two plates, washing the original sample or adding another reagent, and then bringing the plates into a closed configuration.

[0161] In the method of any preceding embodiment, wherein the method further comprises the step of imaging the sample contact area.

[0162] In the device or method of any preceding paragraph (also referred to as "paragraph"), wherein imaging the sample contact area measures a primary sum signal associated with an analyte from the sample contact area.

[0163] In the device or method of any preceding paragraph (also referred to as "paragraph"), wherein imaging the sample contact area measures individual signals resulting from individual binding events between the capture agent and the captured target analyte.

[0164] In the device or method of any preceding paragraph (also referred to as "paragraph"), wherein the sample contact area is imaged to measure (a) a primary sum signal associated with analytes from the sample contact area and (b) individual signals caused by individual binding events between the capture agent and the captured target analytes.

[0165] In the device or method of any preceding paragraph (also referred to as "paragraph"), wherein the presence or concentration of the target analyte in the sample is determined by detecting individual signals resulting from individual binding events between the capture agent and the captured target analyte.

[0166] In the device or method of any preceding paragraph, wherein for an expected target analyte concentration, the volume of each well is configured so that the distribution of the target analyte in (loaded into) each well follows a Poisson distribution.

[0167] In the device or method of any preceding paragraph, wherein for an expected target analyte concentration, the volume of each well is constructed so that the distribution of the target analyte in each well (loading well) is, on average, one target analyte per 2 wells, 3 wells, 5 wells, 10 wells, 20 wells, 0 wells, 50 wells, 75 wells, 100 wells, 150 wells, 200 wells, 300 wells, 500 wells, 1000 wells, 2000 wells, 10000 wells, 100,000 wells, or within a range of any two values.

[0168] In the device or method of any preceding paragraph, wherein in a closed configuration, the average spacing between the inner surface of the first plate and the edge of the microwells in the second plate is less than 1 / 11 (one eleventh), 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 100, 1 / 300, 1 / 500, or within a range of any two values, of the depth of the microwells.

[0169] In the device or method of any preceding paragraph, wherein in the closed configuration, the inner surface of the first plate and the average spacing between the edges of the microwells in the second plate are in substantial contact.

[0170] In the device or method of any preceding paragraph, wherein in a closed configuration, the average spacing between two adjacent holes is less than 5 nm, 10 nm, 30 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, or within a range of any two values.

[0171] A device as in the preceding paragraph, wherein the first plate has an array of holes in the shape of a sphere, rectangle, hexagon and / or any other polyhedron, having a grid of square, hexagon and / or any other grid.

[0172] The manufacturing method of the hole array on the first plate includes, but is not limited to, nanoimprint lithography, photolithography, interference lithography, electron beam lithography, and the like.

[0173] In some embodiments, the holes on the first plate have a spacing (average hole to hole center distance) of 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, 1 mm, or a range between any two values; and a preferred range (spacing) of 10 nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 μm, 1 μm to 10 μm, or 10 μm to 50 μm.

[0174] In some embodiments, the holes on the first plate have a pore size (average length or diameter) of 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, 1 mm, or a range between any two values; and a preferred range of 10 nm-100 nm, 100 nm-500 nm, 500 nm-1 μm, 1 μm-10 μm or 10 μm-50 μm (size).

[0175] In some embodiments, the depth of the holes on the first plate is 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, 1 mm, or a range between any two values; and a preferred range of 10 nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 μm, 1 μm to 10 μm, or 10 μm to 50 μm (depth).

[0176] In some embodiments, the holes have (i) no metal coating (ii) a metal coating on the bottom of the hole (top of the post) (iii) a metal coating on the sidewalls of the hole (sides of the post) (iv) a metal coating on both the bottom and sidewalls of the hole.

[0177] In some embodiments, the coating metal is gold, aluminum, silver, copper, tin, and / or combinations thereof.

[0178] In some embodiments, the pore area ratio (ratio of pore area to total surface area) is 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 99%.

[0179] In some embodiments, the pore edge to pore edge distance is greater than the pore depth, which ensures that the diffusion time from pore edge to pore edge is longer than the diffusion time from pore edge to pore bottom.

[0180] In some embodiments, the dimensions of the wells are designed to ensure that no cross-reactions occur during the assay.

[0181] In some embodiments, the number of wells on the first plate is much greater than the number of molecules in the sample.

[0182] For example, the total number of wells on the first plate is 1-2 times, 2-5 times, 5-10 times, 10-100 times, 100-1000 times, 1000-10000 times of 600, and if the molecular concentration is 1 fM, the volume is 1 μL;

[0183] For example, the total number of wells on the first plate is 1-2 times, 2-5 times, 5-10 times, 10-100 times, 100-1000 times, 1000-10000 times of 600,000 if the concentration of the molecule is 1 pM in a 1 μL volume;

[0184] For example, the total number of wells on the first plate is 1-2 times, 2-5 times, 5-10 times, 10-100 times, 100-1000 times, 1000-10000 times of 600,000,000, if the concentration of the molecule is 1 nM in 1 μL volume;

[0185] In some embodiments, after the nucleic acid capture step, the number of wells is achieved in such a way that the majority of wells capture no more than one target molecule.

[0186] For example, for an orifice of 100 μm, 4 cm 2 The total number of wells on the first plate of size 40000 is 40000. If such a plate is used to measure a 1 fM molecule sample in a 1 μL sample with 600 target molecules, statistically each well will have no more than one molecule.

[0187] In some embodiments, the second plate is an X-plate.

[0188] In some embodiments, the first plate can be any material having a flat or engineered solid surface. Examples of the first plate include, but are not limited to, plastic, silicon, PMMA, gold, and glass. In some embodiments, the second plate can be any material having a flat or engineered solid surface. Examples of the first plate include, but are not limited to, plastic, silicon, PMMA, gold, and glass.

[0189] In some embodiments, the first plate is made of semiconductors including carbon, germanium, selenium, silicon, gallium arsenide (GaAs), gallium nitride (GaN), indium phosphide (InP), zinc selenide (ZnSe) and silicon carbide (SiC); metals include gold, aluminum, silver, copper, tin and / or combinations thereof.

[0190] like Figure 4 As shown, in some embodiments, the surface of the first plate facing the second plate is defined as the inner surface of the first plate; the surface of the second plate facing the first plate is also defined as the inner surface of the second plate. In some embodiments, the inner surface of each plate includes a sample contact area for contacting a sample containing nucleic acid. The sample contact area may occupy part or all of the corresponding inner surface. Figure 4 As shown, the second plate may include a spacer fixed on the inner surface of the second plate. However, it should be noted that in some embodiments, the spacer is fixed on the inner surface of the first plate, while in other embodiments, the spacer is fixed on the inner surfaces of the second plate and the first plate.

[0191] The sample can be any liquid that needs to be tested. In some embodiments, the sample is a body fluid with or without treatment or dilution. For example, the body fluid can be whole blood, plasma, serum, urine, saliva, sweat or breath condensate. In some embodiments, the sample is blood. In certain embodiments, the sample comprises plasma. In certain embodiments, the sample comprises whole blood. In certain embodiments, the sample is blood or plasma that has been diluted with buffer 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000 or 1,000,000 times or in the range of any one between these two values. In some embodiments, the sample contains an analyte, which can be any cell or molecule that can be detected and quantified.

[0192] The term "sample" as used herein refers to a material or material mixture containing one or more analytes 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.), breast milk, cerebrospinal fluid (CSF), cerumen (ear wax), chyle, chyme, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatism, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomitus, urine and exhaled condensate. In a specific embodiment, the sample can be obtained from a subject (e.g., a human), and it can be processed before being used for subject analysis. For example, before analysis, protein / nucleic acid can be extracted from a tissue sample before use, and its method is known. In a specific embodiment, the sample can be a clinical sample, for example, a sample collected from a patient.

[0193] The label is a luminescent label or an optically detectable label, directly or indirectly, before or after it is combined with a capture agent. The label is a label with a Raman scattering, chromaticity, luminescence, fluorescence, electroluminescence, chemiluminescence and / or electrochemiluminescence signal. As used herein, the term "luminescent label" refers to a label that can emit light under external excitation. This can be luminescence. Fluorescent labels (including dye molecules or quantum dots) and luminescent labels (e.g., electroluminescence or chemiluminescent labels) are types of luminescent labels. External excitation is light (photons) for fluorescence, current for electroluminescence, and chemical reactions for chemiluminescence. External excitation can be a combination of the above. The phrase "labeled analyte" refers to an analyte detectably labeled with a luminescent label so that the analyte can be detected by evaluating the presence of the label. The labeled analyte can be directly labeled (i.e., the analyte itself can be directly conjugated to the label, for example, via a strong bond, e.g., a covalent bond or a non-covalent bond), or the labeled analyte can be indirectly labeled (i.e., the analyte is bound by a directly labeled second capture agent).

[0194] In some embodiments, the signal amplification layer is completely or partially present on the bottom or sidewall of the hole or both. When the target analyte or labeling from the amplification layer is 1nm, 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 200nm, 300nm, 400nm, 500nm, 1 μm, 2 μm, 5 μm, 10 μm or the range between any two values; And 0nm to 50nm, 50nm to 100nm, 100nm to 200nm, 200nm to 500nm preferred range, the amplification layer amplifies the signal from the target analyte or the labeling of the target analyte.

[0195] The term "amplification" refers to an increase in the amplitude of a signal, for example, an increase of at least 10-fold, an increase of at least 100-fold, an increase of at least 1,000-fold, an increase of at least 10,000-fold, or an increase of at least 100,000-fold.

[0196] In some embodiments, the inductive amplification layer includes, but is not limited to, U.S. Provisional Patent Application No. 61 / 347,178 filed on May 21, 2010, U.S. Provisional Patent Application No. 61 / 622,226 filed on April 10, 2012, U.S. Provisional Patent Application No. 61 / 708,314 filed on October 1, 2012, U.S. Provisional Patent Application No. 61 / 800,915 filed on March 15, 2013, U.S. Provisional Patent Application No. 61 / 801,933 filed on March 15, 2013, U.S. Provisional Patent Application No. 61 / 802,934 filed on March 15, 2013, Provisional Patent Application No. 61 / 801,096, U.S. Provisional Patent Application No. 61 / 801,424 filed on March 15, 2013, U.S. Provisional Patent Application No. 61 / 794,317 filed on March 15, 2013, U.S. Provisional Patent Application No. 62 / 090,299 filed on December 10, 2014, U.S. Provisional Patent Application No. 62 / 066,777 filed on October 21, 2014, U.S. Provisional Patent Application No. 62 / 234,538 filed on September 29, 2015, U.S. Provisional Patent Application No. 62 / 234,538 filed on June 13, 2015 Utility Patent Application No. 13 / 699,270, U.S. Utility Patent Application No. 13 / 838,600 filed on March 15, 2013, U.S. Utility Patent Application No. 14 / 459,239 filed on August 13, 2014, U.S. Utility Patent Application No. 14 / 459,251 filed on August 13, 2014, U.S. Utility Patent Application No. 14 / 852,412 filed on March 16, 2014, U.S. Utility Patent Application No. 14 / 871,678 filed on September 30, 2015, U.S. Utility Patent Application No. 14 / 431,266 filed on October 5, 2015, U.S. Utility Patent Application No. 14 / 668,750 filed on March 25, 2015, U.S. Utility Patent Application No. 14 / 775,634 filed on September 11, 2015, U.S. Utility Patent Application No. 14 / 775,638 filed on September 11, 2015, U.S. Utility Patent Application No. 14 / 852,417 filed on September 11, 2015, U.S. Utility Patent Application No. 14 / 964 filed on December 9, 2015,394, PCT Application No. (designating the United States) PCT / US2011 / 037455 filed on May 20, 2011, PCT Application No. (designating the United States) PCT / US2013 / 032347 filed on March 15, 2013, PCT Application No. (designating the United States) PCT / US2013 / 062923 filed on October 1, 2013, PCT Application No. (designating the United States) PCT / US2014 / 030108 filed on March 16, 2014, PCT Application No. (designating the United States) PCT / US2014 / 030108 filed on March 14, 2014 / 029675, PCT Application No. (designated US) PCT / US2014 / 028417 filed on March 14, 2014, PCT Application No. (designated US) PCT / US2014 / 029979 filed on March 15, 2014, PCT Application No. (designated US) PCT / US2015 / 056518 filed on October 20, 2015, PCT Application No. (designated US) PCT / US2016 / 054025 filed on September 27, 2016, the entire disclosures of which are incorporated herein by reference for all purposes. ,

[0197] The first plate and the second plate can be moved relative to each other into different configurations. One of the configurations is an open configuration, in which the two plates are partially or completely separated and the spacing between the plates is not limited by a spacer. Figure 4 Shown are plates of open construction, wherein samples can be added to the first plate, the second plate, or both plates. In some embodiments, the inner surface of the respective plates includes a sample contact area that occupies a portion of the entire inner surface. In certain embodiments, a spacer is positioned within the sample contact area. In some embodiments, the spacer is not fixed to any one plate, but is mixed in the sample.

[0198] Another example method for pixelation measurement using a QMAX device

[0199] Figure 5 is an exemplary flow chart showing basic steps in an exemplary process for pixelation measurement using a QMAX device.

[0200] Figure 5 An exemplary flow chart of the process in the "Assay" section is provided. However, it should be noted that the device of the present invention can be used in a variety of assays, including but not limited to measuring the immunoassays herein. For example, although Figure 2 A process is shown for detecting an analyte using an antibody, but the antibody or antibody-expressing cells may be detected and / or quantified using a process and device involving an antigen.

[0201] like Figure 5As shown, in some embodiments, the pixelation assay process includes: (1) depositing the sample on a microplate ( Figure 4 (2) using an X-plate ( Figure 1 (a) covering the first plate with the second plate shown and pressing the two plates together; (3) counting the number of wells to which samples have been added; (4) calculating the sample volume by multiplying the number of wells by the well volume; (5) incubating and capturing the analyte in the isolated wells; (6) amplifying the signal in the isolated wells; (7) counting the wells using the signal; and (8) calculating the concentration of the analyte in the sample.

[0202] In some embodiments, the method of the present invention further comprises incubating the uniform thickness layer for a predetermined period of time before step (5) and after step (4). In certain embodiments, the predetermined period of time is equal to or longer than the time required for the target molecule to diffuse through the uniform thickness layer into the sample.

[0203] In certain embodiments, the predetermined period of time is equal to or longer than the time required for a target molecule to diffuse through a layer of uniform thickness into the sample and be captured by a capture probe.

[0204] In certain embodiments, the predetermined time period is less than 10 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, or 60 minutes, or is within a range between any two values.

[0205] In some embodiments, for the method of the present invention, the sample is deposited on the first plate. In certain embodiments, before the step (5) after step (4), before the step (6) after step (5), the sample is incubated on the first plate for a predetermined time period. In certain embodiments, the predetermined time period is equal to or longer than the time required for the capture antibody and the analyte to reach equilibrium. In certain embodiments, the predetermined time period is less than 10 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes or 60 minutes. Or in the range between any one of the two values.

