Parallel determination of cellular and non-cellular analytes in sample

Abstract

The present disclosure relates to the field of biological / chemical sampling, sensing, assays and applications; the invention specifically relates to parallel determination of cellular and non-cellular analytes in a sample; a device for analyzing target cellular and non-cellular analytes in a sample includes a first plate, a second plate, a plurality of spacers, a plurality of particles, and a trapping agent, the first plate and the second plate being movable relative to each other into an open configuration and a closed configuration; each plate is provided with a sample contact area; compressing the associated sample volume by the two plates into a layer of substantially uniform thickness and substantially stagnated with respect to the plates, and the uniform thickness of the layer being defined by the plates and adjusted by the plates and spacers; wherein the correlated sample volume is a sample portion that analyzes non-cellular analytes and target cells; and wherein the height of the spacer and the geometry of the particles are configured such that in the closed configuration there is no significant overlap between one or more target cells, between particles, and between target cells and particles.

Description

[0001] Cross-references

[0002] This application is a divisional application of the Chinese national phase application with application number 2019800217033 and filing date 2019-01-25. Like the parent application, this application is a national phase application (§371) of international application PCT / US19 / 15309, filed on January 25, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62 / 621,761, filed on January 25, 2018, and International Application No. PCT / US2019 / 013388, filed on January 11, 2019, and relies on and incorporates by reference the entire contents thereof.

[0003] The entire disclosure of any publication or patent document mentioned herein is incorporated by reference in its entirety. Technical Field

[0004] The present disclosure relates to the fields of biological / chemical sampling, sensing, assays and applications. background

[0005] A significant proportion of antibiotics are used to treat conditions that are rarely or completely resistant to antibiotics. Excessive and inappropriate use of antibiotics, leading to the emergence of increasingly drug-resistant bacteria, has become a serious problem worldwide. Individual healthcare practitioners are often faced with the challenge of identifying patients who require, and especially those who do not, require antibiotic treatment. Summary of the Invention

[0006] In certain embodiments, the present disclosure provides a device for determining target cells and non-cellular analytes in a liquid sample, comprising a first plate, a second plate, a plurality of spacers, a plurality of particles and a capture agent, wherein: the first plate and the second plate can be moved relative to each other into different configurations, including an open configuration and a closed configuration; each plate has a sample contact area on its respective surface for contacting a sample containing or suspected of containing one or more target cells and non-cellular analytes. The spacer comprises periodically arranged pillars of uniform height and is fixed to the first plate. The particles have a size of 0.2 μm to 100 μm and are randomly distributed in the sample contact area of ​​the first plate in an open configuration. The capture agent specifically binds to the non-cellular analyte, and the non-cellular analyte is capable of binding to form an optical marker of the labeled non-cellular analyte. wherein an open configuration is a configuration in which: two plates are separated and a sample is deposited on one or both plates; wherein a closed configuration is a configuration configured in the open configuration after sample deposition; wherein in the closed configuration, a relevant sample volume is compressed by the two plates into a layer of substantially uniform thickness and is substantially stagnant relative to the plates, and the uniform thickness of the layer is limited by the plates and regulated by the plates and spacers; wherein the relevant sample volume of the sample is analyzed for non-cellular analytes and target cells; and wherein the uniform thickness and geometry of the particles are configured such that in the closed configuration there is no significant overlap between one or more target cells, between the particles, and between target cells and particles.

[0007] In certain embodiments, the present disclosure provides a device for determining target cells and non-cellular analytes in a liquid sample, the device comprising: a first plate, a second plate, a plurality of spacers, a plurality of particles, and a capture agent, wherein: the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration; each plate has a sample contact area on its respective surface for contacting a sample containing or suspected of containing one or more target cells and non-cellular analytes; the spacer comprises an array of pillars having a uniform height of less than 150 μm fixed to the first plate. The particles have a size of 0.2 μm to 100 μm and are randomly distributed in the sample contact area of ​​the first plate in an open configuration. The capture agent specifically binds to the non-cellular analyte, and the non-cellular analyte is capable of binding to form an optical marker of the labeled non-cellular analyte. wherein the uniform thickness and geometry of the particles are configured such that in a closed configuration there is no significant overlap between one or more target cells, between the particles, and between target cells and particles, and the number of layers of uniform thickness is limited by the plate and regulated by the plate and the spacers; and wherein the relevant sample volume is a portion or the entire volume of the sample, wherein the relevant sample volume is analyzed for non-cellular analytes and target cells, and wherein the number of particles in the relevant sample volume is configured to be at least 10 in the closed configuration.

[0008] In certain embodiments, the present disclosure provides an apparatus for determining target cells and non-cellular analytes in a liquid sample, comprising: a first plate, a second plate, a plurality of spacers, a plurality of particles, a capture agent, an imager, and a processor, wherein: the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration; each plate has a sample contact area on its respective surface for contacting a sample containing one or more target cells and non-cellular analytes. The particles are sized from 0.2 μm to 100 μm and are randomly distributed in the sample in a closed configuration; the spacers comprise an array of pillars having a uniform height of 150 μm fixed to the first plate. A capture agent is attached to the particles, wherein the capture agent specifically binds to the non-cellular analyte, wherein the non-cellular analyte is capable of binding to form an optical marker of the labeled non-cellular analyte. an imager captures at least one image of a sample volume; the processor is configured to process the at least one image to (i) detect the non-cellular analyte by analyzing images of at least 10 particles, and (ii) detect the target cell analyte; wherein the open configuration is a configuration in which: two plates are separated and the sample is deposited on one or both plates; wherein the closed configuration is a configuration in which the sample is deposited on the open configuration; in the closed configuration, the relevant sample volume is compressed by the two plates into a layer of substantially uniform thickness and is substantially stagnant relative to the plates, and the uniform thickness of the layer is limited by the plates and regulated by the plates and spacers; wherein the uniform thickness and geometry of the particles are configured such that in the closed configuration there is no significant overlap between one or more target cells, between particles, and between target cells and particles; and wherein the relevant sample volume is a portion or all of the volume of the sample, and wherein the number of particles in the relevant volume is configured to be at least 10 in the closed configuration; wherein the processor is configured to process the at least one image to (i) detect the non-cellular analyte by analyzing images of at least 10 particles, and (ii) detect the target cell analyte.

[0009] In certain embodiments, the present disclosure provides a device comprising: a first plate; and a second plate. The second plate has a sample contact area on a surface thereof; wherein the first plate and the second plate are movable relative to each other into: an open configuration in which the first plate and the second plate are at least partially separated so that a sample comprising one or more target cells and a non-cellular analyte can be placed therebetween; and a closed configuration in which the first plate is placed on top of the second plate, thereby compressing at least a portion of the sample between the first plate and the second plate into a layer having a uniform thickness; one or more spacers, the one or more spacers comprising a periodic array of posts; and wherein the uniform thickness of the layer is modulated by one or more spacers, a plurality of particles having one or more capture agents disposed on their surfaces, wherein, in the closed configuration, the first plate and the second plate compress at least a portion of the sample such that there is substantially no overlap between (i) the one or more target cells, (ii) the plurality of particles, and (iii) the one or more target cells and the plurality of particles.

[0010] In certain embodiments, one or more spacers are fixed on the surface of the second plate. In certain embodiments, the diameter of the plurality of particles is 0.2 μm to 100 μm. In certain embodiments, when the first plate and the second plate are in an open configuration, the plurality of particles are randomly distributed in the sample contact area of ​​the second plate. In certain embodiments, the capture agent specifically binds to a labeled target analyte, which includes a non-cellular analyte attached to an optical label. In certain embodiments, the one or more spacers have a uniform height. In certain embodiments, the uniform height of the one or more spacers is at least about 0.8 times the particle size. In certain embodiments, the uniform height of the one or more spacers is less than about 200 μm. In certain embodiments, the uniform height of the one or more spacers is between at least about 0.8 times the particle size and about 200 μm. In certain embodiments, the device further includes an imager for obtaining an image of a relevant volume of the sample. In certain embodiments, the relevant volume is a portion or the entire volume of at least a portion of the sample, and wherein the number of particles in the relevant volume is configured to be at least 10 in a closed configuration. The device further includes a processor for processing the image. In certain embodiments, the processing comprises at least one of: (i) detecting a non-cellular analyte by analyzing one or more images of at least 10 particles, and (ii) detecting a target cellular analyte.

[0011] In certain embodiments, the present disclosure provides a method for determining cellular and non-cellular analytes in a liquid sample, comprising: having a device of the present disclosure; having a sample containing or suspected of containing one or more target cells and non-cellular analytes; having an optical tag for the non-cellular analyte capable of binding to the non-cellular analyte to form a label; depositing the sample in a sample contact area of ​​the device in an open configuration, and after step (d) forming the device into a closed configuration, thereby compressing at least a portion of the sample into a layer having a uniform thickness; using an imager to acquire at least one image of a sample volume associated with the sample, wherein the imager is capable of imaging the optical tag and the target cells. Analyzing the at least one image to detect the presence or amount of (i) target cells and / or (ii) non-cellular analytes.

[0012] In certain embodiments, the present disclosure provides a method for determining cellular and non-cellular analytes in a liquid sample, comprising: a device of the present disclosure; a sample containing or suspected of containing one or more target cells and non-cellular analytes; an optical label for the non-cellular analyte capable of binding to the non-cellular analyte to form a label; after step (d), depositing the sample in a sample contact area of ​​the device in an open state, placing the device in a closed state, thereby compressing at least a portion of the sample into a layer of uniform thickness; acquiring at least one image of a sample volume associated with the sample using an imager, wherein the imager is capable of imaging the optical label and target cells; analyzing at least one image using a processor to detect the presence or amount of (i) target cells and / or (ii) non-cellular analytes; wherein the processor is configured to process the at least one image to (i) detect the non-cellular analyte by analyzing images of at least 10 particles, and (ii) detect the target cell analyte.

[0013] In certain embodiments, when detecting a non-cellular analyte, images of at least 10 particles are analyzed by digital counting, wherein the digital counting determines the presence or amount of the non-cellular analyte in the sample. The number or percentage of beads having an optical signal above a threshold, wherein the percentage is the ratio of the number of beads above the threshold to the number below the threshold. In certain embodiments, when detecting a non-cellular analyte, images of at least 10 particles are analyzed by analog counting, wherein the analog counting determines the presence or amount of the non-cellular analyte in the sample from the optical signal. The amplitude of all beads in the relevant sample area is determined. In certain embodiments, the device or method further comprises two or more images of a common area / volume of the sample layer, wherein the common area of ​​the sample layer is the area of ​​the sample containing at least one of the one or more particles. In certain embodiments, the device or method further comprises two or more images, wherein at least one image is a darkfield image and at least one is a brightfield image. In certain embodiments, the thickness of the sample layer is sufficient to produce a monolayer of target cells between the plates. In certain embodiments, the thickness of the sample layer is sufficient to produce no substantial overlap between the one or more target cells between the plates. In certain embodiments, one or more particles have a geometry (shape and size) that is enough to (i) produce a monolayer of particles between the plates and (ii) there is no substantial overlapping between the particles between the plates. In certain embodiments, one or more particles have a density that is enough to (i) produce a monolayer of particles between the plates and (ii) there is no substantial overlapping between the particles between the plates. In certain embodiments, before the sample is deposited in the device, one or more particles are mixed with the sample. In certain embodiments, before the sample is deposited, one or more particles are placed on at least one of the first plate and the second plate, and then mixed with the sample when the sample is deposited on the device. In certain embodiments, one or more particles have various shapes. In certain embodiments, the maximum size of one or more particles is 0.05 μm to 100 μm. In certain embodiments, a non-cellular analyte is detected using a competitive assay, the step comprising providing a step of a labeled competitive detection agent that competes with the non-cellular analyte, if present, which is combined with the capture agent of the analyte. In certain embodiments, non-cellular analytes are detected using a non-competitive assay, the step comprising providing a detection agent for a label specifically bound to another binding site of the non-cellular analyte, wherein the other binding site is a binding group. Different from the binding site of the capture agent. In certain embodiments, one of the images is a direct image, which includes information on topological structure (i.e., geometric shape) and the position of the particle in a common area. Another image is a signal image, which is configured to include a signal from a labeled competitive detection agent as the main signal of the image. In certain embodiments, the sample holder includes a first plate and a second plate, and the first plate and the second plate can be moved relative to each other into different configurations, including an open configuration and a closed configuration.