[0206] In some embodiments, for the method of the present invention, after step (5) and after step (6), the inner surface of the first plate can be washed to remove unbound molecules. For the method, wash before the plate is switched to a closed configuration. In some embodiments, for the method of the present invention, before step (6) and after step (5), before step (7) and after step (6), the plate can be switched to an open configuration (for example, by removing the second plate) and the inner surface of the first plate can be washed. For the method, wash before the plate is switched to a closed configuration. In certain embodiments, such steps reduce nonspecific binding and reduce signal noise. In certain embodiments, each washing step only includes one or more washings. In some embodiments, two washing steps are carried out. In some embodiments, only one washing step is carried out.

[0207] In some embodiments, the inner surface can be washed with a washing solution absorbed in a sponge. In some embodiments, washing is performed by squeezing the sponge to release the washing solution onto the inner surface of the first plate and releasing the sponge to reabsorb the washing solution. In some embodiments, washing improves the limit of detection (LOD) of the detectable signal.

[0208] The amplification method in the (6) amplification step includes but is not limited to:

[0209] A colorimetric enzymatic reaction in which the absorbance signal generated by the substrate is amplified by an enzyme connected to a detection agent; wherein the enzyme comprises horseradish peroxidase; wherein the substrate comprises ABTS or TMB;

[0210] A fluorescence-based enzymatic reaction, wherein the fluorescence signal generated by the substrate is amplified by an enzyme connected to a detection agent; wherein the enzyme comprises horseradish peroxidase; wherein the substrate comprises Amplex red;

[0211] Catalytic amplification. The analyte activates the catalyst, which then produces multiple copies of the reporter molecule.

[0212] Catalytic self-amplification. The analyte activates the catalyst, which leads to the production of reporter molecules. These not only generate the signal, but also activate the catalyst.

[0213] Analyte-induced modification of collective properties. Binding of a single analyte molecule to a receptor affects the properties of neighboring units through signal transduction.

[0214] Multivalent surfaces for binding multiple analyte molecules. Recruiting multiple reporter molecules using multivalent scaffolds such as polymers, dendrimers or nanoparticles amplifies the signal.

[0215] The above-mentioned catalysts include Pd(0)-catalyst, apyrase, potassium permanganate, platinum and the like.

[0216] In certain embodiments, an amplification substrate is added before step (6), and the amplification substrate includes but is not limited to ABTS and TMB.

[0217] In certain embodiments, before the step (7) after step (6), the sample is incubated on the first plate for a predetermined time period. In certain embodiments, the predetermined time period is equal to or longer than the time required for the amplification process. In certain embodiments, the predetermined time period is equal to or longer than the time required for the hole to have a readable signal. In certain embodiments, the predetermined time period is less than 10 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes or 60 minutes, or in the range between any one of the two values.

[0218] Step (3) counting the number of wells loaded with samples; and (7) counting the wells using the signals, various types of "detection methods" include but are not limited to using a fluorescent microscope, a DSLR (digital single-lens reflex camera) and a smart phone.

[0219] In step (4), the volume of the sample is calculated, all the wells are observed and counted, or some of the wells are observed and counted. The total volume of the sample in QMAX is estimated from the product of the number of counts and the well volume.

[0220] Since the number of wells is much larger than the total number of molecules in the sample, statistically each well has no more than one molecule. The total number of molecules in the sample is estimated by counting the number of wells with signals after the amplification step. The final concentration of the analyte in the sample is calculated by dividing the number of molecules by the sample volume.

[0221] Other embodiments of the present invention

[0222] Figure 4 Schematic diagram showing the preparation of a binding site plate (first plate) and a storage plate (second plate) for an exemplary embodiment of the pixelated assay QMAX. The experimental procedure follows Figure 5 Flowchart of the process.

[0223] Specifically, the first plate in this embodiment is a square hole array with a size of 20μm×20μm, a spacing of 100μm, and a depth of 30μm manufactured on a 0.25mm thick acrylic substrate. The first plate was coated with 10μg / mL protein-A in PBS for 2 hours and then washed three times with PBST. The first plate was then coated with 10μg / mL anti-human IgG capture antibody (goat anti-human IgG) dissolved in PBS for 2 hours and then closed with 4% BSA dissolved in PBS for 2 hours. The first plate was then incubated with 100μL protein stabilizer for 2 hours. Excess liquid was removed and the plate was dried at room temperature.

[0224] The second plate in this example is a 0.175 mm thick flat acrylic film. Detection Ab (mouse anti-human IgG) conjugated HRP 10 μg / mL 200 μL was evenly printed and dried thereon at 37°C for 2 hours.

[0225] like Figure 7 As shown, in some embodiments, the first plate comprises a capture antibody that is fully or partially coated on the inner surface of the first plate. In some embodiments, the capture antibody is fully or partially located on the bottom or sidewalls or both of the wells on the first plate.

[0226] In some embodiments, the capture antibody can be applied to the surface by printing, spraying, soaking or any other method of applying a uniform or partial reagent layer. In certain embodiments, the capture antibody is dried on the first plate. It should also be noted that in some embodiments, the capture antibody is coated on the inner surface of the first plate, rather than on the inner surface of the second plate; in some embodiments, the capture antibody is coated on the inner surface of the second plate instead of the first plate; in some embodiments, the capture antibody is coated on the inner surface of both plates. In some embodiments, the capture antibody is a monoclonal antibody, a polyclonal antibody, an engineered antibody (e.g., a single-chain variable fragment (scFv)) or a fragment thereof. In some embodiments, the concentration of the coated capture antibody is 1fg / mL-1g / mL.

[0227] In some embodiments, the capture antibody is constructed to bind to the analyte. For example, when the analyte comprises an antigenic epitope, in certain embodiments, the capture antibody is constructed to specifically bind to the antigenic epitope. In some embodiments, the capture antibody is (a) covalently bound to the surface, or (b) attached to the surface by passive absorption via hydrophobic interactions between the solid surface and non-polar residues on the protein. For example, in Figure 4 In some embodiments shown, the capture antibody is attached to the first plate 10 via Protein A. In certain embodiments, the capture antibody can immobilize the analyte 95 to the inner surface of the first plate.

[0228] Although antibodies can be used to detect antigens, antigens can also be used to detect antibodies. For example, in some embodiments of the present invention, a capture antigen (or epitope) instead of a capture antibody can be coated on the inner surface of each plate (e.g., the first plate). The capture antigen can be attached to the inner surface and used to fix the analyte (e.g., antibody or antibody-expressing cell) on the inner surface.

[0229] like Figure 7As shown, in some embodiments, the first plate includes a blocking agent coated on the inner surface of the first plate. In some embodiments, the blocking agent blocks any unoccupied sites on the solid surface that may cause unwanted nonspecific binding in the assay. In certain embodiments, the blocking agent reduces nonspecific binding. In certain embodiments, the blocking agent can be applied to the surface by printing, spraying, soaking or any other method of applying a uniform reagent layer. In certain embodiments, the blocking agent is dried on the first plate. It should also be noted that in some embodiments, the blocking agent is coated on the inner surface of the first plate, but not on the inner surface of the second plate; in some embodiments, the blocking agent is coated on the inner surface of the second plate instead of the first plate; in some embodiments, the blocking agent is coated on the inner surface of both plates. In some embodiments, the blocking agent is bovine serum albumin (BSA), casein, or total protein from whole milk, etc.

[0230] like Figure 7 As shown, in some embodiments, the first plate includes a stabilizer coated on the inner surface of the first plate. In some embodiments, the stabilizer helps maintain the proper folding of the protein when dry so that the function of the protein is not destroyed during storage. In some embodiments, the stabilizer extends the service life of the reagent, such as, but not limited to, the protein. In some embodiments, the stabilizer can be applied to the surface by printing, spraying, soaking, or any other method of applying a uniform reagent layer. In some embodiments, the stabilizer is dried on the first plate. It should also be noted that in some embodiments, the stabilizer is coated on the inner surface of the first plate, but not on the inner surface of the second plate; in some embodiments, the stabilizer is coated on the inner surface of the second plate, but not on the first plate; in some embodiments, the stabilizer is coated on the inner surfaces of both plates. In some embodiments, the stabilizer is a sugar, such as, but not limited to, sucrose and glucose. In some embodiments, the stabilizer is a polymer. In some embodiments, the stabilizer is glycerol.

[0231] like Figure 7As shown, in some embodiments, the second plate comprises a detection antibody coated on the inner surface of the second plate. In some embodiments, the detection antibody can be applied to the surface by printing, spraying, soaking or any other method of applying a uniform reagent layer. In certain embodiments, the detection antibody is dried on the second plate. It should also be noted that in some embodiments, the detection antibody is coated on the inner surface of the second plate instead of the first plate; in some embodiments, the detection antibody is coated on the inner surface of the first plate instead of the inner surface of the second plate; in some embodiments, the detection antibody is coated on the inner surface of both plates. In some embodiments, the detection antibody is a monoclonal antibody, a polyclonal antibody, an engineered antibody (e.g., a single-chain variable fragment (scFv)) or a fragment thereof. In some embodiments, the concentration of the coated detection antibody is 1fg / mL-1g / mL.

[0232] In some embodiments, the detection antibody is constructed to bind to the analyte. For example, when the analyte comprises an antigenic epitope, in certain embodiments, the detection antibody is constructed to specifically bind to the antigenic epitope. In certain embodiments, the capture antibody and the detection antibody bind to different sites (e.g., epitopes) of the analyte. In certain embodiments, the detection antibody is constructed to specifically bind to the capture antibody-analyte complex. In certain embodiments, the detection antibody is not covalently bound to the inner surface. In certain embodiments, the detection antibody is not attached to the surface by passive absorption via hydrophobic interactions between non-polar residues on the solid surface and the protein. In certain embodiments, the detection antibody 160 can diffuse into the sample after the sample is deposited and the detection antibody contacts the sample liquid.

[0233] In some embodiments, the detection antibody is constructed to produce a detectable signal after being bound to the analyte. For example, in some embodiments, the signal can be a colorimetric signal, a luminescent signal, or a fluorescent signal. For example, in some embodiments, the detection antibody is labeled with a fluorescent marker 165, which produces a signal after the detection antibody 1 is bound to the analyte or the capture antibody-analyte complex. In some embodiments, the fluorescent marker directly labels the detection antibody. In some embodiments, the fluorescent marker 165 labels can bind to the reagent of the detection antibody 160 or the detection antibody-analyte complex. In some embodiments, the second antibody can be conjugated with an optically detectable label, for example, a fluorophore, such as, but not limited to cy5, IR800, SAPEIRDye800CW, Alexa 790, Dylight 800. In some embodiments, the marker on the capture antibody or the detection antibody or the analyte is a nucleic acid. The presence and concentration of quantitative nucleic acids are amplified by real-time PCR.

[0234] In some embodiments, the detection antibody is constructed as a chemical substance capable of amplifying a signal or the signal from the chemical substance can be amplified; wherein the amplification method in the amplification step includes but is not limited to:

[0235] A colorimetric enzymatic reaction in which the absorbance signal generated by the substrate is amplified by an enzyme connected to a detection agent; wherein the enzyme comprises horseradish peroxidase; wherein the substrate comprises ABTS or TMB;

[0236] A fluorescence-based enzymatic reaction, wherein the fluorescence signal generated by the substrate is amplified by an enzyme connected to a detection agent; wherein the enzyme comprises horseradish peroxidase; wherein the substrate comprises Amplex red;

[0237] Catalytic amplification: The analyte activates the catalyst, which then produces multiple copies of the reporter molecule.

[0238] Catalytic self-amplification: The analyte activates the catalyst, which leads to the production of reporter molecules. These not only generate the signal but also activate the catalyst.

[0239] Analyte-induced modification of collective properties. Binding of a single analyte molecule to a receptor affects the properties of neighboring units through signal transduction.

[0240] Multivalent surfaces for binding multiple analyte molecules. Recruiting multiple reporter molecules using multivalent scaffolds such as polymers, dendrimers or nanoparticles amplifies the signal.

[0241] The above-mentioned catalysts include Pd(0)-catalyst, apyrase, potassium permanganate, platinum and the like.

[0242] Although antibodies can be used to detect antigens, antigens can also be used to detect antibodies. For example, in some embodiments of the present invention, a detection antigen (or epitope) instead of a detection antibody can be coated on the inner surface of each plate (e.g., a second plate). Capture antigens can be attached to the inner surface and used to detect analytes (e.g., antibodies or antibody-expressing cells) on the inner surface.

[0243] like Figure 7As shown, in some embodiments, the second plate comprises a stabilizer that stabilizes the protein (e.g., detection antibody) and extends the shelf life of the device. In some embodiments, the stabilizer helps maintain the proper folding of the protein when dry so that the function of the protein is not destroyed during storage. In some embodiments, the stabilizer extends the service life of the reagent, such as, but not limited to, the protein. In some embodiments, the stabilizer can be applied to the surface by printing, spraying, soaking, or any other method of applying a uniform reagent layer. In some embodiments, the stabilizer is dried on the first plate. It should also be noted that in some embodiments, the stabilizer 155 is applied to the inner surface of the first plate instead of the second plate; in some embodiments, the stabilizer is applied to the inner surface of the second plate instead of the first plate; in some embodiments, the stabilizer is applied to the inner surface of both plates. In some embodiments, the stabilizer is a sugar, such as, but not limited to sucrose and glucose. In some embodiments, the stabilizer is a polymer. In some embodiments, the stabilizer is glycerol. In some embodiments, the stabilizer coated on the first plate and the stabilizer coated on the second plate are the same. In some embodiments, the stabilizer coated on the first plate and the stabilizer coated on the second plate are different.

[0244] Figure 8 A schematic diagram of an exemplary embodiment of a pixelated QMAX device in a closed configuration for a capture process is shown. In this method,

[0245] 1) Place 1 μL of antigen (human IgG in PBS) at a concentration of 1 ng / mL to 1 fg / mL on the first plate.

[0246] 2) Press the second plate on top of the liquid by hand.

[0247] 3) Take a picture of the wells on the first plate. Calculate the total sample volume by counting the wells loaded with sample.

[0248] 4) Incubate for 1 minute.

[0249] 5) Strip second plate / wash first plate in PBST for 1 minute, then in water for 1 minute.

[0250] Fig. 9 A schematic diagram of an exemplary embodiment of a pixelated assay QMAX device in a closed configuration for use in an amplification process is shown. In this method,

[0251] 5) Add 3 μL (excess) TMB amplification substrate to the first plate;

[0252] 6) Press the second amplification plate onto the top of the liquid by hand;

[0253] 7) Incubate for 1 minute. During this process, only well-captured antigens are amplified and show signals (color or fluorescence);

[0254] 8) Strip the amplification second plate / wash the first plate in PBST for 1 minute, then wash with water for 1 minute.