[0014] In certain embodiments, the present disclosure provides a method for diagnosing a viral infection or a bacterial infection in a subject, comprising: obtaining a device comprising a first plate, a second plate, and a plurality of particles, the plurality of particles comprising one or more capture agents coupled thereto, wherein the plurality of particles are substantially disposed along at least one monolayer of the first plate and the second plate, wherein the one or more capture agents are capable of associating with an analyte, and wherein the device comprises: an open configuration, wherein the first plate and the second plate are at least partially separated, thereby exposing a sample contact area comprising the plurality of particles, the sample contact area being disposed on at least one of the first plate and the second plate; and a closed configuration, wherein the first plate and the second plate are substantially parallel and comprise a space therebetween. contacting the device with a sample comprising the analyte and one or more cells, wherein the device is in the open configuration; placing the device in the closed configuration so as to contain at least a portion of the sample within the space, wherein the thickness of the at least a portion of the sample is substantially uniform within the space, and wherein, in the closed configuration, the one or more cells within the space are substantially arranged as a monolayer; and capturing one or more images of a common area of ​​the thin sample layer, wherein the common area of ​​the sample layer is an area of ​​the sample comprising at least one of: one of the plurality of particles; and one of the one or more cells.

[0015] In certain embodiments, the method further comprises determining, using a mobile electronic device,: (i) one or more first values ​​corresponding to the amount of the analyte associated with the one or more capture agents; and (ii) one or more second values ​​corresponding to the number of the one or more cells within the sample. In certain embodiments, the plurality of particles is removably coupled to at least one of the first plate and the second plate. In certain embodiments, the plurality of particles is selected from the group consisting of glass beads, magnetic beads, polystyrene beads, latex particles, reporter molecules, fluorescent molecules, dye molecules, non-selective dye molecules, and redox-reactive molecules. In certain embodiments, the plurality of particles comprises a first plurality of particles and a second plurality of particles, wherein the first plurality of particles comprises one or more first capture agents coupled thereto, and wherein the second plurality of particles comprises one or more second capture agents coupled thereto. In certain embodiments, the one or more first capture agents are capable of associating with a first epitope on the analyte, and wherein the one or more second capture agents are capable of associating with a second epitope on the analyte. In certain examples, the first plurality of particles is disposed along the first plate, and the second plurality of particles is disposed along the second plate. In certain embodiments, the first plurality of particles is selected from the group consisting of glass beads, magnetic beads, polystyrene beads, and latex particles. In certain embodiments, the second plurality of particles is selected from reporter molecules, fluorescent molecules, dye molecules, non-selective dye molecules, and redox-responsive molecules. In certain embodiments, at least one of the one or more first capture agents and the one or more second capture agents is capable of specifically associating with the analyte. In certain embodiments, the device further comprises one or more spacers disposed within the space and along at least one of the first plate or the second plate. In certain embodiments, the one or more spacers determine the distance between the first plate and the second plate. In certain embodiments, the length of the one or more spacers along the direction from the first plate to the second plate is at most about 500 microns, at most about 250 microns, at most about 100 microns, at most about 90 microns, at most about 80 microns, at most about 70 microns, at most about 60 microns, at most about 50 microns, at most about 40 microns, at most about 30 microns, at most about 25 microns, at most about 20 microns, at most about 15 microns, at most about 10 microns, at most about 5 microns, at most about 4 microns, at most about 3 microns, at most about 2 microns, at most about 1 micron, at most about 0.5 microns, or at most about 0.1 micron. In certain embodiments, the length of the one or more spacers along the direction from the first plate to the second plate is less than or approximately equal to the average diameter of the one or more cells in the sample.In certain embodiments, the thickness of at least a portion of the sample within the space is at most about 500 microns, at most about 250 microns, at most about 100 microns, at most about 90 microns, at most about 80 microns, at most about 70 microns, at most about 60 microns, at most about 50 microns, at most about 40 microns, at most about 30 microns, at most about 25 microns, at most about 20 microns, at most about 15 microns, at most about 10 microns, at most about 10 microns, at most about 5 microns, at most about 4 microns, at most about 3 microns, at most about 2 microns, at most about 1 micron, at most about 0.5 micron, at most about 0.1 micron, or at most about 0.05 micron. In certain embodiments, the plurality of particles have various shapes. In certain embodiments, the maximum dimension of the plurality of particles is about 500 microns, about 250 microns, about 100 microns, about 90 microns, about 80 microns, about 70 microns, about 60 microns, about 50 microns, about 40 microns, about 100 microns. 30 microns, about 25 microns, about 20 microns, about 15 microns, about 10 microns, about 5 microns, about 4 microns, about 3 microns, about 2 microns, about 1 micron, about 0.5 microns, about 0.1 microns, or about 0.05 microns. In certain embodiments, the one or more cells within the space are substantially arranged as a non-confluent monolayer. In certain embodiments, the one or more cells within the space are in suspension. In certain embodiments, the determining comprises obtaining one or more images of an area of ​​at least a portion of the sample included in the space, wherein the imaged area includes at least one of the plurality of particles. In certain embodiments, the one or more first values ​​correspond to the intensity of energy emitted by the particle. In certain embodiments, the method further comprises contacting the sample with a cellular dye capable of associating with the one or more cells. In certain embodiments, the one or more second values ​​correspond to the intensity of the energy emitted by the cell dye. In certain embodiments, the method further includes using an algorithm to determine the relationship between the one or more first values ​​and the one or more second values. In certain embodiments, the method further includes classifying the infection level of the subject from which the sample was obtained based on the relationship between the one or more first values ​​and the one or more second values. In certain embodiments, the infection is a viral infection, a bacterial infection, or a combination thereof. In certain embodiments, the one or more cells are selected from leukocytes, leukocytes, granulocytes, granulocytes, myeloid cells, lymphoid cells, neutrophils, eosinophils, basophils, lymphocytes, T cells, B cells, natural killer cells, and monocytes. In certain embodiments, the analyte is selected from polypeptides, proteins, marker proteins, fusion proteins, antibodies, small molecules, viral particles, bacteria, C-reactive protein, and any fragment thereof.In certain embodiments, the method further includes contacting at least a portion of the sample with a competitive detection agent capable of associating with the one or more capture agents. In certain embodiments, the competitive detection agent competes with the analyte for binding to the one or more capture agents. In certain embodiments, the method further includes contacting at least a portion of the sample with a supplemental detection agent capable of associating with the analyte. In certain embodiments, the one or more capture agents are capable of associating with a first epitope on the analyte, and wherein the supplemental detection agent is capable of associating with a second epitope on the analyte. In certain embodiments, the method is performed in less than about 10 seconds, less than about 20 seconds, less than about 30 seconds, less than about 40 seconds, less than about 50 seconds, less than about 60 seconds, less than about 120 seconds. Less than about 180 seconds, less than about 240 seconds, less than about 300 seconds, less than about 400 seconds, less than about 500 seconds, less than about 1000 seconds, or less than about 2000 seconds. In certain embodiments, the method does not include contacting at least a portion of the sample with a wash buffer. In certain embodiments, the method is completed without any washing steps. In certain embodiments, the first plate and the second plate are pressed together by hand. In certain embodiments, the non-cellular analyte is a polypeptide, a nucleic acid or a metabolite. In certain embodiments, the sample is a body fluid. In certain embodiments, the body fluid is amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, chyle, endolymph, peripheral lymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomitus, urine and exhaled breath condensate. In certain embodiments, the method further includes diagnosing a disease based on the result. In certain embodiments, the disease is cancer, an inflammatory disease or an infectious disease. In certain embodiments, the method detects a variety of different non-cellular analytes.

[0016] In certain embodiments, the present disclosure provides a kit for determining target cells and non-cellular analytes in a liquid sample, comprising: a first plate, a second plate, one or more particles, and a capture agent, wherein: the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration; each plate has a sample contact area on its respective surface for contacting a sample containing one or more target cells and non-cellular analytes. The particles are sized from 0.2 μm to 100 μm and have a capture agent attached to their surface, wherein the capture agent specifically binds to the non-cellular analyte. wherein the open configuration is a configuration in which: the two plates are separated and the sample is deposited on one or both plates; wherein the closed configuration is a configuration configured in the open configuration after the sample is deposited; in the closed configuration: at least a portion of the sample is compressed into a layer of substantially uniform thickness by the two plates and is substantially stationary relative to the plates, and at least one particle is within at least a portion of the sample; and wherein the uniform thickness and geometry of the particles are configured such that, in the closed configuration, (i) the one or more target cells do not substantially overlap each other, and (ii) the particles do not substantially overlap each other.

[0017] In certain embodiments, the present disclosure provides a kit comprising: a device comprising a first plate; a second plate; a sample contact area disposed on one or both of the first plate and the second plate for contacting a sample comprising one or more particles having a capture agent attached to their surface; wherein the first plate and the second plate are movable relative to each other into an open configuration or a closed configuration, wherein the open configuration is that the first plate and the second plate are at least partially separated so that the sample can be deposited between them; wherein the closed configuration is that the first plate is placed on top of the second plate, thereby compressing at least a portion of the sample between the first plate and the second plate into a layer having a uniform thickness.

[0018] In certain embodiments, the kit further includes instructions for using the device. In certain embodiments, the sample comprises one or more target cells. In certain embodiments, the sample comprises one or more non-cellular analytes. In certain embodiments, the diameter of one or more particles is 0.2 μm to 100 μm. In certain embodiments, the capture agent is combined with the non-cellular analyte. In certain embodiments, at least a portion of the sample is compressed into a layer of substantially uniform thickness by the first plate and the second plate. In certain embodiments, at least a portion of the sample is substantially stagnant relative to the plate. In certain embodiments, the sample thickness in the closed structure is configured to (i) have a monolayer between the plates for one or more target cells, and (ii) there is no substantial overlap between the one or more target cells. In certain embodiments, one or more particles have a geometry (shape and size) configured to (i) have a monolayer between the plates for particles and (ii) there is no substantial overlap between the particles. In certain embodiments, one or more particles have a density configured to (i) have a monolayer between the plates for particles and (ii) there is no substantial overlap between the particles. In certain embodiments, the device also includes a spacer on one or both of the first plate and the second plate. In certain embodiments, the device further comprises a labeled competitive detection agent for competing with the analyte (if present) to bind to the capture agent of the analyte in a competitive assay. In certain embodiments, the device further comprises a labeled detection agent that specifically binds to an additional binding site of the analyte in a non-competitive assay, wherein the additional binding site is a binding site different from the binding site of the capture agent. In certain embodiments, the device further comprises an imager that images target cells and non-cellular analytes when the sample is between the two plates and the plates are in a closed configuration.

[0019] Incorporated by Reference

[0020] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. In some figures, the drawings are drawn to scale. In the figures providing experimental data points, the lines connecting the data points are used only to guide data viewing without other means. For clarity, some elements are enlarged when shown in the drawings. It should be noted that these drawings are not intended to strictly illustrate these elements. The dimensions of the elements should be delineated from the description provided herein and incorporated by reference. The drawings are not intended to limit the scope of the present invention in any way.

[0022] FIG1 provides a schematic diagram illustrating some exemplary methods of the present invention.

[0023] Figure 2 Schematic diagrams showing some exemplary methods of the invention are provided.

[0024] Figure 3 Schematic diagrams showing some exemplary methods of the invention are provided.

[0025] Figure 4 Schematic diagrams are provided showing some exemplary methods of the present invention. Detailed description

[0026] The following detailed description illustrates certain embodiments of the present invention by way of example and not limitation. The section headings and any subheadings used herein, if any, are for organizational purposes only and should not be construed as limiting the invention described in any way. The content under a section heading and / or subheading is not limited to the section heading and / or subheading, but applies to the entire description of the invention.

[0027] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0028] definition

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[0030] As used herein, the term "monolayer" can refer to a layer of cells that is 1 cell thick, or a layer of molecules that is 1 molecule thick. In some embodiments, the monolayer or cells or molecules can be confluent. In some embodiments, the monolayer of cells or molecules (e.g., analyte) can be subconfluent. In some embodiments, the monolayer of cells or molecules can be sparse.

[0031] The terms "link," "bind," "couple," and "attach" are used interchangeably and refer to covalent interactions (e.g., by chemical coupling) or non-covalent interactions (e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, hybridization, etc.), or binding of a molecule or cell to a surface. The terms "specific," "specifically," or "specifically" refer to preferential recognition, contact, and formation of a stable complex between a first molecule and a second molecule as compared to the first molecule and any one of a plurality of other molecules (e.g., there is little to no recognition, contact, or formation of a stable complex between a first molecule, such as a capture agent, and any one of a plurality of other molecules, such as an analyte).

[0032] The analyte or target analyte can be, but is not limited to, a polypeptide, a protein (eg, C-reactive protein), a protein fragment, a labeled protein, a fusion protein, an antibody, an antibody fragment, a small molecule, a viral particle, a cell, or any fragment thereof.

[0033] The subjects described herein can be of any age and can be adults, infants, or children. In some cases, the subject is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 years old, or within a certain range (e.g., between 2 and 20 years old, between 20 and 40 years old, or between 40 and 90 years old). A specific category of subjects that can benefit is a subject having or suspected of having an infection. Another special category of subjects that can benefit is a subject at a higher risk of infection. In addition, the subject treated by any method or composition described herein can be male or female. Any method, device or kit disclosed herein can also be performed on non-human subjects, such as laboratory or farm animals. Non-limiting examples of non-human subjects include dog, goat, guinea pig, hamster, mouse, pig, non-human primate (eg, gorilla, ape, orangutan, lemur, or baboon), rat, sheep, cow, or zebrafish.

[0034] As used herein, "virus" may refer to a living but non-cellular obligate intracellular parasite consisting of DNA or RNA and a protein coat. The diameter of a virus can range from about 20 to about 300 nm. The genome of a Class I virus (Baltimore classification) can have double-stranded DNA. A Class II virus can have single-stranded DNA as its genome. A Class III virus can have double-stranded RNA as its genome. The genome of a Class IV virus can have positive single-stranded RNA, while the genome itself is mRNA. The genome of a Class V virus can be used as a template for mRNA synthesis, and its negative single-stranded RNA may be negative. Class VI and Class VI viruses can have positive single-stranded RNA genomes, but have DNA intermediates not only in replication but also in mRNA synthesis. Viruses can be identified by the diseases they cause in plants, animals, and prokaryotes. Prokaryotic viruses are also called bacteriophages.

[0035] As used herein, "fungi" may refer to a division of eukaryotic organisms that grow in irregular clumps, lack roots, stems, or leaves, and do not contain chlorophyll or other pigments capable of photosynthesis. Each fungal organism can be unicellular to filamentous and have a branching somatic structure (e.g., hyphae) surrounded by a cell wall containing glucan or chitin or both, and containing a true nucleus.

[0036] As used herein, "infection" may refer to the invasion of the body of a multicellular organism by an organism with the potential to cause disease.

[0037] The term "embossing" refers to the integral attachment of the spacers to the panel by impressing (ie, embossing) a piece of material to form the spacers on the panel surface. The material may be a single layer of material or multiple layers of material.

[0038] The term "etched" means that the spacers and the plate are integrally affixed by etching a piece of material to form the spacers on the plate surface. The material can be a single layer of material or multiple layers of material.

[0039] The term "fused to" means that the spacer and the board are integrally fixed by bonding the spacer and the board together, original materials for the spacer and the board are fused to each other, and there is then a clear material boundary fusion between the two materials.

[0040] The term "bonded to" means that the spacer and the plate are integrally fixed by bonding the spacer and the plate through adhesion.

[0041] The term "attached to" means that the spacer and the plate are connected together.

[0042] It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments. From the scope or spirit of this teaching. Any narrated method can be performed in the order of events or in any other order that is logically possible. It will be understood by those skilled in the art that the application of the present invention is not limited to the details of the steps set forth in the description or drawings herein, such as the configuration, arrangement of components, class selection, weighting, predetermined signal limitations, or the like. The present invention can have other embodiments and can be practiced or executed in many different ways.

[0043] Rapid, homogeneous parallel staining of cellular and non-cellular analytes

[0044] A significant proportion of antibiotics are used to treat conditions for which antibiotics have little or no effect. Excessive and inappropriate use of antibiotics, leading to the emergence of increasingly drug-resistant bacteria, has become a serious problem worldwide. Individual healthcare practitioners are often faced with the challenge of identifying patients who require, and especially those who do not, require antibiotic treatment.

[0045] Imaging and / or analyzing cell and non-cellular analyte in parallel can be a useful tool for determining whether a subject suffers from disease (such as virus or bacterial infection).In some embodiments, cell and non-cellular analyte can be imaged and / or analyzed simultaneously (for example, a single image or visual field can be used to determine the amount of cell and non-cellular analyte).In some embodiments, cell and non-cellular analyte can be imaged and / or analyzed simultaneously (for example, a single image or visual field can be used to determine the amount of cell and non-cellular analyte).In some embodiments, series imaging and / or analysis (for example, the first image or the first visual field can be used to determine the quantity of cell, and the quantity of non-cellular analyte can be determined to use the second picture or the second visual field).In some embodiments, a single image or visual field can be used to determine one or more amounts corresponding to cell and / or non-cellular analyte.In some embodiments, two or more images or visual fields can be used to determine one or more amounts corresponding to cell and / or non-cellular analyte.

[0046] Typically, the amount of a cell or non-cell analyte can correspond to the number of cells or non-cell analytes in a sample. However, in some embodiments, the amount of a cell or non-cell analyte can correspond to the concentration of the cell or non-cell analyte in a sample. In some embodiments, the amount of a cell or non-cell analyte can correspond to a portion of the cell or non-cell analyte (e.g., a portion of cells having a given size or shape).

[0047] Analyte can generally be any molecule related to the presence or absence of a disease. Non-limiting examples of analyte include alpha-fetoprotein, prostate specific antigen, cardiac troponin, C-reactive protein (CRP) or human chorionic gonadotropin, carcinogens, influenza virus markers, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, Hanta virus, HTLV I, HTLVII or CMV infection, bacterial infection markers, fungal infection markers or parasitic infection markers. Cell can be any cell of the body. In some embodiments, cell can refer to a cell of the immune system. Non-limiting examples of immune system cells include leukocytes, leukocytes, granulocytes, granulocytes, myeloid cells, lymphoid cells, neutrophils, eosinophils, basophils, lymphocytes, T cells, B cells, natural killer cells and monocytes.

[0048] Embodiments of the present disclosure may include detecting or determining (i) the number of cells in a sample and (ii) the amount of a non-cellular analyte. In some embodiments, the method may further include determining whether the subject has a condition based on the amount of cells and the amount of non-cellular analyte in the sample. In some embodiments, the condition will be a condition that causes a change in the amount of cells or non-cellular analytes in the sample. In some embodiments of the present disclosure, analyzing cells and non-cellular analytes in a sample can be used to determine whether the sample being analyzed was obtained from a subject with an immune system disease. As used herein, an "immune system disease or condition" can refer to a pathological condition caused by a defect in the immune system. If a person is born with a severely defective immune system, they may die from a viral, bacterial, fungal, or parasitic infection. In severe combined immunodeficiency, a lack of enzymes may mean that toxic waste accumulates within immune system cells, killing them and thereby destroying the immune system. A lack of immune system cells also underlies DiGeorge syndrome: improper development of the thymus may mean that the production of T cells is reduced. Most other immune diseases are caused by an excessive immune response or "autoimmunity." For example, asthma, familial Mediterranean fever, and Crohn's disease (inflammatory bowel disease) are all caused by an overreaction of the immune system, while autoimmune polyglandular syndrome and certain aspects of diabetes are caused by the immune system attacking "self" cells and molecules. A key part of the immune system's function is to distinguish between invaders and the body's own cells; if this distinction cannot be made, the reaction against "self" cells and molecules can lead to autoimmune diseases.

[0049] In some embodiments of the present disclosure, cellular and non-cellular analytes in an analytical sample can be used to determine whether the sample being analyzed was obtained from a subject suffering from a metabolic disease. As used herein, a "metabolic disease or disorder" can refer to a pathological condition caused by an error in the metabolic process. Metabolism can be the way the body synthesizes energy from fats, carbohydrates, and proteins in food through enzymatic reactions of minerals and vitamins and synthesizes other molecules it needs. This system has a high tolerance for error: generally, a mutation in one enzyme does not mean that the person can develop a certain disease. Many different enzymes may compete to modify the same molecule, and for multiple metabolic intermediates, there may be more than one way to achieve the same end result. Disease can occur if a key enzyme loses function or the control mechanisms of a metabolic pathway are affected.

[0050] In some embodiments of the present disclosure, analyzing cellular and non-cellular analytes in a sample can be used to determine whether the sample being analyzed was obtained from a subject suffering from a muscle or bone disease. As used herein, "muscle and bone disease or disorder" can refer to a pathological condition caused by defects in genes important for the formation and function of muscle and connective tissue. Connective tissue is used herein as a broad term to include bone, cartilage, and tendon. For example, defects in fibrillin (a connective tissue protein that is important for making tissues strong yet flexible) can cause Marfan syndrome, while catastrophic dysplasia is caused by defects in sulfate transporters in cartilage.

[0051] In some embodiments of the present disclosure, analysis of cellular and non-cellular analytes in a sample can be used to determine whether the sample being analyzed was obtained from a subject suffering from a neurological disorder. Neurological disorders can refer to pathological conditions caused by defects in the nervous system, including the central nervous system, i.e., the brain, and the peripheral nervous system. The brain and nervous system form a complex network of electrical signals responsible for coordinating muscles, sensations, speech, memory, thoughts, and emotions. Several diseases that directly affect the nervous system have a genetic component: some are caused by mutations in a single gene, while others have been shown to have more complex inheritance patterns.