[0255] Other Examples of QMAX Pixelation Measurement Results

[0256] Fig.10 Representative measurements of a pixelated assay with separate wells. (a) The sample volume is estimated by counting the wells loaded with sample in the capture step. (b) The number of molecules in the sample is estimated by counting the number of wells with signal in the amplification step. The final concentration of the analyte in the sample is the number of molecules in the sample volume.

[0257] By counting the number of wells with the amplified signal, the QMAX pixelated assay has molecular-level sensitivity, considering that statistically there is no more than one molecule per well.

[0258] As these examples demonstrate, in some embodiments, the present invention provides a platform for assays that is fast, simple, portable, and requires only as little as 1 μL or less sample, with sensitivity to molecular level concentrations. Using the present invention, assays can be performed in shallow closed spaces with specified parameters, so that sample volume and capture time can be precisely controlled. In some embodiments, the Brownian motion of molecules is confined to a shallow space, so that equilibrium of molecular binding can be reached more quickly. The platform is applicable to any assay performed in a conventional microtiter plate, and therefore has a wide range of applications.

[0259] Example of pixelated detection of homogeneous analytes

[0260] In the device or method of any preceding embodiment, the well generates a signal when the analyte in the sample contacts and reacts with the chemicals and reagents stored in the well. No washing steps are performed in the method.

[0261] In some embodiments, the signal can be a colorimetric signal, a luminescent signal, a fluorescent signal, an absorbance signal, or a micro / nano pattern change.

[0262] In some embodiments, the signal is generated using a chemical reaction, for example, horseradish peroxidase reacts directly with 3,5,3',5'-tetramethylbenzidine (TMB).

[0263] In some embodiments, the signal is generated by a chain chemical reaction, for example, alcohol reacts with alcohol oxidase to produce hydrogen peroxide, which is then reacted with horseradish peroxidase and Amplex Red.

[0264] In some embodiments, after the signal is generated, the signal is further amplified uniformly. For example, the initial signal is generated by nucleic acid. A subsequent polymerase chain reaction (PCR) is performed to generate and amplify the signal in each well.

[0265] In some embodiments, the uniform pixelation assay process comprises: (1) depositing a sample on a microplate ( Figure 4 (2) using an X-plate ( Figure 1 (a) covering the first plate with the second plate shown and pressing the two plates together; (3) counting the number of wells to which samples are added; (4) calculating the sample volume by multiplying the number of wells by the well volume; (5) incubating the analyte in the separate wells and generating signals in the separate wells; (7) counting the signal wells; and (8) back-calculating the concentration of the analyte in the sample.

[0266] In some embodiments, the reagents are dried and evenly coated on the bottom of the microwells.

[0267] In some embodiments, the reagent is in liquid form and is sealed with a membrane on the bottom of the microwell.

[0268] In some embodiments, the reagent is dried and evenly coated on the sidewalls of the microwells.

[0269] In some embodiments, the reagents are dried and evenly spread on other plates that do not have microwells.

[0270] Some examples of colorimetric assays used in the system are given below:

[0271] 1. Glucose colorimetric (fluorescence) determination was performed using glucose oxidase 100 units / ml, horseradish peroxidase 100 units / ml, 4-aminoantipyrine 20 mm and TOOS 20 mm.

[0272] 3,5,3',5'-Tetramethylbenzidine (TMB) 20 mm, Amplex Red 20 mm, Hexokinase 1 unit / ml, ATP 220 g / ml, NAD 400 g / ml.

[0273] 2. Arsenazo III 17μg / ml calcium colorimetric method.

[0274] 3.Bromcresol purple 22μg / ml albumin colorimetric assay.

[0275] 4. Perform colorimetric determination of total protein using 1.34 mg / ml copper sulfate, 3.43 mg / ml sodium potassium tartrate, and 0.28 mg / ml potassium iodide.

[0276] 5. Perform sodium colorimetric assay using ONPG 220 μg / ml and β-galactosidase 0.05 unit / ml.

[0277] 6. Perform potassium colorimetric assay using ADP 220 μg / ml, phosphoenolpyruvate 0.05 unit / ml, pyruvate kinase 0.1 unit / ml, NADH 480 μg / ml, potassium phosphate 13.6 mg / ml, magnesium sulfate 95 μg / ml, FAD 7.85 μg / ml, 4-aminoantipyrine 130 μg / ml, horseradish peroxidase 10 units / ml and TBHBA 1.88 mg / ml.

[0278] 7. Perform chloride colorimetric assay using CNPG 35 30 μg / ml, amylase 0.36 unit / ml, and calcium acetate 250 μg / ml.

[0279] 8. Blood urea nitrogen was measured colorimetrically using urea amidolyase, PEP, ATP, pyruvate kinase, pyruvate oxidase, potassium phosphate, MgCl2, FAD, TBHBA, 4-AAP, and peroxidase.

[0280] 9. Colorimetric determination of creatinine using creatinine amide hydrolase, creatinine amidine hydrolase, sarcosine oxidase, TBHBA, 4-AAP, and peroxidase.

[0281] 10. p-Nitrophenyl phosphate, zinc sulfate, magnesium sulfate alkaline phosphatase colorimetric method.

[0282] 11. Alanine aminotransferase colorimetric assay was performed using L-alanine, α-ketoglutarate, pyruvate oxidase, potassium phosphate, MgCl2, FAD, TBHBA, 4-AAP, and peroxidase.

[0283] 12. Hydrogen peroxide (fluorescence) assay was performed using horseradish peroxidase, 4-aminoantipyrine, TOOS, 3,5,3',5'-tetramethylbenzidine (TMB), and Amplex Red.

[0284] 13. Amylase (colorimetric method) determination was performed using starch, sodium chloride, sodium hydroxide, sodium potassium tartrate, and 3,5-DNS (dinitrosalicylic acid).

[0285] 14. Lactic acid (colorimetric method) determination, using lactate dehydrogenase, NAD+, phosphorylase, INT (iodonitrotetrazolium).

[0286] 15. Determine lactate dehydrogenase using sodium L-lactate, NAD+, diaphorase, and INT (iodonitrotetrazolium) (colorimetric method).

[0287] 16. Glutamine (colorimetric method) is determined using glutamine dehydrogenase, NAD+, phosphorylase, and INT (iodonitrotetrazolium).

[0288] Other embodiments

[0289] 1. A device for analyzing a fluid sample, comprising:

[0290] a first plate, a second plate and a microwell, wherein

[0291] (a) the first plate and the second plate are movable relative to each other into different configurations and have sample contact areas on their respective surfaces for contacting a fluid sample containing a target analyte;

[0292] (b) a second plate having a plurality of microwells in the sample contact region, wherein each microwell has (i) a pore depth of 200 μm or less, (ii) a pore volume substantially smaller than the sample, and (iii) a binding site having a capture agent immobilized at the site, and the capture agent is configured to capture a target analyte;

[0293] wherein one of the configurations is an open configuration, wherein: the average spacing between the inner surface of the first plate and the edge of the microwells in the second plate is at least 250 μm, and the sample is deposited on one or both plates;

[0294] Another of the structures is a closed structure, which is the structure after the sample is deposited in an open structure; in the closed structure, at least a portion of the sample is within the microwell, and the average spacing between the inner surface of the first plate and the edge of the microwell in the second plate is less than 1 / 10 (one tenth) of the depth of the microwell.

[0295] 2. A kit for analyzing a sample, comprising:

[0296] (a) the device of Example 1;

[0297] (b) A sponge configured to release a solution stored in the sponge to the outside and absorb a solution outside the sponge into the inside of the sponge.

[0298] 3. A system for analyzing a sample, comprising:

[0299] (a) The device of claim 1;

[0300] (b) a reader for generating an image of the signal emanating from the binding sites of the second plate;

[0301] (c) a device assembly operably connecting a reading device to the closed configuration of the first and second panels;

[0302] (d) a memory for storing images; and

[0303] (e) Procedure for identifying and counting individual binding events in an image region.

[0304] 4. A method for measuring a fluid sample, comprising:

[0305] (a) obtaining a sample containing a target analyte;

[0306] (b) obtaining the device of Example 1;

[0307] (c) depositing the sample on one or both plates when the plates are configured in an open configuration;

[0308] (d) after (c), moving the two plates of the apparatus of Example 1 into a closed configuration; and

[0309] (e) Reading the sample contact area of ​​the second plate with a reading device to generate a signal image.

[0310] 2-1. The kit as described in Embodiment 2, wherein the kit further comprises a detection agent.

[0311] 2-2. The kit of embodiment 2, wherein the kit further comprises a detection agent and a substrate, and the detection agent and the substrate are constructed to produce a luminescent or color-producing product together.

[0312] 2-2.1. A kit as described in embodiment 2-2, wherein the detection agent is an enzyme linked to a detection agent such as horseradish peroxidase, and the substrate is a color-based ABTS or TMB;

[0313] 2-2.2. The kit of embodiment 2-2, wherein the detection agent is an enzyme linked to the detection agent as horseradish peroxidase, and the substrate is Amplex Red based on fluorescence;

[0314] 2-3. The kit according to embodiment 2, wherein the kit further comprises units, and the binding of a single analyte molecule to the receptor affects the properties of adjacent units through signal transduction (analyte-induced modification of collective properties).

[0315] 2-4. The kit as described in Embodiment 2, wherein the kit further comprises one or more catalysts, and the catalyst comprises Pd(0)-catalyst, apyrase, potassium permanganate or platinum, etc.

[0316] 3-1. A system as described in Embodiment 3, wherein the device component is an adapter connected to a camera of a handheld mobile communication device.

[0317] 3-2. The system of embodiment 3, wherein the signal represents a single target-analyte binding event.

[0318] 3-3. A system as described in Embodiment 3, wherein the device component controls or changes the relative position between the plate and the reading device in at least one of three (x, y, z) orthogonal directions to read the signal.

[0319] 3-4. The system as described in Embodiment 3, wherein the reading device is a CCD camera.

[0320] 3-5. A system as described in embodiment 3, wherein the reading device is a photodetector, and the photodetector includes one or more other optical devices selected from filters, spectrometers, lenses, apertures, beam splitters, mirrors, polarizers, wave plates, and shutters.

[0321] 3-6. The system of embodiment 3, wherein the reading device collects the position, local intensity, local spectrum and local Raman signature of the signal.

[0322] 3-7. A system as described in embodiment 3, wherein the programming includes programming for: (1) determining the local intensity or spectrum or Raman characteristics of the background signal, (2) determining the local signal intensity or spectrum or Raman characteristics of one marker, two markers, three markers, and four or more markers; (3) determining the total number of markers in the imaging area.

[0323] 3-8. A system as described in embodiment 3, wherein identification and counting includes determining any, some or all of local intensity, spectral and Raman characteristics.

[0324] 3-9. The system as described in embodiment 3 further comprises a light source, a power source or a chemical power source for exciting the markings on the surface of the plate.

[0325] 3-10. A system as described in embodiment 3, wherein the system comprises electrodes, electric fields and / or electric field gradients for applying a voltage between the electrodes and the sensing amplification layer to generate an electric field and / or an electric field gradient (a) to move an analyte in a solution placed on the surface of the plate to a capture agent on the sensing amplification layer.

[0326] 3-11. The system of embodiment 3, wherein the system comprises an electrode for applying a bias voltage between the signal amplification layer and another electrode to further improve sensitivity.

[0327] 3-12. A system as described in embodiment 3, wherein the reading device is an electrical or mechanical or biological probe, which collects position, local electrical, local mechanical, local biological and local optical interactions between the plate and the reading device.

[0328] 3-13. The system of embodiment 13, wherein the reading device is a camera of a handheld mobile communication device.

[0329] 4-1. The method of embodiment 4, wherein the method further comprises a washing step to remove any biological material not bound to the capture agent.

[0330] 4-1. The method of embodiment 4, wherein the method does not comprise any step of washing to remove any biological material not bound to the capture agent.

[0331] 4-2. A method as described in Embodiment 4, wherein the method further comprises the step of adding a detection agent.

[0332] 4-3. The method of embodiment 4, wherein the method further comprises the following steps: (i) adding a detection agent, (ii) washing to remove any unbound detection agent, and (iii) adding a substrate to produce color.

[0333] 4-4. The method of embodiment 4, wherein the reading in step (e) is performed when the plate is in a closed configuration and the microwells have substrates.

[0334] 4-5. The method of embodiment 5, wherein the method is a homogeneous assay that reads the signal without using a washing step to remove any biological material or label that is not bound to the capture agent at the binding site.

[0335] 5. The device, kit, system or method of any preceding embodiment, wherein the binding area has a signal amplification layer and the capture agent is immobilized on the signal amplification layer.

[0336] 6. A device, kit, system or method as described in the above embodiments, wherein the micropore spacing is 1nm to 10nm, 10nm to 100nm, 100nm to 500nm, 500nm to 1μm, 1μm to 5μm, 5μm to 50μm, 50μm to 500μm, 500μm to 1mm or 1mm to 5mm; preferably in the range of 1μm-10μm, 10μm-50μm, or 50μm-500μm.

[0337] 7. A device, kit, system or method as described in any of the preceding embodiments, wherein the micropore size (length or diameter) is 1nm to 10nm, 10nm to 100nm, 100nm to 500nm, 500nm to 1μm, 1μm to 5μm, 5μm to 50μm, 50μm to 500μm, 500μm to 1mm or 1mm to 5mm; preferably in the range of 0.5μm-5μm, 5μm-25μm or 25μm-300μm.

[0338] 8. The device, kit, system or method of any of the preceding embodiments, wherein the micropore depth is 1 nm to 10 nm, 10 nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 μm, 1 μm to 5 μm, 5 μm to 50 μm, 50 μm to 500 μm, 500 μm to 1 mm or 1 mm to 5 mm; preferably 0.5 μm-5 μm, 5 μm-25 μm or 25 μm-300 μm;

[0339] 9. The device, kit, system or method of any preceding embodiment, wherein the microwell has a spherical, rectangular, hexagonal and / or any other polyhedral shape.

[0340] 10. The device, kit, system or method of the preceding embodiments, wherein the microwells have a lattice of square, hexagonal and / or any other lattice.

[0341] 11. A device, kit, system or method as described in any of the preceding embodiments, wherein the microwells are non-spaced and have an even spacing in Example 6 above.

[0342] 12. A device, kit, system or method as described in any of the preceding embodiments, wherein the micropore area ratio (the ratio of micropore area to total surface area) is at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or within a range between any two values.

[0343] 13. The device, kit, system or method of any preceding embodiment, wherein the material of the plate is polystyrene, PMMA, PC, COC, COP or another plastic.

[0344] 14. A device, kit, system or method as described in any of the preceding embodiments, wherein the micropore distance (spacing minus size) is greater than the micropore depth; and is configured to ensure that the diffusion time of the analyte from one micropore to another is longer than the incubation time (diffusion time of the micropore depth).

[0345] 15. The device, kit, system or method of any preceding embodiment, wherein one or both plates comprise a spacer permanently affixed to the inner surface of the respective plate.