[0052] In some embodiments of the present disclosure, analysis of cells (e.g., white blood cells) and non-cellular analytes (e.g., carcinogens) in a sample can be used to determine whether the sample being analyzed is obtained from a subject suffering from cancer. The subject can be suffering from any type of cancer. Examples of cancer can include, but are not limited to, adrenal cancer, anal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, bronchial cancer, and liver cancer. Cardiovascular system, cervical cancer, colon cancer, colorectal cancer, digestive system cancer, endocrine system cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal tumors, kidney cancer, hematopoietic system malignancies, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, muscular system cancer, myelodysplastic syndrome (MDS), myeloma, nasal cavity cancer, nasopharyngeal cancer, nervous system cancer, lymphatic system cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penis cancer, pituitary tumor, prostate cancer, rectal cancer, renal pelvis cancer, reproductive system cancer, reservoir cancer of the respiratory system, sarcoma, salivary gland cancer, skeletal system cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, laryngeal cancer, thymus cancer, thyroid cancer, tumor, urinary system cancer, uterine cancer, vaginal cancer, or vulvar cancer. The term "lymphoma" can refer to any type of lymphoma, including B-cell lymphoma (e.g., diffuse large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system lymphoma) or T-cell lymphoma (e.g., precursor T-lymphocytic lymphoma or peripheral T-cell lymphoma). The term "leukemia" can refer to any type of leukemia, including acute leukemia or chronic leukemia. Types of leukemia include acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, acute undifferentiated leukemia, or chronic lymphocytic leukemia. In some cases, a cancer patient does not have a specific type of cancer. For example, in some cases, a patient may have a cancer other than breast cancer.

[0053] The capture agent with reporter molecule can be used for detecting cell and / or non-cellular analyte.For example, α-pyridinium orange can be used for the leukocyte in dyeing sample.The limiting examples of reporter molecule include cyanine 5 (Cy5), histidine (His), V5, FLAG, influenza hemagglutinin (HA), Myc, VSV-G and thioredoxin (Trx), glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and automatic fluorescent protein, including blue fluorescent protein (BFP).For example, the anti-CRP antibody (such as capture agent) putatively conjugated to GFP can be used for determining the presence or amount of CRP in sample.

[0054] A staining agent (e.g., dye) can be used to make cells (e.g., nucleated cells) detectable. For example, a sample can be stained with apyridine orange (AO), which is a dye that can stain living cells and certain components of the cytoplasm. The absorption peak of apyridine orange is at 490 nm, and it emits at 520 nm when bound to DNA. Other fluorescent dyes can be used, such as Hoechst 33258 and Hoechst 33342. Generally, any fluorescent dye that non-specifically stains cells, cytoplasm, cellular nucleic acid material, or the nucleus itself can be used. These dyes are referred to herein as "non-specific fluorescent dyes." In some embodiments, a capture agent or stain can be applied to one or more surfaces of the first plate and / or the second plate. It is contemplated that applying a capture agent or stain to one or more surfaces of the first plate or the second plate can be used to reduce the number of steps that a user performs in order to analyze a sample. For example, if the first plate is coated with a cell staining reagent, the user only needs to deposit the sample onto a QMAX card or a Q quality card (e.g., within the sample contact area). After contacting the cell staining reagent, the cells in the sample can be stained automatically (e.g., without user intervention). The number of steps that reduce user execution can reduce error (such as human error), improve the accuracy of data analysis, and reduce the time required for analysis sample.In some embodiments, capture agent can be coated on one or more surfaces of the first plate, and staining agent can be coated on one or more surfaces of the second plate.In another embodiment, capture agent and staining agent can be coated on one or more surfaces of the first plate or the second plate.In another embodiment, capture agent and staining agent can be coated on one or more surfaces of the first plate and the second plate.

[0055] As an example, CRP (C-reactive protein) is a protein that is normally present in very low concentrations (<1 μg / mL) in the blood of healthy people. In bacterial infections, CRP concentrations increase significantly (>10 μg / mL), while viral infections typically cause only very modest CRP elevations or no elevation at all. Therefore, CRP can be used as a biomarker to clinically distinguish bacterial from viral infections. Other biomarkers in this category include white blood cell (WBC) count, lymphocyte (LYM) count, and PCT (calcitonin).

[0056] The quantity of analyzing cell and non-cellular analyte can include determining the quantity of cell (such as leukocyte), determining the quantity (such as CRP) of non-cellular analyte, and comparing these values ​​with one or more reference values, to determine whether the subject of obtaining analysis sample therefrom suffers from disease.In some embodiments, the amount of analyzing cell and non-cellular analyte can include determining the ratio of the amount of cell and the amount of non-cellular analyte, and comparing this ratio with reference value, to determine whether the sample obtained from analyzing subject is conditional.In certain embodiments, the measurement result (such as, the quantity of cell or the quantity of non-cellular analyte) obtained from healthy subjects or the subject suffering from disease can be stored in a database, and is used to determine reference value, for comparing with future measurement.

[0057] Example 1

[0058] Rapid, homogeneous parallel staining of cellular and non-cellular analytes

[0059] Here, we describe a one-step combined blood test that reports CRP, WBC, and LYM levels or counts within 60 seconds. The combination of the three tests (CRP, WBC, and LYM) yields high sensitivity and specificity, allowing differentiation between bacterial and viral infections and therefore being valuable for antibiotic administration.

[0060] In the local reading method, as shown in Figure 1, one or more particles will be measured for the following two measurements: (a) The signal from the particle area (SP). It can be from the entire particle area or from a specified area of ​​the particle area. (b) The signal from the area around the particle (local background SB). It can be from the entire area around the particle or a specified area. The definition of "surrounding" can be a distance from the outer surface of the particle of 0.01D, 0.1D, 0.2D, 0.5D, 1D, 2D, 5D, 10D, 50D or a range between any two values. Where "D" is the average diameter of the particle. The true measurement signal (SA) of each particle can be determined as SA = SP-SB. The measurement signal from each CROF (SCROF) can be the average of multiple particles. It can be all particles on the entire CROF or all particles in a specified area of ​​the CROF (for example, SCROF = average (SA1, SA2, SA3...SAn))

[0061] In this experiment, the device for immunoassay includes a first plate and a second plate. Figure 3As shown in Figure 1, capture antibodies are first conjugated to magnetic beads. Next, as shown in Figure 2, a single X-plate is coated with antibody-coupled beads mixed with a WBC staining reagent. As shown in Panel 3, a second X-plate is then coated with a labeled anti-CRP detection antibody. In Panel 4, after each X-plate has been prepared, the Q-card is ready to receive a drop of blood. As shown in Figure 5, after a drop of whole blood has been applied, the X-plates are quickly pressed together to dissolve the reagents and initiate the assay. Subsequently, as shown in Figure 6, after 30 seconds, the assay can be analyzed for WBC count and CRP concentration.

[0062] -The experiments were conducted using the following materials:

[0063] - Anti-human CRP rabbit polyclonal antibody (Abcam)

[0064] -PureProteome NHS FlexiBind Magnetic Beads (Millipore)

[0065] -Human CRP Analyte (Fitzgerald)

[0066] -4% bovine serum albumin

[0067] -PBS (phosphate buffered saline)

[0068] -PBS-Tween 20 (PBST)

[0069] -X-plate with 10μm spacers

[0070] -Whole blood

[0071] -cridine orange

[0072] Perform the experiment according to the following procedure:

[0073] 1. Conjugation of anti-CRP capture antibody to beads: NHS-activated beads (10 μm diameter micropores) were coupled to anti-CRP mouse monoclonal capture antibody (Fitzgerald) according to the manufacturer's protocol.

[0074] 2. Bead Blocking Treatment: Block the antibody-coupled beads overnight with 4% BSA in PBS. The next day, wash the beads six times with PBST before use.

[0075] 3. First plate coating: 1 μL of antibody-conjugated beads (bead concentration 107–108 / mL) from step 2 with or without 25 μg / mL cridine orange (AO, WBC staining reagent) was dropped onto the Q card and air-dried at room temperature.

[0076] 4. Homogenized BEST assay. 1 μL of fresh whole blood and 1 μL of Cy5-labeled anti-CRP mouse monoclonal detection antibody (Fitzgerald) were mixed and then dropped onto the same area of ​​the Q card occupied by the dried, antibody-conjugated magnetic beads. The mixture was immediately covered with a second X-plate containing 10 μm columns. The assay was incubated for 30 seconds before imaging.

[0077] 5. Imaging. Without washing, use your iPhone 6s to capture two fluorescence images at different wavelengths to observe the staining signal of WBCs or the signal from the anti-CRP detection antibody.

[0078] In another experiment, a device for immunoassay includes a first plate and a second plate. Figure 4 As shown in the first image, capture antibodies are first conjugated to magnetic beads. Next, as shown in the second image, one X-plate is coated with antibody-conjugated beads. As shown in the third image, a second X-plate is then coated with labeled anti-CRP detection antibodies. In the fourth image, after each X-plate is prepared, the Q-card is ready to receive a drop of blood mixed with WBC staining reagent. As shown in the fifth image, after adding a drop of whole blood mixed with the WBC staining reagent, the X-plates are quickly pressed together to dissolve the reagents and initiate the assay. Subsequently, as shown in the sixth image, after 30 seconds, the assay can be analyzed for WBC count and CRP concentration.

[0079] QMAX System

[0080] A) QMAX card

[0081] Details of the QMAX card are described in detail in various publications, including International Application No. PCT / US2016 / 046437 (Essenlix Docket No. ESSN-028WO), which is incorporated herein by reference for all purposes.

[0082] I.Board

[0083] In the present invention, the plates of the CROF are generally made of any material that: (i) is capable of adjusting the thickness of a portion or the entire volume of the sample in conjunction with the spacers, and (ii) does not significantly adversely affect the sample, assay, or objective to which the plate is applied. However, in certain embodiments, the specific materials used for the plates (and therefore their properties) may be used to achieve certain objectives.

[0084] In certain embodiments, the two panels have the same or different parameters for each of the following parameters: panel material, panel thickness, panel shape, panel area, panel flexibility, panel surface properties, and panel optical transparency.

[0085] (i) Plate material. The plate is made of a single material, a composite material, multiple materials, a multilayer material, an alloy, or a combination thereof. Each material used for the plate may be an inorganic material, an organic material, or a mixture, where examples of the materials are given in the paragraphs of Mat-1 and Mat-2.

[0086] Mat-1: The inorganic materials of the plate include but are not limited to glass, quartz, oxides, silicon dioxide, silicon nitride, hafnium oxide (HfO), aluminum oxide (AlO), semiconductors: (Si, GaAs, GaN, etc.), metals (such as gold, silver, copper, aluminum, Ti, Ni, etc.), ceramics or any combination thereof.

[0087] Mat-2: Organic materials used for insulators include but are not limited to polymers (such as plastics) or amorphous organic materials. The polymer material for the spacer includes, but is not limited to, acrylate polymers, vinyl polymers, olefin polymers, cellulosic polymers, non-cellulosic polymers, polyester polymers, nylon, cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), polycarbonate (PC), cyclic olefin polymer (COP), liquid crystal polymer (LCP), polyamide (PA), polyethylene (PE), polyimide (PI), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyetheretherketone (PEEK), polyethersulfone (PES), polyethylene phthalate (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polydimethylsiloxane (PDMS), rubber or any combination thereof.