[0346] 15-1. The device, kit, system or method of embodiment 15, wherein the spacers have a predetermined substantially uniform height equal to or less than 200 microns and a predetermined spacer pitch;

[0347] 16. The device, kit, system or method of any preceding embodiment, wherein the average spacing between the plates in the closed configuration is 100 μm or less.

[0348] 17. The device, kit, system or method of any preceding embodiment, wherein the average spacing between the plates in the closed configuration is 50 μm or less.

[0349] 15. The device, kit, system or method of any preceding embodiment, wherein the device further comprises a hinge connecting the first plate and the second plate and configured to allow the plates to rotate about the hinge into different configurations.

[0350] 19. The device, kit, system or method of any preceding embodiment, wherein at least one of the plates is flexible.

[0351] 20. A method for measuring a fluid sample, comprising:

[0352] (a) obtaining a sample containing a target analyte;

[0353] (b) obtaining the device of Example 1;

[0354] (c) depositing the sample on one or both plates when the plates are configured in an open configuration;

[0355] (d) after (c), moving the two plates of the apparatus of Example 1 into a closed configuration; and

[0356] (e) Reading the sample contact area of ​​the second plate with a reading device to generate a signal image.

[0357] 20-1. The method of embodiment 20 further comprises: (f) quantifying the signal in the image area to provide an estimate of the amount of one or more analytes in the sample.

[0358] 20-2. A method as described in embodiment 20-1, wherein step (f) includes identifying and counting individual binding events between analytes and capture agents in the image area, thereby providing an estimate of the amount of one or more analytes in the sample.

[0359] 20-3. A method as described in embodiment 20-1, wherein step (f) includes quantifying a primary sum signal in the image area to provide an estimate of the amount of one or more analytes in the sample.

[0360] 20-4. A method as described in embodiment 20, wherein the sample contact area of ​​the second plate has a reagent storage site.

[0361] 20-5. A method as described in embodiment 20, wherein the sample contact area of ​​the second plate has a reagent storage site, and the storage site is approximately above the binding site on the first plate in the closed structure.

[0362] 20-6. A method as described in embodiment 20, wherein the sample contact area in the first plate further includes a reagent storage site.

[0363] 20-7. A method as described in embodiment 20, wherein the sample contact area in the first plate further includes a reagent storage site, wherein the reagent storage site and the binding site are not at the same position of the sample contact area.

[0364] 20-8. A method as described in embodiment 20-7, wherein the reagent in the reagent storage site is a detection agent that binds to the target analyte.

[0365] 20-9. A method as described in embodiment 20, wherein the method further comprises the step of labeling the target analyte with a detection agent.

[0366] 20-10. The method of embodiment 20-9, wherein the detection agent comprises a label.

[0367] 20-11. The method of embodiment 20-9, wherein both the capture agent and the detection agent are bound to the target analyte to form a sandwich.

[0368] 20-12. A method as described in embodiment 20, wherein the method further comprises measuring the volume of the sample in the area imaged by the reading device.

[0369] 20-13. The method of embodiment 20, wherein the first plate comprises a plurality of binding sites, each binding site comprising:

[0370] (i) adjacent to the relevant signal amplification layer, and

[0371] (ii) Capture agents attached to the proximity-dependent signal amplification layer.

[0372] 20-14. A method as described in embodiment 20, wherein the target analyte is a protein, a peptide, DNA, RNA, a nucleic acid, a small molecule, a cell or a nanoparticle.

[0373] 20-15. The method of any of the preceding method embodiments, wherein the capture agent specifically binds to the target analyte.

[0374] 20-16. The method of any of the preceding method embodiments, wherein the image displays the location, local intensity, and local spectrum of the signal.

[0375] 20-17. The method of any of the preceding method embodiments, wherein the signal is a luminescent signal selected from the group consisting of fluorescent, electroluminescent, chemiluminescent, and electrochemiluminescent signals.

[0376] 20-18. The method of any of the preceding method embodiments, wherein the signal is a Raman scattering signal.

[0377] 20-20. The method of any of the preceding method embodiments, wherein the signal is a force due to a local electrical, local mechanical, local biological, or local optical interaction between the plate and the reader.

[0378] 20-21. The method according to any of the preceding method embodiments, wherein before step (b), it further comprises a step of labeling the target analyte with a label before or after the target analyte binds to the capture agent.

[0379] 20-22. A method as described in any of the preceding method embodiments, wherein the reading step (b) is performed by applying a bias voltage between the signal amplification layer and another electrode to provide greater sensitivity.

[0380] 20-23. A method as described in any of the foregoing method embodiments, wherein the identification and counting step (c) comprises: (1) determining the local intensity of the background signal, (2) determining the local signal intensity of one marker, two markers, three markers, and four or more markers; and (3) determining the total number of markers in the imaging area.

[0381] 20-24. A method as described in any of the aforementioned method embodiments, wherein the identification and counting step (c) comprises: (1) determining the local spectrum of the background signal, (2) determining the local signal spectrum of one marker, two markers, three markers, and four or more markers; (3) determining the total number of markers in the imaging area.

[0382] 20-25. A method as described in any of the aforementioned method embodiments, wherein the identification and counting step (c) comprises: (1) determining the local Raman characteristics of the background signal, (2) determining the local signal Raman characteristics of one marker, two markers, three markers and four or more markers; (3) determining the total number of markers in the imaging area.

[0383] 20-26. The method of any of the preceding method embodiments, wherein the identifying and counting steps comprise determining one or more of a local intensity, a spectrum, and a Raman signature.

[0384] 20-27. The method of any of the preceding method embodiments, wherein the binding step (a) is accelerated by applying an electric field to the plate, thereby moving the analyte to the sensing amplification layer.

[0385] 20-28. The method of any of the preceding method embodiments, wherein the proximity-related signal amplification layer comprises D2PA.

[0386] 20-29. A method as described in any of the preceding embodiments, wherein the adjacent related signal amplification layer comprises one or more metal disks and a significantly flat metal film, wherein most of the metal disks have a spacing from the metal film, and the spacing and size of the disks are smaller than the wavelength of the light used for sensing.

[0387] 20-30. The method of embodiments 20-29, wherein the metal disk has a shape selected from the group consisting of a circle, a polygon, a pyramid, an ellipse, an elongated strip, or any combination thereof.

[0388] 20-31. A method as described in embodiments 20-29, wherein the pitch is 0.5 to 30 nm, and wherein the disk has an average lateral dimension in the range of 20 nm to 250 nm.

[0389] 20-32. The method of any of the preceding method embodiments, wherein the capture agent is attached to the sensing amplification layer via a molecular linking layer that links the capture agent to the sensing amplification layer.

[0390] 20-33. The method of any of the preceding method embodiments, wherein the signal is an optical signal.

[0391] 20-34. The method of any of the preceding method embodiments, wherein the signal is generated by a fluorescent label that binds to the bound analyte either before or after the analyte binds to the capture agent.

[0392] 20-35. The method according to any of the preceding method embodiments, wherein the average distance between two adjacent signals read in the reading step (c) to form a signal image is greater than 10 nm.

[0393] 20-36. The method of any of the preceding method embodiments, wherein the signal is a signal generated by Raman scattering.

[0394] 20-37. The method of any of the preceding method embodiments, wherein the capture agent is an antibody.

[0395] 20-38. The method of any of the preceding method embodiments, wherein the capture agent is a polynucleotide.

[0396] Other additional embodiments

[0397] As used herein, a "capture component" is any molecule, other chemical / biological entity, or solid support modification disposed on a solid support that can be used to specifically attach, bind, or otherwise capture a target molecule or particle (e.g., an analyte molecule or a dissociated species) such that the target molecule / particle becomes fixed relative to the capture component and the solid substrate. As used herein, "immobilization" refers to being captured, attached, bound, or attached to prevent the dissociation or loss of the target molecule / particle, but does not require absolute fixity relative to the capture component or the solid substrate. The capture components that are useful or potentially useful for implementing certain aspects and embodiments of the present invention are discussed in more detail below. At least some of the analyte molecules can become fixed relative to the capture component when exposed to a substrate comprising a plurality of capture components, thereby forming a plurality of fixed complexes. For example, in certain embodiments, substantially all of the plurality of analyte molecules can become fixed relative to the capture component such that substantially each fixed complex comprises a capture component and an analyte molecule.

[0398] As used herein, "binding ligand" is any molecule, particle, etc. that specifically binds or additionally specifically binds to analyte molecules, immobilized complexes and / or dissociated substances or binds or additionally binds to another molecule or particle (e.g., another binding ligand) of analyte molecules, immobilized complexes and / or dissociated substances. In certain embodiments, the binding ligand can convert the precursor marker molecule into a marker, as discussed in more detail below. More than one type of binding ligand can be used in any given assay method, for example, a first type of binding ligand and a second type of binding ligand. In one embodiment, the first binding ligand can bind to the analyte molecule, and the second binding ligand can bind to the first binding ligand. When the substrate is exposed to multiple types of binding ligands, in some cases, at least some of the multiple immobilized complexes can additionally include at least one of each type of binding ligand. In certain embodiments, the binding ligand can be exposed to the substrate after capturing the analyte molecule so that the binding ligand is bound to the immobilized complex. In other embodiments, the binding ligand can bind to the analyte molecule to form a complex, which is then captured by the substrate to form an immobilized complex. In other embodiments, the binding ligand may bind to a dissociated species that is formed when the immobilized complex or a portion thereof is released from the substrate.

[0399] In some embodiments, the fixed complex comprises a cleavable bond. As used herein, a "cleavable bond group" is capable of being easily (i.e., under conditions that are harmless to the integrity of the other parts of the fixed complex) and selectively cleaved when exposed to a dissociating agent. The cleavable bond when cleaved by exposure to a dissociating agent forms a dissociated substance. A specific example of a cleavable bond that can be cleaved using β-mercaptoethanol is a disulfide bond. The cleavable bond and the corresponding dissociating agent that can cause the cleavage of the cleavable bond are discussed in more detail below.

[0400] In some embodiments, a plurality of molecules can be released from the first substrate by being exposed to a dissociating agent. For example, a substrate comprising a plurality of capture components can be exposed to a sample comprising a plurality of analyte molecules or particles so that the analyte molecules or particles are combined with the capture components to form a plurality of complexes, and the complex is fixed relative to the substrate. Each immobilized complex can include at least one capture component and at least one analyte molecule or particle. A plurality of immobilized complexes are exposed to a reducing agent (e.g., β-mercaptoethanol, dithiothreitol, tris(2-carboxyethyl)phosphine, etc.) causing at least a portion of at least some of the plurality of immobilized complexes to dissociate from the substrate to form a plurality of dissociated substances. At least some dissociated substances can be detected to determine the presence of analyte molecules or particles in a fluid sample and / or to measure the amount or concentration of analyte molecules or particles in a fluid sample, as discussed in more detail herein. A reducing agent may or may not be removed from a solution comprising a dissociated substance before detecting the dissociated substance, as discussed in more detail herein. In some embodiments, a dissociating agent is a reducing agent (e.g., β-mercaptoethanol). In some embodiments, a dissociating agent has substantially no specific affinity to the capture component. That is, the dissociating agent does not cause the release of the dissociated species by interacting with the capture component and releasing analyte molecules associated with the capture component by competitive binding.

[0401] In some embodiments, a plurality of dissociated substances can be formed by cracking cleavable bonds. For example, each immobilized complex can include at least one cleavable bond (e.g., a disulfide bond). The cleavable bond can be located in a capture component, an analyte molecule, or a binding ligand, and can be cleaved to form a plurality of dissociated substances. In one embodiment, the cleavable bond is a disulfide bond, which can be cleaved by exposing the fixed complex to a reducing agent in some cases.

[0402] In some embodiments, at least a portion of the immobilized complex comprises an enzyme component. That is, at least one of the capture component, the analyte molecule, or any other component of the immobilized complex (e.g., a binding ligand) comprises an enzyme component. In some cases, the enzyme component can be in the portion of the immobilized complex that dissociates from the first substrate to form the dissociated species. For example, Fig. 9 Exemplary embodiments of assays are described in which the binding ligand comprises a moiety (eg, an enzyme component) as discussed in more detail herein.

[0403] In certain embodiments, the scheme may include the use of at least one binding ligand, at least a portion of which comprises at least a portion of the dissociated material transferred from the first substrate to the second substrate (e.g., the binding ligand can be fixed before or after the molecule or particle is released from the first substrate). In some embodiments, the binding ligand comprises a cleavable bond (e.g., a disulfide bond) and / or dissociates from the first substrate by exposure to a reducing agent. In some embodiments, at least one binding ligand comprises an enzyme component. For example, the binding ligand or at least a portion thereof forms at least a portion of the dissociated material transferred from the first substrate to the second substrate, and may also comprise a portion (e.g., an enzyme component or an enzyme substrate) that can convert a precursor marker molecule (e.g., an enzyme substrate) into a marker (e.g., a detectable product). After the dissociated material is transferred to or in the second substrate and the dissociated material is optionally captured, the second substrate may be exposed to a variety of precursor marker molecules, wherein a variety of precursor marker molecules are converted into a variety of marker molecules when exposed to the binding ligand. The measurement of the concentration of the analyte molecules or particles in the fluid sample may then be determined based on the measurement of the marker molecules on or in the second substrate.

[0404] A method for detecting an analyte molecule or particle in a QMAX device, comprising:

[0405] (a) obtaining a sample containing a plurality of analyte molecules or particles;

[0406] (b) obtaining a QMAX device, the QMAX device comprising:

[0407] A first plate, a second plate and a spacer, wherein:

[0408] i. The plates are movable relative to each other into different configurations;

[0409] ii. One or both plates are flexible;

[0410] iii. One or two plates have multiple reaction vessels;

[0411] iv. Each plate comprises an inner surface having a sample contact area for contacting a blood sample;

[0412] v. One or both plates contain multiple capture components;

[0413] vi. One or both plates contain a spacer permanently affixed to the sample contact area of ​​the respective plate;

[0414] vii. The spacer has:

[0415] (1) a predetermined substantially uniform height having a value selected in the range of 1 μm to 80 μm,

[0416] (2) a column shape having a substantially uniform cross-section and a flat top surface;

[0417] (3) The ratio of width to height is equal to or greater than 1;

[0418] (4) a predetermined fixed, non-random spacer spacing in the range of 10 μm to 200 μm (micrometers); and

[0419] (c) depositing the sample on one or both plates, exposing the plate comprising the plurality of capture components to the sample comprising the plurality of analyte molecules or particles, such that the analyte molecules or particles associate with the capture components to form a plurality of complexes, each complex being immobilized relative to the plate and comprising at least one capture component and at least one analyte molecule or particle;

[0420] (d) dissociating at least a portion of each complex to form a plurality of dissociated species that are not immobilized relative to the plate;

[0421] (e) separating the plurality of dissociated species across a plurality of reaction vessels;

[0422] (f) determining the presence or absence of a dissociated species in at least one reaction vessel;

[0423] (g) determining the number of multiple reaction vessels and / or the portion of the multiple reaction vessels that contain or do not contain the dissociated species, wherein the multiple dissociated species are separated so that a statistically significant portion of the reaction vessels do not contain the dissociated species and a statistically significant portion of the reaction vessels contain at least one dissociated species.

[0424] A method for determining a measurement of the concentration of analyte molecules or particles in a fluid sample, comprising:

[0425] capturing a plurality of analyte molecules or particles on a first plate;

[0426] releasing a plurality of molecules or particles from a first plate;

[0427] detecting molecules or particles released from the first plate on or within a second plate comprising a plurality of reaction vessels;

[0428] and determining a measurement of the concentration of analyte molecules or particles in the fluid sample based on the detection of molecules or particles released from the first plate on or within the second plate, wherein the concentration of analyte molecules or particles in the fluid sample is determined by determining the number or number of a plurality of reaction vessels that contain or do not contain molecules or particles released from the first plate.

[0429] A method or device as described in any of the preceding embodiments, wherein the number or quantity of the plurality of reaction vessels containing the dissociated substance is related to the concentration of analyte molecules or particles in the sample.

[0430] A method or apparatus as in any of the preceding embodiments, further comprising an act of determining a concentration of analyte molecules or particles in the fluid sample.

[0431] A method or apparatus as described in any preceding embodiment, wherein the plate comprises a plurality of beads.

[0432] A method or device as in any preceding embodiment, wherein the beads are magnetic.

[0433] A method or device as described in any of the preceding embodiments, wherein the plate comprises a microtiter plate.

[0434] A method or apparatus as described in any of the preceding embodiments, wherein the plurality of reaction vessels are formed when at least a portion of the sealing assembly is mated with at least a portion of the second plate.

[0435] A method or apparatus as described in any of the preceding embodiments, wherein a plurality of reaction vessels are defined on a planar second plate.

[0436] A method or apparatus as described in any of the preceding embodiments, wherein each of the plurality of reaction vessels has a volume of about 10-100 picoliters.

[0437] A method or apparatus as described in any of the preceding embodiments, wherein each of the plurality of reaction vessels comprises at least one dissociated species capture assembly.

[0438]

[00136] A method or apparatus as in any of the preceding embodiments, further comprising immobilizing at least one of the plurality of dissociated species relative to the at least one dissociated species capture component.

[0439] A method or apparatus as described in any of the preceding embodiments, wherein each of the plurality of reaction vessels is exposed to at least one precursor labeling agent molecule.

[0440] A method or apparatus as described in any of the preceding embodiments, wherein at least one precursor labeling agent molecule is converted to a labeling agent molecule when contained in a reaction vessel containing a dissociating species.

[0441] A method or apparatus as described in any of the preceding embodiments, wherein the presence or absence of dissociated species in the reaction vessel is determined by determining the presence or absence of labeling agent molecules in the reaction vessel.

[0442] A method or device as described in any of the preceding embodiments, wherein the plate is exposed to a plurality of first binding ligands.

[0443] A method or device as in any of the preceding embodiments, wherein a first binding ligand associates with each of the plurality of analyte molecules or particles in the exposing to form at least a portion of the plurality of complexes.

[0444] A method or device as described in any of the preceding embodiments, wherein each first binding ligand comprises an enzyme component.

[0445] A method or device as described in any of the preceding embodiments, wherein the first binding ligand comprises a cleavable bond.

[0446] A method or apparatus as described in any of the preceding embodiments, wherein a plurality of dissociated species are formed by cleaving at least some of the cleavable bonds.

[0447] A method or device as in any of the preceding embodiments, wherein at least one of the plurality of dissociated species comprises at least a portion of the first binding ligand.

[0448] A method or apparatus as in any preceding embodiment, wherein the plurality of dissociated species are formed by exposing the plate to electromagnetic radiation.

[0449] A method or apparatus as in any preceding embodiment, wherein the plurality of dissociated species are formed by exposing the plate to a dissociating agent.

[0450] A method or device as described in any of the preceding embodiments, wherein the dissociating agent comprises at least one of a pH agent, a salt agent, a denaturing agent, a reducing agent, a chemical agent or an enzyme.

[0451] A method or device as in any preceding embodiment, wherein the analyte molecule or particle is a protein.

[0452] A method or device as described in any of the preceding embodiments, wherein the capture component is an antibody.

[0453] The method or apparatus as described in any of the preceding embodiments further comprises sealing a plurality of reaction vessels.

[0454] A method or device as described in any of the preceding embodiments, wherein the first plate comprises a plurality of first capture components.

[0455] A method or device as in any of the preceding embodiments, wherein at least one of the plurality of analyte molecules or particles is captured by being specifically immobilized relative to at least one of the plurality of first capture components.

[0456] A method or device as described in any of the preceding embodiments, further comprising the act of exposing the plurality of analyte molecules or particles captured on the first plate to a plurality of first binding ligands.

[0457] A method or device as in any of the preceding embodiments, wherein at least one of the plurality of first binding ligands becomes immobilized relative to each of at least a portion of the plurality of analyte molecules or particles captured on the first plate.

[0458] A method or apparatus as in any preceding embodiment, wherein the act of releasing comprises exposing the plate to electromagnetic radiation.

[0459] A method or apparatus as in any preceding embodiment, wherein the act of releasing comprises exposing the plate to a dissociating agent.

[0460] A method or device as described in any of the preceding embodiments, wherein the second plate comprises a plurality of second capture components.

[0461] A method or device as in any of the preceding embodiments, wherein each of at least a portion of the plurality of molecules or particles released from the first plate becomes immobilized relative to at least one second capture component on the second plate.

[0462] The method or apparatus as described in any of the preceding embodiments further comprises an action of sealing at least a portion of the plurality of reaction vessels.

[0463] A method or device as described in any of the preceding embodiments, wherein the measurement of the concentration of analyte molecules or particles in the fluid sample is determined at least in part by analyzing the Poisson distribution of the number or quantity of multiple reaction vessels containing analyte molecules or particles released from the plate.

[0464] A method or apparatus as in any preceding embodiment, wherein less than about 80% of the total number of the plurality of reaction vessels contain at least one analyte molecule or particle released from the plate.

[0465] A method or apparatus as in any of the preceding embodiments, wherein the second plate comprises a planar surface and a sealing assembly comprising a plurality of microwells, and the plurality of reaction vessels are formed when at least a portion of the planar plate is mated with at least a portion of the sealing assembly.

[0466] Color-coded beads for multiplexing:

[0467] A device or method as described in any preceding embodiment, wherein the label is a bead containing a colored barcode.

[0468] A device or method as described in any preceding embodiment, wherein the beads having a color barcode contain a reagent having affinity for a single analyte.

[0469]

[00136] A device or method as in any preceding embodiment, wherein the number of beads of each type of barcode that capture a particular type of analyte is statistically significant.

[0470] A device or method as described in any of the preceding embodiments, wherein the markers are beads having different geometric sizes, wherein the sizes include but are not limited to spheres, cubes, cuboids, and tetrahedrons.

[0471] A device or method as described in any of the above embodiments, wherein the microwells have different geometric shapes, and each microwell of a certain shape can only accommodate beads of one geometric size.

[0472] A device or method as in any preceding embodiment, wherein beads of different geometric sizes contain capture agents for different analytes.

[0473]

[00136] A device or method as in any preceding embodiment, wherein the number of beads of each individual geometric size that capture a particular analyte is statistically significant.

[0474] A device or method as in any preceding embodiment, wherein the ratio of the number of labels to the number of spacers / pillars can be used for quantification.

[0475] A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample on a QMAX card, comprising:

[0476] The QMAX card was measured using beads as markers;

[0477] A measure of the analyte concentration in the sample is determined based on determining the ratio of the number of beads bound to the analyte molecules to the number of spacers (pillars).

[0478] Other embodiments of the present invention and related disclosures

[0479] The present invention includes various embodiments that can be combined in various ways as long as the various components do not contradict each other. The embodiments should be considered as a single invention document: each application refers to the other applications as a reference, and is also cited as a whole for all purposes rather than as a discrete independent document. These embodiments include not only the disclosure in the current document, but also the documents cited, incorporated or claiming priority here.

[0480] (1) Definition

[0481] Terms used to describe the devices, systems, and methods disclosed herein are defined in this application or in PCT Application (designating the United States) Nos. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0482] The terms "CROF card (or card)", "COF card", "QMAX card", "Q card", "CROF device", "COF device", "QMAX device", "CROF board", "COF board" and "QMAX board" are interchangeable, except that in some embodiments, the COF card does not include a gasket; and these terms refer to a device that includes a first board and a second board that are movable relative to each other into different configurations (including an open configuration and a closed configuration), and the device includes a gasket to adjust the spacing between the boards (except for some embodiments of COF). The term "X board" refers to one of the two boards in a CROF card, wherein the gasket is fixed to the board. Further description of COF cards, CROF cards and X boards is described in provisional application serial number 62 / 456065 filed on February 7, 2017, the entire contents of which are incorporated herein for all purposes.

[0483] (2) Q card, gasket and uniform sample thickness

[0484] Device disclosed herein, system and method can include or use for sample detection, analysis and quantitative Q card, gasket and uniform sample thickness embodiment.In some embodiments, Q card includes gasket, which helps to make at least a portion of sample become highly uniform layer.The structure, material, function, variation and size of gasket and the uniformity of sample layer are disclosed herein, or in PCT application (designated U.S.) No. PCT / US2016 / 045437 and PCT / US0216 / 051775, respectively submitted on August 10, 2016 and September 14, 2016, the U.S. Provisional Application No. 62 / 456065 submitted on February 7, 2017, the U.S. Provisional Application No. 62 / 456287 submitted on February 8, 2017, the U.S. Provisional Application No. 62 / 456504 submitted on February 8, 2017, list, describe and summarize, all the contents of all these applications are incorporated herein for all purposes.

[0485] (3) Hinges, slots, recessed edges and sliding parts

[0486] The devices, systems and methods disclosed herein may include or use Q cards for sample detection, analysis and quantification. In some embodiments, the Q card includes hinges, notches, grooves and slides, which help to facilitate the operation of the Q card and the measurement of the sample. The structure, material, function, variation and size of the hinge, notch, groove and slide are disclosed herein, or are listed, described and summarized in PCT applications (designated U.S.) Nos. PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065 filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287 filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504 filed on February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0487] (4) Q card, sliding parts and mobile phone detection system

[0488] Device, system and method disclosed herein may include or use Q card for sample detection, analysis and quantitative. In some embodiments, Q card is used together with the sliding piece that enables the card to be read by the mobile phone detection system. The structure, material, function, change, size and connection of Q card, sliding piece and mobile phone detection system are disclosed herein, or are listed, described and summarized in PCT application (designated U.S.) No. PCT / US2016 / 045437 and PCT / US0216 / 051775 submitted on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065 submitted on February 7, 2017, U.S. Provisional Application No. 62 / 456287 submitted on February 8, 2017 and U.S. Provisional Application No. 62 / 456504 submitted on February 8, 2017, and the entire contents of all these applications are incorporated herein for all purposes.

[0489] (5) Detection method

[0490] Device, system and method disclosed herein can include or be used for various types of detection methods. Detection methods are disclosed herein, or are listed, described and summarized in PCT applications (designated U.S.) Nos. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, respectively, and the entire contents of all these applications are incorporated herein for all purposes.

[0491] (6) Tags

[0492] The devices, systems and methods disclosed herein can employ various types of labels for analyte detection. Labels are disclosed herein or listed, described and summarized in PCT Applications (designated U.S.) Nos. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, the entire contents of all of which are incorporated herein for all purposes.

[0493] (7) Analyte

[0494] The devices, systems and methods disclosed herein can be applied to operate and detect various types of analytes (including biomarkers). Analytes are disclosed herein, or listed, described and summarized in PCT Applications (designated U.S.) Nos. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0495] (8) Application (field and sample)

[0496] Device, system and method disclosed herein can be used for various applications (fields and samples). The application is disclosed herein, or listed, described and summarized in PCT application (designated U.S.) No. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, the entire contents of all these applications are incorporated herein for all purposes.

[0497] (10) Cloud

[0498] The devices, systems and methods disclosed herein may employ cloud technology for data transmission, storage and / or analysis. Relevant cloud technologies are disclosed herein or listed, described and summarized in PCT Applications (designated U.S.) Nos. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0499] Spacer Fill Factor

[0500] The term "spacer fill factor" or "fill factor" refers to the ratio of the spacer contact area to the total board area, where the spacer contact area is the contact area of ​​the top surface of the spacer contacting the inner surface of the board in the closed configuration, and the total board area is the total area of ​​the inner surface of the board contacted by the spacer flat top. Since there are two boards and each spacer has two contact surfaces, each contacting one board, the fill factor is the minimum.

[0501] For example, if the spacer is a column with a flat top, almost uniform cross-section, and 2 μm height with a square (10 μm×10 μm), and the spacer is a spacer with a spacing of 100 μm, the filling factor of the spacer is 1%. If in the above example, the bottom of the column spacer is a square shape of 15 μm×15 μm, the filling factor is still defined as 1%.

[0502] A method or device as in any of the preceding embodiments, wherein the spacer has a columnar shape and a nearly uniform cross-section.

[0503] A method or apparatus as in any of the preceding embodiments, wherein the spacer pitch (SD) is equal to or less than about 120 μm (micrometers).

[0504] A method or apparatus as in any of the preceding embodiments, wherein the spacer pitch (SD) is equal to or less than about 100 μm (micrometers).

[0505] A method or device as in any of the preceding embodiments, wherein the fourth power of the spacer spacing (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible board (ISD 4 / (hE)) is 5×10 6 μm 3 / GPa or less.

[0506] A method or device as in any of the preceding embodiments, wherein the fourth power of the spacer spacing (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible board (ISD 4 / (hE)) is 5×10 5 μm 3 / GPa or less.

[0507] A method or device as described in any of the preceding embodiments, wherein the spacers have a columnar shape, a substantially flat top surface, a predetermined substantially uniform height and a predetermined constant inter-spacer distance, the inter-spacer distance being at least about 2 times larger than the size of the analyte, wherein the Young's modulus of the spacer multiplied by the filling factor of the spacer is equal to or greater than 2 MPa, wherein the filling factor is the ratio of the spacer contact area to the total plate area, and wherein for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1 (one).

[0508] A method or apparatus as in any of the preceding embodiments, wherein the spacers have a columnar shape, a substantially flat top surface, a predetermined substantially uniform height, and a predetermined constant inter-spacer distance that is at least about 2 times greater than the size of the analyte, wherein the Young's modulus of the spacers multiplied by the packing factor of the spacers is equal to or greater than 2 MPa, wherein the packing factor is the ratio of the spacer contact area to the total plate area, and wherein, for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1 (one), wherein the fourth power of the spacer spacing (ISD) divided by the thickness (h) of the flexible plate and the Young's modulus (E) (ISD) 4 / (hE)) is 5×10 6 μm 3 / GPa or less.