[0088] In some embodiments, the plates are each independently made of at least one of glass, plastic, ceramic, and metal. In some embodiments, each plate independently comprises at least one of glass, plastic, ceramic, and metal.

[0089] In some embodiments, one plate is different from another plate in lateral area, thickness, shape, material, or surface treatment. In some embodiments, one plate is the same as another plate in lateral area, thickness, shape, material, or surface treatment.

[0090] The material of the panels can be rigid, flexible or anything in between. Rigidity (ie, stiffness) or flexibility is relative to a given pressure used to move the panels into a closed configuration.

[0091] In certain embodiments, the choice of a rigid or flexible plate is determined by the need to control the uniformity of the sample thickness in the closed configuration.

[0092] In some embodiments, at least one of the two plates is transparent (to light). In some embodiments, at least a portion or portions of one or both plates are transparent. In some embodiments, the plates are opaque.

[0093] (ii) Plate Thickness. In certain embodiments, at least one plate has an average thickness of 2 nm or less, 10 nm or less, 100 nm or less, 500 nm or less, 1000 nm or less, 2 μm (micrometers) or less, 5 μm or less, 10 micrometers or less, 20 micrometers or less, 50 micrometers or less, 100 micrometers or less, 150 micrometers or less, 200 micrometers or less, 300 micrometers or less, 500 micrometers or less, 800 micrometers or less, 1 mm (millimeter) or less, 2 mm or less, 3 mm or less, or a range between any two values.

[0094] In certain embodiments, the average thickness of at least one plate is at most 3 mm (millimeters), at most 5 mm, at most 10 mm, at most 20 mm, at most 50 mm, at most 100 mm, at most 500 mm, or a range between two values.

[0095] In some embodiments, the thickness of the plate is not uniform across the plate. Using different plate thicknesses at different locations can be used to control plate bending, folding, sample thickness adjustment, etc.

[0096] (iii) Plate shape and area. Generally, the plate can have any shape that allows for compressed, open flow of the sample and adjustment of sample thickness. However, in certain embodiments, a specific shape may be advantageous. The plate shape can be circular, oval, rectangular, triangular, polygonal, annular, or any combination of these shapes.

[0097] In some embodiments, the two plates can have the same size or shape, or different sizes or shapes. The area of ​​the plates depends on the application. The area of ​​the plates can be up to 1 square millimeter, up to 10 square millimeters, up to 100 square millimeters, up to 1 square centimeter, up to 5 square centimeters, up to 10 square centimeters, up to 100 square centimeters, up to 500 square centimeters, up to 1000 square centimeters, up to 5000 square centimeters, up to 10,000 square centimeters, or up to 10,000 square centimeters, or any permutation therebetween. The shape of the plates can be rectangular, square, circular, or other shapes.

[0098] In some embodiments, at least one of the panels is in the form of a strip (or ribbon) having a width, a thickness, and a length. The width can be at most 0.1 cm, at most 0.5 cm, at most 1 cm, at most 5 cm, at most 10 cm, at most 50 cm, at most 100 cm, at most 500 cm, at most 1000 cm, or any range between any two values. The length can be any desired length. The strip can be rolled into a roll.

[0099] (iv) Plate surface flatness. In many embodiments, the inner surface of the plate is flat or remarkably flat, flat. In certain embodiments, the two inner surfaces are parallel to each other in the closed configuration. A flat inner surface can facilitate quantification and / or control of sample thickness by simply using a predetermined spacer height in the closed configuration. For non-flat inner surfaces of the plate, not only the spacer height needs to be known, but also the exact topology of the inner surface needs to be known to quantify and / or control the sample thickness in the closed configuration. Knowing the surface topology requires additional measurements and / or corrections, which can be complex, time consuming, and expensive.

[0100] The flatness of the panel surface is relative to the final sample thickness (the final thickness is the thickness in the closed configuration) and is often characterized in terms of "relative surface flatness," which is the ratio of the final sample thickness to the final sample thickness.

[0101] In some embodiments, the relative surface is less than 0.01%, 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 30%, less than 50%, less than 70%, less than 80%, less than 100%, or a range between any two of these values.

[0102] (v) Plate Surface Parallelism. In some embodiments, the two surfaces of the plate are substantially parallel to each other. In some embodiments, the two surfaces of the plate are not parallel to each other.

[0103] (vi) Plate Flexibility. In some embodiments, the plates are flexible under the compression of the CROF process. In some embodiments, both plates are flexible under the compression of the CROF process. In some embodiments, one plate is rigid and the other plate is flexible under the compression of the CROF process. In some embodiments, both plates are rigid. In some embodiments, both plates are flexible, but with different degrees of flexibility.

[0104] (vii) Optical transparency of the panels. In some embodiments, the panels are optically transparent. In some embodiments, both panels are optically transparent. In some embodiments, one panel is optically transparent while the other panel is opaque. In some embodiments, both panels are opaque. In some embodiments, both panels are optically transparent, but with different light transmittances. The optical transparency of a panel can refer to a portion or the entire area of ​​the panel.

[0105] (viii) Surface wetting properties. In certain embodiments, the inner surface of the plate wets (i.e., has a contact angle less than 90 degrees) the sample, transfers the liquid, or both. In certain embodiments, both plates have inner surfaces that wet the sample, transfer the liquid, or both. They have the same or different wettability. In certain embodiments, a plate has an inner surface that wets the sample, transfers the liquid, or both. The inner surface of the other plate does not wet (i.e., has a contact angle equal to or greater than 90 degrees). Wetting of the inner surface of a plate can refer to a portion or the entire area of ​​the plate.

[0106] In some embodiments, the inner surface of the plate has other nano- or microstructures to control the lateral flow of the sample during CROF. Nano- or microstructures include, but are not limited to, channels, pumps, etc. Nanostructures and microstructures are also used to control the wetting properties of the inner surface.

[0107] II. Spacer

[0108] (i) Function of the spacer. In the present invention, the spacer is configured to have one or any combination of the following functions and characteristics: the spacer is configured to (1) control the thickness of the sample or the relevant volume of the sample in conjunction with the plate (preferably, the thickness control is precise, uniform, or both within the relevant area); (2) allow the sample to have a compressed regulated open flow (CROF) on the surface of the plate; (3) not occupy a significant surface area (volume) in a given sample area (volume); (4) reduce or increase the effect of particle or analyte sedimentation in the sample; (5) change and / or control the wettability of the inner surface of the plate; (6) determine the position, size ratio and / or information related to the plate, or (7) any combination of the above.

[0109] (ii) Spacer structure and shape. To achieve the desired sample thickness reduction and control, in certain embodiments, spacers are fixed to their respective plates. Generally, the spacers can have any shape as long as they can adjust the sample thickness during the CROF process, but certain shapes are preferred to achieve certain functions (e.g., better uniformity, degree of overshoot in pressing, etc.).

[0110] The spacer can be a single spacer or a plurality of spacers (e.g., an array). Certain embodiments of the plurality of spacers are arrays of spacers (e.g., pillars) where the distance between the spacers is periodic or aperiodic, or periodic or aperiodic in certain regions of the plate, or has different distances in different regions of the plate.

[0111] There are two types of spacers: open and closed. Open spacers allow sample flow through the spacer (i.e., the sample flows through the spacer, such as a pillar). Closed spacers prevent sample flow (i.e., the sample cannot flow through the spacer, such as a ring-shaped spacer, where the sample is inside the ring). Both types of spacers use their height to adjust the final sample thickness in a closed configuration.

[0112] In some embodiments, the spacers are only open spacers. In some embodiments, the spacers are only closed spacers. In some embodiments, the spacers are a combination of open spacers and closed spacers.

[0113] The term "columnar spacer" means that the spacer has a cylindrical shape, and the cylindrical shape may refer to an object having a height and side shape that allows a sample to flow around it during compressed open flow.

[0114] In some embodiments, the lateral shape of the columnar spacer is a shape selected from the group consisting of: (i) a circle, an ellipse, a rectangle, a triangle, a polygon, a ring, a star, a letter shape (e.g., an L-shape, a C-shape, letters A to Z), a number shape (e.g., shapes such as 0, 1, 2, 3, 4, ... to 9); (ii) a shape from group (i) having at least one rounded corner; (iii) a shape from group (i) having a jagged or rough edge; (iv) any combination of (i), (ii), and (iii). For multiple spacers, different spacers may have different lateral shapes and sizes and different distances from adjacent spacers.

[0115] In certain embodiments, the spacers can be and / or include columns, posts, beads, spheres, and / or other suitable geometric shapes. The lateral shape and dimensions of the spacers (i.e., the dimensions transverse to the surface of the corresponding plates) can be anything, except that in certain embodiments, the following limitations apply: (i) the geometry of the spacers does not cause significant errors in measuring sample thickness and volume; and (ii) the geometry of the spacers does not prevent sample flow between the plates (i.e., it is not a closed form). However, in certain embodiments, it is desirable for some spacers to be closed spacers to restrict sample flow.

[0116] In some embodiments, the shape of the spacer has rounded corners. For example, a rectangular spacer has one, several, or all chamfered corners (e.g., rounded rather than 90-degree angles). Rounded corners generally make the spacer easier to manufacture and, in some cases, less damaging to the biological material.

[0117] The sidewalls of the pillars can be straight, curved, angled, or have different shapes in different sections of the sidewalls. In some embodiments, the spacers are pillars having various lateral shapes, sidewalls, and pillar height to pillar side area ratios. In a preferred embodiment, the spacers have a pillar shape designed to allow open flow.

[0118] (iii) Spacer material. In the present invention, the spacer is generally made of any material that can be used with the two plates to adjust the thickness of the relevant volume of the sample. In some embodiments, the material used for the spacer is different from the material used for the plates. In some embodiments, the material used for the spacer is at least partially the same as the material used for at least one of the plates.

[0119] The spacer is made of a single material, a composite material, multiple materials, a multilayer material, an alloy, or a combination thereof. Each material used for the spacer can be inorganic, organic, or a mixture. Examples of these materials are given in the sections of Mat-1 and Mat-2. In a preferred embodiment, the spacer is made of the same material as the plate used in the CROF.

[0120] (iv) Mechanical strength and flexibility of the spacer. In certain embodiments, the mechanical strength of the spacer is sufficient such that during compression and in the closed configuration of the panel, the height of the spacer is the same or significantly the same as the height of the panel in the open configuration. In certain embodiments, the difference in the spacer between the open and closed configurations can be characterized and predetermined.

[0121] The spacer material can be rigid, flexible, or anything in between. Rigidity is relative to the applied pressure used to bring the panels into the closed configuration: if the spacer deforms by no more than 1% of its height under pressure, the spacer material is considered rigid; otherwise, it is considered flexible. When the spacer is made of a flexible material, the final sample thickness in the closed configuration can still be determined based on the applied pressure and mechanical properties of the spacer.

[0122] (v) Spacers within the sample. To achieve the desired reduction and control of sample thickness, and in particular to obtain good sample thickness uniformity, in certain embodiments, spacers are placed within the sample or within the relevant volume of the sample. In certain embodiments, one or more spacers are within the sample or within the relevant volume of the sample, with appropriate spacing between the spacers. In certain embodiments, at least one spacer is within the sample, at least two spacers are within the sample or within the relevant volume of the sample, or at least "n" spacers are within the sample or within the relevant volume of the sample, where "n" can be determined by the sample thickness uniformity or the desired sample fluidity during CROF.