[0509] A device as described in any of the preceding device embodiments, wherein the ratio of the inter-spacer distance of the spacers to the average width of the spacers is 2 or greater, and the filling factor of the spacers multiplied by the Young's modulus of the spacers is 2 MPa or greater.

[0510] A method or device as described in any of the preceding embodiments, wherein the analyte is a protein, a peptide, a nucleic acid, a synthetic compound or an inorganic compound.

[0511] A method or device as described in any of the preceding embodiments, wherein the sample is a biological sample, and the biological sample is selected from amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma or serum), breast milk, cerebrospinal fluid (CSF), cerumen (ear wax), chyle, chyme, endolymph, perilymph, feces, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatism, saliva, exhaled condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomitus and urine.

[0512] A method or device as described in any of the preceding embodiments, wherein the spacer is columnar and the ratio of the width to the height of the column is equal to or greater than 1.

[0513] A method as in any preceding embodiment, wherein the sample deposited on one or both plates has an unknown volume.

[0514] A method or apparatus as described in any of the preceding embodiments, wherein the spacers are in the form of columns, and the columns have a substantially uniform cross-section.

[0515] A method or device as described in any of the preceding embodiments, wherein the sample is used to detect, purify and quantify compounds or biomolecules associated with the stage of certain diseases.

[0516] A method or device as described in any of the preceding embodiments, wherein the sample is related to infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, psychiatric disorders, neuropsychiatric disorders, lung diseases, kidney diseases and other and organic diseases.

[0517] A method or device as described in any of the preceding embodiments, wherein the sample involves detection, purification and quantification of microorganisms.

[0518] A method or device as described in any of the preceding embodiments, wherein the sample involves viruses, fungi and bacteria from the environment (eg, water, soil or biological samples).

[0519] A method or device as described in any of the preceding embodiments, wherein the sample involves detection or quantification of compounds or biological samples (eg, toxic waste, anthrax) that pose a threat to food safety or national security.

[0520] A method or apparatus as in any preceding embodiment, wherein the sample is related to quantification of a vital parameter in a medical or physiological monitor.

[0521] A method or device as in any preceding embodiment, wherein the sample is related to glucose, blood, oxygen level, total blood cell count.

[0522] A method or device as described in any of the preceding embodiments, wherein the sample is associated with the detection and quantification of specific DNA or RNA from a biological sample.

[0523] A method or apparatus as described in any of the preceding embodiments, wherein the sample is associated with sequencing and comparison of genetic sequences in chromosomal and mitochondrial DNA for genomic analysis.

[0524] A method or apparatus as described in any preceding embodiment, wherein the sample relates to detecting a reaction product, for example during drug synthesis or purification.

[0525] A method or device as described in any of the preceding embodiments, wherein the sample is cells, tissues, body fluids and feces.

[0526] A method or device as described in any of the preceding embodiments, wherein the sample is a sample in the fields of human, veterinary, agricultural, food, environmental and pharmaceutical testing.

[0527] A method or device as described in any of the preceding embodiments, wherein the sample is a biological sample selected from hair, nail, ear wax, breath, connective tissue, muscle tissue, neural tissue, epithelial tissue, cartilage, cancer sample or bone.

[0528] An apparatus or method as described in any of the preceding embodiments, wherein the spacing between the spacers is in the range of 5 m to 120 m.

[0529] An apparatus or method as described in any of the preceding embodiments, wherein the spacing between the spacers is in the range of 120 m to 200 m.

[0530] The device as described in any of the preceding device embodiments, wherein the flexible plate has a thickness in the range of 20 μm to 250 μm and a Young's modulus in the range of 0.1 to 5 GPa.

[0531] The device as described in any of the preceding device embodiments, wherein for the flexible sheet, the thickness of the flexible sheet multiplied by the Young's modulus of the flexible sheet is in the range of 60 to 750 GPa-μm.

[0532] A device as described in any of the preceding device embodiments, wherein the uniform thickness sample layer is at least 1 mm 2 It is uniform over the lateral area.

[0533] A device as described in any of the preceding device embodiments, wherein the uniform thickness sample layer is at least 3 mm 2 It is uniform over the lateral area.

[0534] A device as described in any of the preceding device embodiments, wherein the uniform thickness sample layer is at least 5 mm 2 It is uniform over the lateral area.

[0535] A device as described in any of the preceding device embodiments, wherein the uniform thickness sample layer is at least 10 mm 2 It is uniform over the lateral area.

[0536] A device as described in any of the preceding device embodiments, wherein the uniform thickness sample layer is at least 20 mm 2 It is uniform over the lateral area.

[0537] A device as described in any of the preceding device embodiments, wherein the uniform thickness sample layer is 20 mm 2 Up to 100mm 2 The lateral area within the range is uniform.

[0538] The device as in any preceding device embodiment, wherein the uniform thickness sample layer has a thickness uniformity of up to + / - 5% or better.

[0539] The device as in any preceding device embodiment, wherein the uniform thickness sample layer has a thickness uniformity of up to + / - 10% or better.

[0540] The device as in any preceding device embodiment, wherein the uniform thickness sample layer has a thickness uniformity of up to + / - 20% or better.

[0541] The device as in any preceding device embodiment, wherein the uniform thickness sample layer has a thickness uniformity of up to + / - 30% or better.

[0542] The present invention can be used for various applications in various fields where it is necessary to determine the presence and / or quantification of one or more analytes in a sample. For example, the present invention can be used to detect atoms, molecules, proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds, organic compounds, bacteria, viruses, cells, tissues, nanoparticles, etc. The sample can be a sample from a variety of fields, including but not limited to humans, veterinary medicine, agriculture, food, environment, health, health care, beauty and other fields.

[0543] Embodiments of the present invention

[0544] An apparatus for performing a digital assay, comprising:

[0545] A first plate, a second plate and a microwell, wherein:

[0546] (e) the first plate and the second plate are movable relative to each other into different configurations and have sample contact areas on their respective surfaces for contacting a fluid sample containing an analyte;

[0547] (f) the second plate has a plurality of microwells in the sample contacting region, wherein each microwell has (i) a predetermined and known geometry, (ii) a pore depth of 200 μm or less, and (iii) a volume substantially smaller than the fluid sample,

[0548] wherein one of the configurations is an open configuration, wherein: the average spacing between the inner surface of the first plate and the edges of the microwells in the second plate is greater than the depth of the wells, and the sample is deposited on one or both plates; and

[0549] Another of the structures is a closed structure, which is a structure after the sample is deposited in the open structure; in the closed structure, at least a portion of the sample is within the microwell, and the average spacing between the inner surface of the first plate and the edge of the microwell in the second plate is less than 1 μm or less than 1 / 10 (one tenth) of the depth of the microwell.

[0550] An apparatus comprising a thermal cycler and the device as described in embodiment 1.

[0551] An apparatus comprises a thermal cycler, the device as described in embodiment 1 and a reader for real-time PCR.

[0552] A method for separating a fluid sample, comprising:

[0553] Obtaining a device or apparatus as described in any of the preceding embodiments,

[0554] When the plates are in an open configuration, the sample is deposited on one or both plates, wherein the deposition is in the form of a single or multiple droplets of the sample, wherein at least one of the droplets has a volume that occupies more than two microwells; and the plates are closed to the closed configuration to separate the samples in the microwells.

[0555] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the analyte is a protein, a peptide, a nucleic acid, a virus, a bacterium, a cell, a nanoparticle, a molecule, a synthetic compound or an inorganic compound.

[0556] The device, apparatus or method of any preceding embodiment further comprises a spacer configured to adjust a spacing between the first plate and the second plate.

[0557] The device, apparatus or method of any of the preceding embodiments further comprises a binding site located on the inner surface of the one or both plates, wherein the binding site comprises a capture agent immobilized at the site, and the capture agent is constructed to specifically capture an analyte in the sample.

[0558] The device, apparatus or method as described in any of the preceding embodiments further comprises a surface amplification layer, wherein the surface amplification layer is located on the inner surface of the one or two plates, wherein the surface amplification layer comprises a capture agent fixed at the site, and the capture agent is configured to specifically capture the analyte in the sample, wherein the surface amplification layer amplifies optical signals from the analyte or a label attached to the analyte, and they are amplified more strongly when they are near the surface amplification layer than when they are micrometers or more away.

[0559] A device, apparatus or method as described in any of the preceding embodiments, wherein the amplification factor of the surface amplification layer is adjusted to make the optical signal from a single label directly or indirectly bound to the capture agent visible.

[0560] A device, apparatus or method as described in any of the preceding embodiments, wherein the amplification factor of the surface amplification layer is adjusted to make the optical signal from a single label directly or indirectly bound to the capture agent visible.

[0561] A device, apparatus or method as described in any of the preceding embodiments, wherein the device further comprises a reagent in the microwell in the tight structure of the plate, wherein when there is binding between the analyte and the detection agent, the reagent will produce a plurality of luminescent components in the well, and the detection agent specifically binds to the analyte.

[0562] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the spacing between the first plate and the second plate in the closed configuration is configured such that the saturation binding time of the target analyte to the capture agent is 300 seconds or less.

[0563] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the spacing between the first plate and the second plate in the closed configuration is configured such that the saturation binding time of the target analyte to the capture agent is 60 seconds or less.

[0564] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the amplification factor of the surface amplification layer is adjusted to make the optical signal from a single label visible.

[0565] A device, an apparatus or a method as described in any preceding embodiment, wherein the capture agent is a nucleic acid.

[0566] A device, an apparatus or a method as described in any preceding embodiment, wherein the capture agent is a protein.

[0567] A device, an apparatus or a method as described in any preceding embodiment, wherein the capture agent is an antibody.

[0568] A device, an apparatus or a method as described in any preceding embodiment, wherein the capture agent is an aptamer.

[0569] A device, an apparatus or a method as described in any preceding embodiment, wherein the capture agent is an aptamer.

[0570]

[00136] The device, apparatus or method of any of the preceding embodiments, further comprising a storage location located on the inner surface of the one or both plates, wherein the storage location comprises a reagent soluble in a liquid.

[0571] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the reagent is used to amplify the analyte in the sample.

[0572] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the reagent amplifies the analyte by polymerase chain reaction (PCR).

[0573] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the reagent is a detection agent.

[0574] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the volume of each well is configured for the expected target analyte concentration so that the distribution of the target analyte in each well loaded follows a Poisson distribution.

[0575] A device, apparatus or method as described in any of the preceding embodiments, wherein the volume of each well is constructed for the expected target analyte concentration so that the distribution of the target analyte in each well to which the sample is loaded is on average one target analyte per 2 wells, 3 wells, 5 wells, 10 wells, 20 wells, 0 wells, 50 wells, 75 wells, 100 wells, 150 wells, 200 wells, 300 wells, 500 wells, 1000 wells, 2000 wells, 10000 wells, 100,000 wells, or within the range of any two values.

[0576] A device, an apparatus or a method as described in any of the foregoing embodiments, wherein the volume of each well is preferably constructed according to the expected target analyte concentration so that the distribution of the target analyte in each well to which the sample is added is on average one target analyte per 10 wells, 20 wells, 0 wells, 50 wells, 75 wells, 100 wells, or within the range of any two values.

[0577] An apparatus, device or method as described in any of the preceding embodiments, wherein in the closed structure, the average spacing between the inner surface of the first plate and the edge of the micropores in the second plate is less than 1 / 11 (one eleventh), 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 100, 1 / 300, 1 / 500 of the micropore depth, or within the range of any two values.

[0578] A device, an apparatus or a method as described in any of the preceding embodiments, wherein in the closed configuration, the inner surface of the first plate is in substantial contact with the edges of the microwells in the second plate.

[0579] A device, an apparatus or a method as described in any of the preceding embodiments, wherein in the closed structure, the average spacing between two adjacent holes is less than 5nm, 10nm, 30nm, 50nm, 100nm, 200nm, 500nm, 1μm, 2μm, 5μm, 10μm, 20μm, 50μm, 100μm, or within a range of any two values.

[0580] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the micropores have a shape selected from circular, rectangular, hexagonal and / or any other polyhedron having a square, hexagonal and / or any other grid.

[0581] An apparatus, an equipment or a method as described in any of the foregoing embodiments, wherein the holes on the first plate have a range of at least 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, 1 mm or any of the values; and a spacing (average hole-to-hole center distance) in a preferred range of 10 nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 μm, 1 μm to 10 μm or 10 μm to 50 μm.

[0582] An apparatus, an equipment or a method as described in any of the foregoing embodiments, wherein the holes on the first plate have a size of 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, 1 mm, or a range between any of the values; and a pore size (average length or diameter) in a preferred range of 10 nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 μm, 1 μm to 10 μm or 10 μm to 50 μm.

[0583] An apparatus, device or method as described in any of the preceding embodiments, wherein the holes on the first plate have a range of at least 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, 1 mm or any of the values; and a depth of a preferred range of 10 nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 μm, 1 μm to 10 μm or 10 μm to 50 μm. An apparatus, device or method as described in any of the preceding embodiments, wherein the holes have (i) no metal coating, (ii) a metal coating on the bottom of the hole (the top of the column), (iii) a metal coating on the sidewall of the hole (the side of the column) and / or (iv) a metal coating on both the bottom and sidewall of the hole.

[0584] The device, apparatus or method of any preceding embodiment, wherein the metal is gold, aluminum, silver, copper, tin and / or any combination thereof.

[0585] In the device, apparatus or method of any of the foregoing embodiments, the pore area ratio (the ratio of the pore area to the total surface area) is 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 99%.

[0586] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the distance from hole edge to hole edge is greater than the hole depth, which ensures that the diffusion time from hole edge to hole edge is longer than the diffusion time from hole edge to hole bottom.

[0587] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the size of the wells is designed to ensure that no cross reactions occur during the analytical process.

[0588] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the number of wells on the first plate is much greater than the number of molecules in the sample.

[0589] A device, an apparatus or a method as described in any of the preceding embodiments, wherein if the concentration of the molecule is about 1 fM / μL, then the total number of wells on the first plate is 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000 times, 1000 to 10000 times of 600.

[0590] A device, an apparatus or a method as described in any of the preceding embodiments, wherein if the concentration of the molecule is about 1 pM / μL, the total number of wells on the first plate is 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000 times, 1000 to 10000 times of 600,000.

[0591] A device, an apparatus or a method as described in any of the preceding embodiments, wherein if the concentration of the molecule is about 1 nM / μL, then the total number of wells on the first plate is 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000 times, 1000 to 10000 times of 600,000,000.

[0592] A device, apparatus or method as described in any preceding embodiment, wherein the number of pores allows no more than one target molecule to be placed in a pore after closing the device.

[0593] A device, an apparatus or a method as described in any preceding embodiment, wherein at least one of the plates comprises an amplification surface.

[0594] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the device further comprises a thin sealing layer located between the first plate and the second plate, wherein in the closed configuration, the sealing layer is configured to prevent the sample or analyte in one microwell from moving to other microwells.

[0595] A device, apparatus or method as in any preceding embodiment, wherein the device further comprises a fixture, wherein in a closed configuration, the embodiment is configured to prevent a sample or analyte in one microwell from moving to other microwells.

[0596] In the device, apparatus or method as described in any of the preceding embodiments, the signal amplification layer comprises a metal material layer.

[0597] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the signal amplification layer comprises a metal material layer and a dielectric material layer located on top of the metal material layer, wherein the capture agent is located on the dielectric material.

[0598] The device, apparatus or method as described in any of the preceding embodiments, wherein the metal material layer is a uniform metal layer, a nanostructured metal layer or a combination thereof.

[0599] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the amplification layer comprises a metal material layer and a dielectric material layer on top of the metal material layer, wherein the capture agent is on the dielectric material and the dielectric material layer has a thickness of 0.5nm, 1nm, 5nm, 10nm, 20nm, 50nm, 00nm, 200nm, 500nm, 1000nm, 2μm, 3μm, 5μm, 10μm, 20μm, 30μm, 50μm, 100μm, 200μm, 500μm or within a range of any two values.

[0600] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the sample is deposited entirely on the one or both plates, and the closing step diffuses the sample onto and into at least some of the microwells.

[0601] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the method comprises depositing the samples together on a plate, pressing the second plate and separating the samples into wells, counting the wells to which the samples are applied, calculating the volume of the samples, counting the wells with signals, and calculating the concentration of the analyte in the samples.

[0602] A device, an apparatus or a method as described in any preceding embodiment, wherein the method comprises identifying which wells have not been loaded with sample.

[0603] A device, apparatus or method as described in any of the preceding embodiments, further comprising the step of measuring a signal associated with the target analyte in each of the microwells when the plate is in a closed configuration.

[0604] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the method comprises amplification, wherein the amplification makes the analyte more observable than the analyte without the amplification, and wherein the amplification comprises chemiluminescence, luminescence, nucleic acid amplification, ELISA (enzyme-linked immunosorbent assay), light enhancement using a plasmonic structure, or a chemical reaction.

[0605] The device, apparatus or method of any preceding embodiment further comprises counting the number of wells comprising the target analyte.

[0606] A device, an apparatus or a method as described in any preceding embodiment, wherein statistically each well will have no more than one target analyte molecule.

[0607] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the distribution of the target analyte in each well to which the sample is loaded follows a Poisson distribution.

[0608] The device, apparatus or method of any preceding embodiment further comprises determining the concentration of the target analyte in the sample.

[0609] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the target analyte is a protein, a nucleic acid, a small molecule, a cell or a particle.

[0610] A device, an apparatus or a method as described in any preceding embodiment, wherein the target analyte is a nucleic acid and the method comprises amplifying the nucleic acid.

[0611] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the amplification is performed by polymerase chain reaction (PCR).

[0612] A device, an apparatus or a method as described in any preceding embodiment, wherein the target analyte is analyzed using a binding assay.

[0613] The device, apparatus or method of any preceding embodiment, further comprising washing unbound target analyte from the device.

[0614] A device, apparatus or method as described in any of the preceding embodiments, wherein the method further comprises: partially or completely separating the two plates after the two plates have been closed; washing the original sample or adding another reagent; and then bringing the plates into a closed configuration.

[0615] A device, an apparatus or a method as described in any preceding embodiment, wherein the washing is performed using a sponge.

[0616] The device, apparatus or method of any preceding embodiment, wherein the method further comprises imaging the sample contact area.

[0617] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the imaging of the sample contact area measures the primary sum signal associated with the analyte from the sample contact area.

[0618] A device, an apparatus or a method as in any preceding embodiment, wherein said imaging of said sample contact area measures individual signals resulting from said individual binding events between capture agent and captured target analyte.

[0619] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the imaging of the sample contact area measures both (a) the aggregate signal associated with the analyte from the sample contact area and (b) the individual signals caused by the individual binding events between the capture agent and the captured target analyte.

[0620] A device, an apparatus or a method as described in any of the preceding embodiments, wherein the presence or concentration of the target analyte in the sample is determined by detecting the individual signals caused by the individual binding events between the capture agent and the captured target analyte.

[0621] A device, apparatus or method as described in any of the preceding embodiments, wherein the method comprises (i) identifying the empty wells by imaging the wells in a bright field image and / or by imaging prior to the amplification step, and (ii) quantifying the analyte concentration by subtracting the empty wells in a volume calculation, thereby subtracting the empty wells when determining the actual sample volume.

[0622] use

[0623] Among them, the present method can be used to detect and / or measure diseases such as cancer, infection or inflammatory diseases (see, for example, Tables 1-3 of WO2017058827), autoantibody epitopes (see Table 4 of WO2017058827), allergen epitopes (see Table 5 of WO2017058827), infectious agents (see, for example, Table 6 of WO2017058827), miRNA (see, for example, Table 7 of WO2017058827), environmental markers (see, for example, Table 8 of WO2017058827), For example, Table 8 of WO2017058827), food markers (see, for example, Table 9 of WO2017058827), small molecules, such as metabolites or drugs (e.g., THC-COOH (11-nor-9-carboxy-THC)), one or more molecules in cell-free DNA (cfDNA), including circulating tumor DNA (ctDNA), one or more molecules in RNA-free (cfRNA) cells, and cells, for example, circulating tumor cells, viruses or bacteria, etc.

[0624] In some embodiments, sample is body fluid or its processed form.Although several other body fluids can be used in the present method, interested body fluid includes blood plasma, saliva and urine.Body fluid includes but is not limited to amniotic fluid, aqueous humor, vitreous humor, blood (for example, whole blood, fractionated blood, blood plasma, serum etc.), breast milk, cerebrospinal fluid (CSF), cerumen (ear wax), chyle, chyme, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatism, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomitus and urine.In some embodiments, sample can be obtained from experimenter (for example, people), and can be processed before being measured for experimenter.For example, before analysis, protein can be extracted from tissue sample before starting the present method.In specific embodiments, sample can be clinical sample, for example, sample collected from patient.

[0625] Depending on the target analyte, the methods of the invention may have a sensitivity of at least 5 fM, 10 fM, 50 fM, 100 fM, 0.5 pM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM or 100 nM.

[0626] Without wishing to be bound to any particular use, the present method has a particular use in analyzing plasma. Plasma can be obtained non-invasively, and it contains a variety of different low-abundance proteins that are diagnostic, prognostic or therapeutic (see generally Anderson et al., Molecular & Cellular Proteomics 2002 1: 845-867 and Anderson et al., Clinical Chemistry 2010 56: 177-185). Therefore, in some embodiments, the present method can be used for quantifying any one or combination (e.g., 2, 3, 4, 5 or more) of the following proteins in plasma: acid phosphatase, IgG, alanine aminotransferase (ALT or SGPT), IgM, albumin, inhibin-A, aldolase, insulin. Alkaline phosphatase (ALP), insulin-like growth factor-I (IGF-I), alpha-1-acid glycoprotein (serum mucin), insulin-like growth factor-II (IGF-II), alpha-1-antitrypsin, IGFBP-1, alpha-2-antiplasmin, IGFBP-3, alpha-2-HS-glycoprotein, interleukin-2 receptor (IL-2R), alpha-2-macroglobulin, isocitrate dehydrogenase, alpha-fetoprotein (tumor marker), kappa light chain, amylase, lactate dehydrogenase heart part (LDH-1), amylase, liver part of lactate dehydrogenase (LLDH), ACE, lactoferrin, antithrombin III (ATIII), A light chain, apolipoprotein A1, lipase, apolipoprotein B, Lp(a), aspartate aminotransferase (AST or SGOT), lipoprotein-associated phospholipase A2 (LP-PLA2), 3-2 microglobulin, LH, 3-thromboglobulin, lysozyme, biotinidase, macrophage migration inhibitory factor (MIF), myeloperoxidase (MPO), cancer antigens 125 (CA125), myoglobin, cancer antigen 15-3 (CA15-3), osteocalcin, cancer antigen, human epididymis protein (HE4), parathyroid hormone, carcinoembryonic antigen (CEA), phosphohexose isomerase, ceruloplasmin, plasminogen, cholinesterase, plasminogen activator inhibitor (PAI), complement C1, prealbumin, complement C1 inhibitor, NTproBNP, complement C1Q, procalcitonin (PCT), complement C3, prolactin, complement C4, serum properdin factor B, Complement C5, prostatic acid phosphatase (PAP), CRP, prostate-specific antigen (PSA), creatine kinase-BB (CKBB), protein C, creatine kinase-MM (CKMM), protein S, cystatin C, pseudocholinesterase, erythropoietin, pyruvate kinase, factor IX antigen, renin, factor X, retinol binding protein (RBP), factor XIII, sex hormone binding globulin, ferritin, soluble mesothelin-related peptide, fibrinogen, sorbitol dehydrogenase (SDH), fibronectin,Thyroglobulin, FSH, TSH, GGT, thyroxine-binding globulin (TBG), haptoglobin, tissue plasminogen activator (T-PA), human chorionic gonadotropin (hCG), transferrin, hemopexin, transferrin receptor (TFR), her-2 / neu protein, troponin T (TnT); human growth hormone (HGH), TnI (heart), human placental lactogen (HPL), trypsin, IgA, urokinase, IgD, von Willebrand factor, IgE, nucleotidase, IgG subclass 4, ADAMTS13 activity and inhibitor, inhibin B (infertility), adenosine deaminase, IGFBP-2, adiponectin, intercellular adhesion molecule 1, pituitary glycoprotein hormone subclass interferon-β, α-galactosidase, interferon-α, EIA, α-N-acetylglucosaminidase, interleukin-1 receptor antagonist, amyloid 13-protein, interleukin-1 soluble receptor type II, angiotensinogen, interleukin-1α, anti-Müllerian hormone (AMH), interleukin-113, 3-glucuronidase, interleukin-2, 3-N-acetylglucosaminidase, interleukin-3, calprotectin, interleukin-4, cancer antigen 72-4, interleukin-5 cholecystokinin, interleukin-6, complement C2, interleukin-7, complement C4 binding protein, interleukin-8, complement C6, interleukin-9, complement C7 level, interleukin-10, complement C8 level, interleukin-11, complement C9 level , interleukin-12, corticosteroid binding globulin (corticosteroid transfer protein), interleukin-13, CYFRA21-1 (soluble cytokeratin fragment), interleukin-14, dopa decarboxylase, interleukin-15, elastase, interleukin-16, eosinophil cationic protein, interleukin-17, epidermal growth factor, interleukin-18, epidermal growth factor receptor (EGFR), kallikrein, factor II, leptin, factor V, leucine aminopeptidase, factor VII, mannose-binding lectin, factor VIII, neuron-specific enolase (NSE), factor XI, neurophysin, factor XII, pancreatic inhibin, fibroblast growth factor (FGF2), pepsinogen I, gastric inhibitory poly peptide (GIP), pepsinogen II, glial cell-derived neurotrophic factor (GDNF), glutathione peroxidase, proteasome activity, plasma-based Leumeta, granulocyte colony-stimulating factor, S-100B protein, granulocyte-macrophage colony-stimulating factor, soluble CD30, growth hormone binding protein; squamous cell carcinoma antigen, hemoglobin, thyrotropin-releasing hormone (TRH), heparin cofactor II, transforming growth factor-131, hexosaminidase A and total hexosaminidase, tumor necrosis factor receptor 1, high molecular weight kininogen, tumor necrosis factor receptor 2, human growth hormone-releasing hormone (HGH-RH), tumor necrosis factor-α, IgG subclass 1, tumor necrosis factor-13,IgG subclass 2, vascular endothelial growth factor (VEGF), IgG subclass 3, and vitamin D binding protein.

[0627] Obviously, the method can also be used to identify microbial (e.g., bacterial or viral) pathogens in clinical samples, for example, cell surface proteins or secreted proteins. In these embodiments, the capture agent can target proteins or other parts from the pathogen. If a circle is detected, the subject can be diagnosed as infected by the pathogen. The microorganisms that can be identified using the present method, composition and kit include but are not limited to: viruses, yeast, Gram (+) bacteria, Gram (-) bacteria, Enterobacteriaceae, Enterococcus bacteria, Staphylococcus bacteria and Campylobacter bacteria, Escherichia coli (E. coli), various strains of Escherichia coli (E. coli), such as K12-MG1655, CFT073, O157: H7ED1933, O157: H7VT2-Sakai, etc., Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, coagulase-negative Staphylococci, and various Candida species including white Candida albicans, Candida tropicalis, Candida dubliniensis, Candida visnatus, Candida parapsilosis, Klebsiella pneumoniae, various mycobacteria such as Mycobacterium tuberculosis, Mycobacterium bovis, BCG, Mycobacterium scrofula, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium gordonii, Mycobacterium ulcerans, Mycobacterium genus, Mycobacterium phage, Mycobacterium simianum, Mycobacterium fortuitum, Mycobacterium malmoe, Mycobacterium enteritidis, Mycobacterium haemophilus, Mycobacterium africanum, Listeria species, Chlamydia species, Mycoplasma species, Salmonella species, Brucella species, Yersinia species, etc. Therefore, the method can identify microorganisms to the level of genus, species, subspecies, strain or variant of microorganisms.

[0628] In some embodiments, the results of the method can be diagnostic (e.g., can provide a diagnosis of a disease or condition or type or stage of a disease or condition, etc.), prognostic (e.g., indicating a clinical outcome, e.g., survival or death within a time frame), or therapeutic (e.g., indicating which treatment will be most effective). In some embodiments, the method can be used to analyze a panel of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more analytes that are independently present at higher or lower concentrations relative to a control (e.g., an internal control), wherein the identity of the analytes and their abundance collectively correlate with a phenotype.

[0629] The method can be used to analyze a patient sample. In this embodiment, the method can include: (a) quantifying one or more analytes in the sample using the above method, and (b) providing a report indicating correlation with the phenotype. This embodiment can further include making a diagnosis, prognosis or treatment based on the report. The report can indicate a normal range for the analyte.

[0630] In some embodiments, the method may include creating a report as described above (which may be forwarded in electronic form from a remote location) and forwarding the report to a physician or other medical professional to determine whether the patient has a phenotype (e.g., cancer, etc.) or to identify an appropriate therapy for the patient. The report may be used as a diagnosis to determine whether the subject has a disease or condition, e.g., cancer. In certain embodiments, the method may be used to determine the stage or type of cancer, identify metastatic cells, or monitor a patient's response to treatment.

[0631] In any embodiment, the report may be forwarded to a "remote location", where "remote location" means a location other than the location where the image was examined. For example, the remote location may be another location (e.g., an office, a laboratory, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc. Thus, when one item is indicated as being "remote" from another, it means that the two items may be in the same room but separate, or at least in different rooms or different buildings, and may be at least one mile, ten miles, or at least one hundred miles apart. "Communicating" information means sending data representing the information as an electrical signal over an appropriate communication channel (e.g., a private or public network). "Forwarding" an item means any means of bringing the item from one location to the next, whether by physically transporting the item or otherwise (where possible), and at least in the case of data includes physically transporting a medium carrying the data or transmitting the data. Examples of communication media include radio or infrared transmission channels and network connections to another computer or networked device, as well as Internet or email transmissions and information recorded on a website, etc. In certain embodiments, the report may be analyzed by an MD or other qualified medical professional, and a report based on the image analysis results may be forwarded to the patient from whom the sample was obtained.