[0123] (vi) Spacer height. In some embodiments, all spacers have the same predetermined height. In some embodiments, the spacers have different predetermined heights. In some embodiments, the spacers may be divided into groups or regions, each group or region having its own spacer height. And in some embodiments, the predetermined height of the spacers is the average height of the spacers. In some embodiments, the spacers have approximately the same height. In some embodiments, a certain number of spacers have the same height.

[0124] The height of the spacer is selected based on the desired specified final sample thickness and residual sample thickness. The spacer height (predetermined spacer height) and / or sample thickness is 3 nm or less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1 μm or less, 2 μm or less, 3 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm or less, 2 mm or less, 4 mm or less, or a range between two values.

[0125] The spacer height and / or sample thickness is from 1 nm to 100 nm in one preferred embodiment, from 100 nm to 500 nm in another preferred embodiment, from 500 nm to 1000 nm in a separate preferred embodiment, from 1 μm (i.e., 1000 nm) to 2 nm, from 1 μm in another preferred embodiment, from 2 μm to 3 μm in another preferred embodiment, from 3 μm to 5 μm in another preferred embodiment, from 5 μm to 10 μm in another preferred embodiment, from 10 μm to 50 μm in another preferred embodiment, and from 100 μm to 100 μm in a separate preferred embodiment.

[0126] In certain embodiments, the spacer height and / or sample thickness is (i) equal to or slightly larger than the smallest dimension of the analyte, or (ii) equal to or slightly larger than the largest dimension of the analyte. "Slightly larger" means it is approximately 1% to 5% larger, and any number in between can be used.

[0127] In certain embodiments, the spacer height and / or sample thickness is greater than the smallest dimension of the analyte (eg, the analyte has an anisotropic shape), but less than the largest dimension of the analyte.

[0128] For example, a red blood cell has a disc shape with a minimum dimension of 2 μm (disc thickness) and a maximum dimension of 11 μm (disc diameter). In one embodiment of the present invention, the spacer is selected so that the inner surface spacing of the plate in the relevant area is 2 μm (equal to the minimum dimension) in one embodiment, 2.2 μm in another embodiment, or 3 (50) 1% larger than the minimum dimension but smaller than the maximum dimension of the red blood cell in another embodiment. Such an embodiment has certain advantages in blood cell counting. In one embodiment, for red blood cell counting, by making the inner surface spacing 2 or 3 μm, and making any number between the two values, the undiluted whole blood sample is confined to a spacing within which each red blood cell (RBC) does not overlap with other cells, so that the red blood cells can be accurately counted visually. (Excessive overlap between RBCs can lead to serious errors in counting).

[0129] In certain embodiments of the present invention, when the plate is in a closed configuration, the plate and spacers are used to adjust not only the thickness of the sample, but also the orientation and / or surface density / configuration of analytes / entities in the sample. When the plate is in a closed configuration, the thinner the sample, the fewer analytes / entities per unit surface area (i.e., the lower the surface concentration).

[0130] (vii) Spacer lateral dimensions. For open spacers, the lateral dimensions can be characterized by the lateral dimensions in two orthogonal directions (x and y) (sometimes referred to as width). The lateral dimensions of the spacer in each direction may be the same or different. In some embodiments, the lateral dimension in each direction (x or y) is...

[0131] In certain embodiments, the ratio of the lateral dimensions in the x- to y-directions is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10,000, or a range between any two of these values. In certain embodiments, different ratios are used to adjust the flow direction of the sample; for example, the larger the ratio, the larger the direction of flow (larger the dimension).

[0132] In certain embodiments, the different lateral dimensions of the spacers in the x and y directions serve as a means of (a) using the spacers as scale marks to indicate the orientation of the plate, (b) using the spacers to create more sample flow in a preferred direction, or both.

[0133] In a preferred embodiment, period, width and height.

[0134] In some embodiments, all spacers have the same shape and size. In some embodiments, each spacer has a different lateral dimension.

[0135] For closed spacers, in some embodiments, the internal lateral shape and dimensions are selected based on the total volume of the sample to be enclosed by the closed spacer, where the volume size has been described in the present disclosure; in some embodiments, the external shape and dimensions are selected based on the required strength to support the pressure of the liquid against the spacer and the compressive pressure of the squeeze plate.

[0136] (viii) Aspect ratio of height to average lateral dimension of the pillar spacers. In certain embodiments, the aspect ratio of the height to average lateral dimension of the pillar spacers is 100,000, 10,000, 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.0001, 00001, or a range between any two values.

[0137] (ix) Spacer Height Accuracy. The height of the spacers should be precisely controlled. The relative accuracy of the spacers (i.e., the ratio of the deviation from the desired spacer height) is 0.001% or less, 0.01% or less, 0.1% or less, 0.5% or less, 1% or less, 2% or less, 5% or less, 8% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, 99.9% or less, or any range therebetween.

[0138] (x) Spacer spacing. The spacers may be a single spacer or a plurality of spacers in the relevant area of ​​the plate or sample. In certain embodiments, the spacers on the plate are configured and / or arranged in an array, and the array may be periodic, aperiodic, or periodic at some locations on the plate and aperiodic at others.

[0139] In certain embodiments, the periodic array of spacers has a lattice that is square, rectangular, triangular, hexagonal, polygonal, or any combination thereof, where combination means that different locations of the plate have different lattices of spacers.

[0140] In certain embodiments, the inter-spacer distance of the spacer array is periodic in at least one direction of the array (ie, uniform inter-spacer distance). In certain embodiments, the distance between the spacers is configured to improve uniformity between the plate spacings in the closed configuration.

[0141] The distance between adjacent spacers (i.e., the distance between spacers) is 1 μm or less, 5 μm or less, 10 μm or less, 20 μm, 30 μm or less, 40 μm or less, 50 μm or less, 60 μm or less, 70 μm or less, 80 μm or less, 90 μm or less, 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, or between two values.

[0142] In some embodiments, the distance between the spacers is 400, 500, 1 mm, 2 mm, 3 mm, 5 mm, 7 mm, 10 mm, or any range between any two of the above values. In some embodiments, the distance between the spacers is 10 mm, 20 mm, 30 mm, 50 mm, 70 mm, 100 mm, or any range between any two values.

[0143] The distance between adjacent spacers (i.e., the distance between the spacers) is selected so that for given characteristics of the plate and the sample, in certain embodiments, the variation in sample thickness between two adjacent spacers in the closed configuration of the plate is, at most, 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or any range between two values; or in certain embodiments, at most 80%, 100%, 200%, 400%, or a range between any two values.

[0144] Obviously, in order to maintain a given sample thickness variation between two adjacent spacers, a closer spacer spacing is required when using a more flexible plate.

[0145] Specifies the precision of the interval spacing distance.

[0146] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 2 to 4 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 1 μm to 100.

[0147] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 2 to 4 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 100 μm to 250.

[0148] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 4 to 50 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 1 μm to 100 μm.

[0149] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 4 to 50 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 100 μm to 250 μm.

[0150] The period of the spacer array is from 1 nm to 100 nm in one preferred embodiment, from 100 nm to 500 nm in another preferred embodiment, from 500 nm to 1000 nm in a separate preferred embodiment, from 1 μm (i.e., 1000 nm) to 2 μm in another preferred embodiment, from 2 μm to 3 μm in a separate preferred embodiment, from 3 μm to 5 μm in another preferred embodiment, from 5 μm to 10 μm in a separate preferred embodiment, from 10 μm to 50 μm in another preferred embodiment, from 50 μm to 100 μm in a separate preferred embodiment, from 100 μm to 175 μm in a separate preferred embodiment, and from 175 μm to 300 μm in a separate preferred embodiment.

[0151] (xi) Spacer Density. The spacers are arranged on each panel at a surface density of greater than 1 per 1 μm², greater than 1 per 10 μm², greater than 1 per 100 μm², greater than 1 per 500 μm², greater than 1 per 1000 μm², greater than 1 per 5000 μm², greater than 1 per 0.01 mm², greater than 1 per 0.1 mm², greater than 1 per 1 mm², greater than 1 per 5 mm², greater than 1 per 10 mm², greater than 1 per 100 mm², greater than 1 per 1000 mm², greater than 1 per 10,000 mm², or a range between these values.

[0152] (3) The spacer is configured so as not to occupy a large surface area (volume) in a given sample area (volume);

[0153] (xii) Spacer Volume to Sample Volume Ratio. In many embodiments, the ratio of the spacer volume (i.e., the volume of the spacer) to the sample volume (i.e., the volume of the sample), and / or the ratio of the volume of the spacer within the relative volume of the container, is controlled to achieve certain advantages. Advantages include, but are not limited to, uniformity of sample thickness control, uniformity of analyte, and sample flow characteristics (i.e., flow rate, flow direction, etc.).

[0154] In some embodiments, the ratio of the spacer volume (r) to the sample volume, and / or the ratio of the volume of the spacer within the relevant volume of the sample to the relevant volume of the sample is at most 100%, at most 99%, at most 70%, at most 50%, at most 30%, at most 10%, at most 5%, at most 3%, at most 1%, at most 0.1%, at most 0.01%, at most 0.001%, or a range between any of these values.

[0155] (xiii) Spacers fixed to the panels. The spacing distance between the spacers and the orientation of the spacers, which are critical in the present invention, are preferably maintained during the process of moving the panels from the open configuration to the closed configuration, and / or are preferably predetermined before the panels are moved from the open configuration to the closed configuration.

[0156] In certain embodiments of the present disclosure, a spacer is secured to one of the panels prior to closing the panels into the closed configuration. The term "spacer secured with its respective panel" means that the spacer is attached to a panel and remains attached during use of the panel.

[0157] An example of "a spacer being fixed to its respective plate" is when the spacer is integrally formed from one material of the plate and the position of the spacer relative to the plate surface does not change. An example of "a spacer not being fixed to its respective plate" is when the spacer is bonded to the plate with an adhesive, but when the plate is used, the adhesive fails to hold the spacer in its original position on the plate surface (that is, the spacer moves from its original position on the plate surface).

[0158] In some embodiments, at least one spacer is secured to its respective plate. In some embodiments, two spacers are secured to their respective plates. In some embodiments, a majority of spacers are secured to their respective plates. In some embodiments, all spacers are secured to their respective plates.

[0159] In certain embodiments, the spacers are integrally secured to the plate.

[0160] In certain embodiments, the spacers are secured to their respective plates by one or any combination of the following methods and / or configurations: attaching, bonding, fusing, embossing, and etching.

[0161] The term "embossing" refers to the integral attachment of the spacers to the panel by impressing (ie, embossing) a piece of material to form the spacers on the panel surface. The material may be a single layer of material or multiple layers of material.

[0162] The term "etched" means that the spacers and the plate are integrally affixed by etching a piece of material to form the spacers on the plate surface. The material can be a single layer of material or multiple layers of material.

[0163] The term "fused to" means that the spacer and the board are integrally fixed by bonding the spacer and the board together, original materials for the spacer and the board are fused to each other, and there is then a clear material boundary fusion between the two materials.

[0164] The term "bonded to" means that the spacer and the plate are integrally fixed by bonding the spacer and the plate through adhesion.

[0165] The term "attached to" means that the spacer and the plate are connected together.

[0166] In some embodiments, the spacers and plates are made of the same material. In other embodiments, the spacers and plates are made of different materials. In another embodiment, the spacers and plates are integrally formed. In another embodiment, one end of the spacer is fixed to its corresponding plate, while the other end is open to accommodate different configurations of the two plates.