[0632] Notes:

[0633] Other embodiments according to the inventive subject matter of the present disclosure are described in the following enumerated paragraphs.

[0634] 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 "detection agent" includes a single detection agent and multiple detection agents, and reference to a "reagent" includes a single reagent and multiple reagents.

[0635] As used herein, the terms "adapted to" and "configured" mean that an element, component, or other subject matter is designed and / or intended to perform a given function. Thus, the use of the terms "adapted to" and "configured" should not be interpreted to mean that a given element, component, or other subject matter is simply "capable of" performing a given function. Similarly, a subject matter stated as being configured to perform a particular function may additionally or alternatively be described as being operable to perform that function.

[0636] As used herein, the use of the phrase "for example," the phrase "as an example," and / or simply the terms "example" and "exemplary," when referring to one or more components, features, details, structures, embodiments, and / or methods according to the present disclosure is intended to convey that the described components, features, details, structures, embodiments, and / or methods are illustrative, non-exclusive examples of components, features, details, structures, embodiments, and / or methods according to the present disclosure. Thus, the described components, features, details, structures, embodiments, and / or methods are not intended to be limiting, required, or exclusive / exhaustive; and other components, features, details, structures, embodiments, and / or methods, including structurally and / or functionally similar and / or equivalent components, features, details, structures, embodiments, and / or methods, are also within the scope of the present disclosure.

[0637] As used herein, the phrases "at least one" and "one or more" with respect to a list of more than one entity refer to any one or more entities in the list of entities, and are not limited to at least one of each and every entity specifically listed in the list of entities. For example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") may refer to A alone, B alone, or A and B in combination.

[0638] As used herein, the term "and / or" placed between a first entity and a second entity means one of (1) the first entity, (2) the second entity, and (3) the first entity and the second entity. Multiple entities listed using "and / or" should be interpreted in the same manner, i.e., "one or more" of the entities so conjoined. In addition to the entities specifically identified by the "and / or" clause, other entities may optionally be present, whether related or unrelated to those entities specifically identified.

[0639] When numerical ranges are mentioned herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other endpoint is excluded. Both endpoints should be assumed to be included unless otherwise stated. In addition, unless otherwise stated or obvious to one of ordinary skill in the art from the context and understanding.

[0640] In the event that any patent, patent application, or other reference is incorporated herein by reference and (1) defines a term in a manner inconsistent with the unincorporated portion of the present disclosure or the other incorporated reference and / or (2) is otherwise inconsistent with the unincorporated portion of the present disclosure or the other incorporated reference, the unincorporated portion of the present disclosure shall control and the term or the disclosure incorporated therein shall control solely with respect to the reference in which the term was defined and / or incorporated disclosure originally existed.

Claims

1. A device for performing a digital assay on a liquid sample containing or suspected of containing an analyte, comprising: a first plate, a second plate, a spacer, and microwells, wherein: (a) the first plate and the second plate are movable relative to each other into an open configuration and a closed configuration and have a sample contact area on their respective inner surfaces for contacting a fluid sample having a volume; (b) the second plate has the microwells in the sample contacting region, wherein each microwell has (i) a predetermined and known geometry, (ii) a pore depth of 200 μm or less, and (iii) a volume substantially smaller than the volume of the fluid sample, and (c) the spacers have a columnar shape and a uniform height, the spacers being secured within the microwells, or on the inner surface of the first plate, or both, and being configured to maintain a substantially uniform distance between the inner surface of the first plate and the bottom of the wells when in a closed configuration; wherein at least one of the first plate and the second plate is a flexible plate, the spacer has a separation distance (ISD), and the flexible plate has a Young's modulus (E) and a thickness (h), Where ISD is divided by the thickness of the flexible board (h) and the fourth power of Young's modulus (E) (ISD 4 / (hE)) is 5x10 6 μm 3 / GPa or less, and 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; wherein in the open configuration: the average spacing between the inner surface of the first plate and the edges of the microwells in the second plate is greater than the depth of the wells, and the sample is deposited on one or both plates; as well as wherein in the closed configuration, the closed configuration is formed after the sample is deposited in the open configuration; at least a portion of the sample is within the microwell, and the height of the spacer is selected so that the average spacing between the inner surface of the first plate and the edge of the microwell in the second plate is 1 / 2 or less of the depth of the microwell; wherein if the analyte concentration is about 1 fM / μL, the total number of microwells on the second plate is 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000 times, or 1000 to 10000 times greater than 600; If the analyte concentration is about 1 pM / μL, the total number of microwells on the second plate is 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000 times, or 1000 to 10000 times greater than 600,000; If the analyte concentration is about 1 nM / μL, the total number of wells on the second plate is 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000 times, 1000 to 10000 times greater than 600,000,000. The number of the microwells allows no more than one target molecule to be placed in each well after closing the device.

2. An apparatus comprising a thermal cycler and the device as claimed in claim 1.

3. An apparatus comprising a thermal cycler, the device as claimed in claim 1 and a reader for real-time PCR.

4. A method for separating a fluid sample, comprising: Obtain the device according to claim 1, depositing the sample on the one or both plates when the plates are in an open configuration, wherein the deposition is in the form of a single or multiple droplets of the sample, wherein at least one of the droplets has a volume that occupies more than two microwells; and The plate is closed into the closed configuration to separate the samples in the microwells.

5. The method of claim 4, wherein the analyte is a protein, a peptide, a nucleic acid, a virus, a bacterium, a cell, a nanoparticle, a molecule, a synthetic compound or an inorganic compound.

6. The device of claim 1, further comprising a second spacer configured to adjust a spacing between the first plate and the second plate.

7. The device of claim 1, further comprising a binding site located at the sample contact area of ​​the one or both plates, wherein the binding site comprises a capture agent immobilized at the site, and the capture agent is configured to specifically capture the analyte in the sample.

8. The device of claim 7, further comprising a surface amplification layer, the surface amplification layer being located on the sample contact area of ​​the one or both plates, wherein the surface amplification layer comprises a capture agent immobilized at the binding site, and the capture agent is configured to specifically capture the analyte in the sample, wherein the surface amplification layer amplifies an optical signal from the analyte or a label attached to the analyte, and the analyte is amplified more strongly when it is near the surface amplification layer than when it is micrometers or more away.

9. The device of claim 8, wherein the amplification factor of the surface amplification layer is adjusted to make the optical signal from a single label directly or indirectly bound to the capture agent visible.

10. The device of claim 7, wherein the device further comprises a reagent in the microwell of the second plate in the closed configuration, wherein the reagent produces a plurality of luminescent components in the microwell when the capture agent specifically binds to the analyte.

11. The device of claim 7, wherein the average spacing in the closed configuration is configured such that a saturation binding time of the analyte to the capture agent is 300 seconds or less.

12. The device of claim 7, wherein the average spacing in the closed configuration is configured such that a saturation binding time of the analyte to the capture agent is 60 seconds or less.

13. The device of claim 8, wherein the amplification factor of the surface amplification layer is adjusted to make the optical signal from a single label visible.

14. The device of claim 7, wherein the capture agent is a nucleic acid.

15. The device of claim 7, wherein the capture agent is a protein.

16. The device of claim 7, wherein the capture agent is an antibody.

17. The device of claim 7, wherein the capture agent is an aptamer.

18. The device of claim 10, wherein the capture agent is an aptamer.

19. The device of claim 1, further comprising a storage location located on the inner surface of the one or both plates, wherein the storage location comprises a reagent soluble in a liquid.

20. The device of claim 10, wherein the reagent is a reagent that amplifies an analyte in the sample.

21. The device of claim 10, wherein the reagent amplifies the analyte by polymerase chain reaction (PCR).

22. The device of claim 10, wherein the reagent is a detection agent.

23. The device of claim 1, wherein each microwell has a volume, and the volume of each microwell is configured such that the distribution of the target analyte in each well filled with the sample follows a Poisson distribution for the concentration of the expected analyte.

24. The device of claim 1 , wherein each microwell has a volume, and the volume of each microwell is configured for the concentration of an expected analyte so that the distribution of the analyte in each well filled with the sample is on average one target analyte per 2 microwells, 3 microwells, 5 microwells, 10 microwells, 20 microwells, 0 microwells, 50 microwells, 75 microwells, 100 microwells, 150 microwells, 200 microwells, 300 microwells, 500 microwells, 1000 microwells, 2000 microwells, 10000 microwells, 100,000 microwells, or within a range of any two values.

25. The device of claim 1, wherein the volume of each microwell is constructed based on the concentration of the expected analyte, so that the distribution of the target analyte in each microwell filled with the sample is an average of one target analyte per 10 microwells, 20 microwells, 30 microwells, 50 microwells, 75 microwells, 100 microwells, or within the range of any two values.

26. A device as described in claim 1, wherein in the closed structure, the average spacing between the inner surface of the first plate and the edge of the microwells in the second plate is less than 1 / 2, 1 / 3, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 11, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 100, 1 / 300 or 1 / 500 of the depth of the microwells.

27. The device of claim 1, wherein in the closed configuration, an inner surface of the first plate contacts edges of microwells in the second plate.

28. The device of claim 1, wherein in the closed configuration, the average distance between two adjacent microwells is less than 5 nm, 10 nm, 30 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, or 100 μm.

29. The device of claim 1, wherein the microwells have a shape selected from a circular, rectangular, hexagonal, or square grid.

30. The device of claim 1, wherein the microwells on the first plate have a hole-to-hole center distance of at least 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, or 1 mm on average.

31. The device of claim 1, wherein the microwells on the second plate have an average length or diameter of 1 nm.

32. The device of claim 1, wherein the microwells on the second plate have a depth of at least 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, or 1 mm.

33. The device of claim 1, wherein the micropores have (i) No metal coating, (ii) a metal coating at the bottom of the hole, (iii) a metal coating on the sidewalls of the hole, or (iv) There is a metal coating on both the bottom and the side walls of the hole.

34. The device of claim 33, wherein the metal is gold, aluminum, silver, copper, tin, or any combination thereof.

35. The device of claim 1, wherein the micropores have a pore area, and a ratio of the pore area to the total area of ​​the inner surface of the second plate is 40% to 50%, 50% to 60%, or 60% to 70%.

36. The device of claim 1, wherein the microwells have well edges, and the distance between two adjacent well edges is greater than the well depth, which ensures that the diffusion time from one well edge to another well edge is longer than the diffusion time from the well edge to the well bottom.

37. The device of claim 1, wherein the size of the microwells is designed to ensure that no cross-reaction occurs during the analysis.

38. The device of claim 1, wherein the number of microwells on the second plate is much greater than the number of analytes in the sample.

39. The device of claim 1, wherein the micropores have a pore area, and the ratio of the pore area to the total area of ​​the inner surface of the second plate is 70% to 80%, 80% to 90%, or 90% to 99%.

40. The device of claim 1, wherein at least one of the plates comprises an amplification surface.

41. The device of claim 1, wherein the device further comprises a thin sealing layer between the first plate and the second plate, wherein in a closed configuration, the sealing layer is configured to prevent the sample or analyte in one microwell from migrating into other microwells.

42. The device of claim 1, wherein the device further comprises a clamp, wherein in a closed configuration, the clamp is configured to prevent a sample or analyte in one microwell from migrating to other microwells.

43. The device of claim 8, wherein the surface amplification layer comprises a layer of metallic material.

44. The device of claim 8, wherein the surface amplification layer comprises a layer of metal material and a dielectric material located on top of the metal material layer, wherein the capture agent is located on the dielectric material.

45. The device of claim 44, wherein the metallic material is a uniform metallic layer, a nanostructured metallic layer, or a combination thereof.

46. ​​The device of claim 8, wherein the surface amplification layer comprises a layer of metal material and a dielectric material layer on top of the metal material layer, wherein the capture agent is above the dielectric material layer, and the dielectric material layer has a thickness of 0.5 nm to 500 μm.

47. The method of claim 4, wherein the sample is deposited entirely on the one or both plates and the closing step diffuses the sample through and into at least some of the microwells.

48. The method of claim 4, wherein the method comprises depositing the sample on a plate, pressing the second plate and separating the sample into wells, counting the wells filled with the sample, calculating the volume of the sample, counting the wells with signals, and calculating the concentration of the analyte in the sample.

49. The method of claim 4, wherein the method comprises identifying which wells are not filled with the sample.

50. The method of claim 4, further comprising the step of measuring a signal associated with a target analyte in each of said microwells when said plate is in a closed configuration.

51. The method of claim 4, wherein the method comprises amplification, wherein the amplification renders the analyte more observable than the analyte without the amplification, and wherein the amplification comprises chemiluminescence, nucleic acid amplification, ELISA, light enhancement using a plasmonic structure.

52. The method of claim 50, further comprising counting the number of wells containing the target analyte.

53. The method of claim 50, wherein statistically each well will have no more than one target analyte molecule.

54. The method of claim 50, wherein the distribution of the target analyte in each well filled with the sample follows a Poisson distribution.

55. The method of claim 50, further comprising determining the concentration of the target analyte in the sample.

56. The method of claim 50, wherein the target analyte is a protein, a nucleic acid, a small molecule, a cell, or a particle.

57. The method of claim 50, wherein the target analyte is a nucleic acid and the method comprises amplifying the nucleic acid.

58. The method of claim 57, wherein the amplification is performed by polymerase chain reaction (PCR).

59. The method of claim 50, wherein the target analyte is analyzed using a binding assay.

60. The method of claim 50, further comprising washing unbound target analyte from the device.

61. The method of claim 4, wherein the method further comprises: partially or completely separating the two plates after the two plates have been closed; flushing away the original sample or adding another reagent; and then bringing the plates into a closed configuration.

62. The method of claim 60, wherein the washing is performed using a sponge.

63. The method of claim 4, wherein the method further comprises imaging the sample contact area.

64. The method of claim 63, wherein the imaging of the sample contact area is measuring a one-time total signal associated with the analyte from the sample contact area.

65. The method of claim 61, wherein imaging of the sample contact area is by measuring individual signals resulting from individual binding events between the capture agent and the captured target analyte.

66. The method of claim 61, wherein the imaging of the sample contact area is a measurement of (a) a one-time aggregate signal from the sample contacting region associated with the analyte, and (b) Individual signals resulting from individual binding events between capture agent and captured target analyte, both.

67. The method of claim 66, wherein the presence or concentration of the target analyte in the sample is determined by detecting the individual signals resulting from the individual binding events between a capture agent and the captured target analyte.

68. A method as claimed in claim 51, wherein the method includes (i) identifying empty wells by imaging the wells in a bright field image and / or by imaging prior to the amplification step, and (ii) quantifying the analyte concentration by subtracting the empty wells in the volume calculation, thereby subtracting the empty wells when determining the actual sample volume.

Images