[0167] In other embodiments, each spacer is independently at least one of attached to, bonded to, fused to, embossed in, and etched in the respective plate. The term "independently" means that one spacer is secured to its respective plate by the same or different methods selected from the group consisting of attaching, bonding, fusing, embossing, and etching on the respective plate.

[0168] In some embodiments, the distance between at least two spacers is predetermined ("predetermined spacer distance" means that the distance is known to a user when the board is used).

[0169] In certain embodiments of all of the methods and apparatus described herein, additional spacers are present in addition to the fixed spacers.

[0170] (xiv) Specific Sample Thickness. In the present invention, it was observed that a larger plate retention force (i.e., the force holding the two plates together) can be achieved by using a smaller plate spacing (for a given sample area) or a larger sample area (for a given sample), or both.

[0171] In certain embodiments, at least one plate is transparent in an area surrounding the area of ​​interest, each plate having an inner surface configured to contact the sample in a closed configuration; the inner surfaces of the plates are substantially parallel to each other in the closed state; the inner surfaces of the plates are substantially planar except for locations having spacers; or any combination thereof.

[0172] The spacers can be fabricated on the plate in various ways using photolithography, etching, imprinting (nanoimprinting), deposition, stripping, fusing, or a combination thereof. In some embodiments, the spacers are directly imprinted or embossed on the plate. In some embodiments, the spacers are imprinted into a material (e.g., plastic) deposited on the plate. In some embodiments, the spacers are fabricated by directly imprinting the surface of a CROF plate. Nanoimprinting can be accomplished using a roll-to-roll technique using a roller imprinter, or it can be rolled into a flat nanoimprint. This approach has significant economic advantages, thereby reducing costs.

[0173] In some embodiments, the spacers are deposited on the plate. Deposition can be by evaporation, sticking, or peeling. In the sticking process, the spacers are first made on a carrier and then the spacers are transferred from the carrier to the plate. In the lift-off process, a removable material is first deposited on the plate and a hole is created in the material; the bottom of the hole exposes the plate surface, and then an isolating material is deposited into the hole, and the removable material is then removed, leaving only the spacer on the plate surface. In some embodiments, the spacers deposited on the plate are fused to the plate. In some embodiments, the spacers and the plate are manufactured in a single process. Single processes include embossing (i.e., embossing, molding) or compositing.

[0174] In certain embodiments, at least two of the spacers are secured to the respective plates by different fabrication methods, and optionally, wherein the different fabrication methods include at least one of deposition, bonding, fusion, imprinting, and etching.

[0175] In certain embodiments, one or more spacers are secured to the respective plates by a manufacturing method of bonding, fusing, embossing or etching, or any combination thereof.

[0176] In certain embodiments, the manufacturing method for forming such an integrated spacer on the board includes methods of being bonded, fused, stamped, or etched, or any combination thereof.

[0177] (xv)ISD^4 / hE

[0178] In certain embodiments of the present disclosure, a device for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a first plate. In certain embodiments of the present disclosure, a device for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a second plate. In certain embodiments of the present disclosure, a device for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a spacer. In certain embodiments, the plates can be moved relative to each other into different configurations. In certain embodiments, one or both plates are flexible. In certain embodiments, each plate includes an inner surface having a sample contact area for contacting a fluid sample. In certain embodiments, each plate includes a force area on its respective outer surface for applying pressure to press the plates together. In certain embodiments, one or both of the two plates include a spacer permanently fixed to the inner surface of each plate. In certain embodiments, the spacer has a predetermined, substantially uniform height equal to or less than 200 microns and a predetermined fixed spacing. In certain embodiments, the fourth power of the spacer distance (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD4 / (hE)) is 5x106μm3 / GPa. In certain embodiments, at least one spacer is within the sample contact area. In certain embodiments, one configuration is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the two plates is not limited by the spacer, and the sample is deposited on one or both plates. In certain embodiments, another configuration is a closed configuration, which is constructed after the sample is deposited in an open configuration and the plates are forced to a closed configuration by applying pressure on the force area. In the closed state, at least a portion of the sample is compressed by the two plates into a highly uniform layer and is essentially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the contact area of ​​the sample. Two plates, regulated by the plates and the spacer.

[0179] In certain embodiments of the present disclosure, a method for forming a thin layer of a fluid sample having a uniform predetermined thickness by extrusion may include obtaining an apparatus of the present disclosure. In certain embodiments of the present disclosure, a method for forming a thin layer of a fluid sample having a uniform predetermined thickness by pressing may include: depositing a fluid sample on one or both plates when the plates are configured into an open configuration. In certain embodiments, the open configuration is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not limited by a spacer. In certain embodiments of the present disclosure, a method for forming a thin layer of a fluid sample having a uniform predetermined thickness by pressing may include forcing the two plates into a closed configuration, wherein: at least a portion of the sample is compressed by the two plates. A layer of substantially uniform thickness is produced, wherein the uniform thickness of the layer is limited by the sample contacting surfaces of the plates and regulated by the plates and the spacers.

[0180] In certain embodiments of the present disclosure, an apparatus for analyzing a fluid sample may include a first plate. In certain embodiments of the present disclosure, an apparatus for analyzing a fluid sample may include a second plate. In certain embodiments of the present disclosure, an apparatus for analyzing a fluid sample may include a spacer. In certain embodiments, the plates are movable relative to each other into different configurations. In certain embodiments, one or both plates are flexible. In certain embodiments, each plate has a sample contact area on its respective inner surface for contacting the fluid sample. In certain embodiments, one or both plates include spacers, and the spacers are affixed to the inner surface of the respective plate. In certain embodiments, the spacers have a predetermined, substantially uniform height equal to or less than 200 microns, and the distance between the spacers is predetermined. In certain embodiments, the Young's modulus of the spacers multiplied by the filling factor of the spacers is at least 2 MPa. In certain embodiments, at least one spacer is within the sample contact area. In certain embodiments, one configuration is an open configuration, wherein the two plates are partially or completely separated, the spacing between the two plates is not limited by the spacer, and the sample is deposited on one or both plates. In some embodiments, another configuration is a closed configuration, which is configured after the sample is deposited in an open configuration; in the closed state: at least a portion of the sample is compressed by two plates into a highly uniform layer, wherein the uniform thickness of the layer is limited by the sample contacting surfaces of the plates and by the plates and the spacer.

[0181] In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by extrusion may include obtaining an apparatus of the present disclosure. In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include: depositing a fluid sample on one or two plates when the plates are configured into an open configuration. In certain embodiments, an open configuration is a configuration in which two plates are partially or completely separated and the spacing between the plates is not limited by a spacer. In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by extrusion may include forcing two plates into a closed configuration. In certain embodiments, at least a portion of the sample is compressed by two plates into a layer of substantially uniform thickness, wherein the uniform thickness of the layer is limited by the sample contacting surfaces of the plates and regulated by the plates and the spacer.

[0182] In certain embodiments of the present disclosure, an apparatus for analyzing a fluid sample may include a first plate. In certain embodiments of the present disclosure, an apparatus for analyzing a fluid sample may include a second plate. In certain embodiments, the plates are movable relative to each other into different configurations. In certain embodiments, one or both plates are flexible. In certain embodiments, each plate has a sample contact area on its respective surface for contacting a sample containing an analyte. In certain embodiments, one or both plates includes a spacer permanently affixed to the plate within the sample contact area, wherein the spacer has a predetermined, substantially uniform height and a predetermined, fixed spacer distance that is at least approximately twice as large as the size of the analyte, up to 200 μm, and wherein at least one spacer is within the sample contact area. In certain embodiments, one configuration is an open configuration, wherein the two plates are separated, the spacing between the two plates is not restricted by the spacer, and the sample is deposited on one or both plates. In certain embodiments, another configuration is a closed configuration, which is configured in the open configuration after sample deposition. In the closed state, at least a portion of the sample is compressed by the two plates into a highly uniform layer, wherein the uniform thickness of the layer is limited by the sample contact surfaces of the plates and regulated by the plates and the spacer.

[0183] In certain embodiments of the present disclosure, a method of forming a thin layer of a fluid sample having a uniform predetermined thickness by extrusion may include obtaining an apparatus of the present disclosure. In certain embodiments of the present disclosure, a method of forming a thin layer of a fluid sample having a uniform predetermined thickness by pressing may include: depositing the fluid sample on one or both of two plates. When the plates are configured into an open configuration, wherein the open configuration is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not limited by a spacer. In certain embodiments of the present disclosure, a method of forming a thin layer of a fluid sample having a uniform predetermined thickness by pressing may include forcing the two plates into a closed configuration, wherein: at least a portion of the sample is compressed by the two plates into a layer having a substantially uniform thickness, wherein the uniform thickness of the layer is limited by the sample contacting surfaces of the plates and regulated by the plates and the spacers.

[0184] In certain embodiments of the present disclosure, an apparatus for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a first plate. In certain embodiments of the present disclosure, an apparatus for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a second plate. In certain embodiments of the present disclosure, an apparatus for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include spacers. In certain embodiments, the plates are movable relative to each other into different configurations. In certain embodiments, one or both plates are flexible. In certain embodiments, each plate includes a sample contact area on its respective inner surface for contacting and / or compressing the fluid sample. In certain embodiments, each plate includes an area on its respective outer surface for applying a force to press the plates together. In certain embodiments, one or both of the two plates include spacers permanently affixed to the inner surface of each plate. In certain embodiments, the spacers have a predetermined, substantially uniform height equal to or less than 200 microns, a predetermined width, and a predetermined, fixed spacing. In certain embodiments, the ratio of the inter-spacer distance to the spacer width is 1.5 or greater. In certain embodiments, at least one spacer is within the sample contact area. In certain embodiments, one configuration is an open configuration, in which the two plates are partially or completely separated, the spacing between the two plates is not limited by a spacer, and the sample is deposited on one or both plates. In certain embodiments, another configuration is a closed configuration, which is deployed in an open configuration after sample deposition. In the closed state, at least a portion of the sample is compressed into a highly uniform layer by the two plates and is essentially stagnant relative to the plates, where the uniform thickness of the layer is limited by the contact area of ​​the sample between the two plates, regulated by the plates and the spacer.

[0185] In certain embodiments of the present disclosure, a method for forming a thin layer of a fluid sample having a uniform predetermined thickness by pressing with imprecise pressure may include obtaining an apparatus of the present disclosure. In certain embodiments of the present disclosure, a method for forming a thin layer of a fluid sample having a uniform predetermined thickness by pressing with imprecise pressure may include obtaining a fluid sample. In certain embodiments of the present disclosure, a method for forming a thin layer of a fluid sample having a uniform predetermined thickness by pressing with imprecise pressure may include: depositing the sample on one or two plates; or depositing the sample on one or two plates. When the plates are configured in an open configuration, wherein the open configuration is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not limited by a spacer. In certain embodiments of the present disclosure, the method for forming a thin layer of a fluid sample having a uniform predetermined thickness by pressing with imprecise pressure may include forcing the two plates into a closed configuration, wherein: at least a portion of the sample is compressed into a layer of substantially uniform thickness by the two plates, wherein the uniform thickness of the layer is limited by the sample contacting surfaces of the plates and regulated by the plates and the spacer.

[0186] In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and a flat top surface for contacting another plate. In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and a flat top surface for contacting another plate having a substantially uniform cross-section. In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and having a flat top surface for contacting another plate, wherein the flat top surface of the column has a variation of less than 10 nm. In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and having a flat top surface for contacting another plate, wherein the flat top surface of the column has a variation of less than 50 nm. In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and having a flat top surface for contacting another plate, wherein the flat top surface of the column has a variation of less than 50 nm. In some embodiments, the spacer has a pillar shape with its foot secured to one plate and having a flat top surface for contacting another plate, wherein the flat top surface of the pillar has a variation within a range of less than 10 nm, 20 nm, 30 nm, 100 nm, 200 nm, or any two values.

[0187] In certain embodiments, the Young's modulus of the spacer multiplied by the filling factor of the spacer is at least 2 MPa. In certain embodiments, the sample includes an analyte, and the predetermined constant spacing distance is at least about twice the size of the analyte, up to 200 μm. In certain embodiments, the sample includes an analyte, the predetermined constant spacing distance is at least about 2 times greater than the size of the analyte, up to 200 μm, and the Young's modulus of the spacer multiplied by the filling factor of the filler is at least 2 MPa.

[0188] In certain embodiments, the fourth power of the spacer distance (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible board (ISD^4 / (hE)) is less than or equal to 5x10^6 μm^3 / GPa. In certain embodiments, the fourth power of the spacer distance (ISD) divided by the thickness and Young's modulus of the flexible board (ISD^4 / (hE)) is 1x10^6 μm^3 / GPa. In certain embodiments, the fourth power of the spacer distance (ISD) divided by the thickness and Young's modulus of the flexible board (ISD^4 / (hE)) is 5x10^5 μm^3 / GPa. In certain embodiments, the Young's modulus of the spacer multiplied by the filling factor of the spacer is at least 2 MPa, and the fourth power of the inter-spacer distance (ISD) divided by the thickness and Young's modulus of the flexible board (ISD^4 / (hE)) is 1x10^5 μm^3 / GPa. In certain embodiments, the Young's modulus of the spacer multiplied by the spacer's filling factor is at least 2 MPa, and the fourth power of the inter-spacer distance (ISD) divided by the thickness of the flexible board and the Young's modulus (ISD^4 / (hE)) is 1x10^4 μm^3 / GPa. In certain embodiments, the Young's modulus of the spacer multiplied by the spacer's filling factor is at least 20 MPa.

[0189] In certain embodiments of the present disclosure, the ratio of the spacing between the spacers to the average width of the spacers is 2 or greater. In certain embodiments, the ratio of the spacing between the spacers to the average width of the spacers is 2 or greater, and the Young's modulus of the spacer multiplied by the filling factor of the spacer is at least 2 MPa. In certain embodiments, the distance between the spacers is at least about 2 times the size of the analyte, up to 200 μm. In certain embodiments, the ratio of the distance between the spacers to the width of the spacer is 1.5 or greater. In certain embodiments, the ratio of the width to the height of the spacer is 1 or greater. In certain embodiments, the ratio of the width to the height of the spacer is 1.5 or greater. In certain embodiments, the ratio of the width to the height of the spacer is 2 or greater. In certain embodiments, the ratio of the width to the height of the spacer is greater than 2, 3, 5, 10, 20, 30, 50, or within the range of any two of these values.

[0190] In certain embodiments, the force that squeezes the two plates into a closed structure is an imprecise squeezing force. In certain embodiments, the force that presses the two plates into a closed structure is an imprecise pressing force provided by a human hand. In certain embodiments, forcing the two plates to compress at least a portion of the sample into a layer of substantially uniform thickness includes using a compliant press, pressing the area of ​​at least one plate in parallel or sequentially to press the plates together to form a closed structure, wherein the compliant press produces a substantially uniform pressure on the plate on at least a portion of the sample, and the press expands at least a portion of the sample laterally between the sample contact surfaces of the plate. And wherein the closed structure is a structure in which the spacing between the plates in the layer of uniform thickness area is adjusted by spacers. And wherein the reduced sample thickness reduces the time for mixing the reagents on the storage position with the sample. In certain embodiments, the pressing force is an imprecise force, the magnitude of which is (a) unknown and unpredictable when the force is applied, or (b) unknown and cannot be predicted within equal accuracy or better than 20% of the average pressing force applied. In some embodiments, the compressive force is an imprecise force whose magnitude is (a) unknown and unpredictable, or (b) unknown and cannot be predicted within equal accuracy or better than 30% of the average compressive force applied at the time the force is applied. In some embodiments, the compressive force is an imprecise force whose magnitude is (a) unknown and unpredictable, or (b) unknown and cannot be predicted within equal accuracy or better than 30% of the average compressive force applied at the time the force is applied; and wherein the uniform thickness variation of the highly uniform thickness layer is 20%. In some embodiments, the compressive force is an imprecise force whose magnitude cannot be determined with an accuracy equal to or better than 30%, 40%, 50%, 70%, 100%, 200%, 300%, 500%, 1000%, 2000%, or a range therebetween at the time the force is applied.

[0191] In certain embodiments of the present disclosure, the thickness of the flexible sheet is in the range of 10 μm to 200 μm. In certain embodiments, the thickness of the flexible sheet is in the range of 20 μm to 100 μm. In certain embodiments, the thickness of the flexible sheet is in the range of 25 μm to 180 μm. In certain embodiments, the thickness of the flexible sheet is in the range of 200 μm to 260 μm. In certain embodiments, the thickness of the flexible sheet is equal to or less than 250 μm, 225 μm, 200 μm, 175 μm, 150 μm, 125 μm, 100 μm, 75 μm, 50 μm, 25 μm, 10 μm, 5 μm, 1 μm, or a range between two values. In certain embodiments, the viscosity of the sample is 0.1 to 4 (mPa·s). In certain embodiments, the thickness of the flexible sheet is in the range of 200 μm to 260 μm. In certain embodiments, the flexible sheet has a thickness in the range of 20 μm to 200 μm and a Young's modulus in the range of 0.1 to 5 GPa.

[0192] In certain embodiments of the present disclosure, sample deposition is deposition directly from the subject to the plate without using any transfer device. In certain embodiments, during deposition, the amount of unknown sample deposited on the plate. In certain embodiments, the method also includes an analysis of the sample. In certain embodiments, the analysis includes calculating the volume of the relevant sample volume by measuring the lateral area of ​​the relevant sample volume and calculating the volume from the lateral area and the predetermined spacer height. In certain embodiments, the pH value at the sample position between the two plates in the closed structure is determined by the volume of the position and by analyzing the image obtained from the position. In certain embodiments, the determination made by analyzing the image uses artificial intelligence and machine learning.

[0193] In certain embodiments, the analyzing step (e) comprises measuring: i. imaging; ii. luminescence selected from photoluminescence, electroluminescence, and electrochemiluminescence, iii. surface Raman scattering, iv. electrical impedance selected from resistance, capacitance, and inductance, or any combination of vi-iv. In certain embodiments, the analysis comprises reading, image analysis, or counting analytes, or a combination thereof. In certain embodiments, the sample comprises one or more analytes, and one or both of the plate sample contact surfaces comprise one or more binding sites, each binding site immobilizing a corresponding analyte. In certain embodiments, one or both of the plate sample contact surfaces comprise one or more storage sites, each storage site storing one or more reagents, wherein the one or more reagents are dissolved and diffused in the sample. In certain embodiments, one or both of the plate sample contact surfaces comprise one or more amplification sites, each amplification site being capable of amplifying a signal from the analyte or the marker of the analyte when the analyte or marker is within 500 nm of the amplification site. In certain embodiments, i. one or both plate sample contact surfaces contain one or more binding sites, each binding site binding to and immobilizing a corresponding analyte; or ii. one or both plate sample contact surfaces include one or more storage locations, each storing one or more reagents; wherein the one or more reagents are dissolved and diffused in the sample, and wherein the sample contains one or more analytes; or iii. one or more amplification sites, each capable of amplifying a signal from the analyte or the marker of the analyte when the analyte or marker is 500 nm away from the amplification site; or iv. any combination of i to iii.

[0194] In certain embodiments, the liquid sample is a biological sample selected from amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), skin (cerumen), chyle, bell, endolymph, perilymph, feces, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, exhaled breath condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomitus, and urine.

[0195] In certain embodiments, the uniform thickness of the layer in the closed configuration is less than 150 μm. In certain embodiments, compression is provided by a pressurized liquid, a compressed gas, or a conformal material. In certain embodiments, the analysis comprises counting cells in the uniform thickness layer. In certain embodiments, the analysis comprises performing an assay in the uniform thickness layer. In certain embodiments, the assay is a binding assay or a biochemical assay. In certain embodiments, the total volume of the deposited sample is less than 0.5 μL. In certain embodiments, multiple drops of sample are deposited on one or both plates.

[0196] In some embodiments, the distance between the spacers is in a range of 1 μm to 120 μm. In some embodiments, the distance between the spacers is in a range of 120 μm to 50 μm. In some embodiments, the distance between the spacers is in a range of 120 μm to 200 μm. In some embodiments, the thickness of the flexible board is in a range of 20 μm to 250 μm, and the Young's modulus is in a range of 0.1 to 5 GPa. In some embodiments, for the flexible board, the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in a range of 60 to 750 GPa-μm.

[0197] In certain embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 1 mm². In certain embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 3 mm². In certain embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 5 mm². In certain embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 10 mm². In certain embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 20 mm². In certain embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​20 mm² to 100 mm². In certain embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / -5% or better. In certain embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / -10% or better. In certain embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / -20% or better. In certain embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / -30% or better. In certain embodiments, the sample layer of uniform thickness has a thickness uniformity of up to + / - 40% or better. In certain embodiments, the sample layer of uniform thickness has a thickness uniformity of up to + / - 50% or better.

[0198] In certain embodiments, the spacers are pillars having a cross-sectional shape selected from the group consisting of circular, polygonal, circular, square, rectangular, elliptical, oval, or any combination thereof. In certain embodiments, the spacers have a cylindrical shape with a substantially flat top surface and a substantially uniform cross-section, wherein, for each spacer, the ratio of the spacer's lateral dimension to its height is at least 1. In certain embodiments, the spacing between the spacers is periodic. In certain embodiments, the spacers have a fill factor of 1% or greater, where the fill factor is the ratio of the spacer contact area to the total plate area. In certain embodiments, the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 20 MPa, where the fill factor is the ratio of the spacer contact area to the total plate area. In certain embodiments, the spacing between the two plates in the closed configuration is less than 200 μm. In certain embodiments, the spacing between the two plates in the closed configuration is a value selected from 1.8 μm to 3.5 μm. In certain embodiments, the spacers are affixed to the plate by direct embossing of the plate or injection molding of the plate. In some embodiments, the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP, or another plastic. In some embodiments, the spacers have a cylindrical shape, and the sidewall corners of the spacers have a rounded shape with a radius of curvature of at least 1 μm. In some embodiments, the spacers have a density of at least 1000 / mm2. In some embodiments, at least one of the plates is transparent. In some embodiments, the mold used to make the spacers is made from a mold containing features that are made by: (a) directly performing reactive ion etching or ion beam etching, or (b) by replicating or multiple replicating reactive ion etching or ion beam etching of features.

[0199] In some embodiments, the spacers are configured to have a fill factor in the range of 1% to 5%. In some embodiments, the surface variation is relative to the spacer height, and the ratio of the columnar top surface variation to the spacer height is less than 0.5%, 1%, 3%, 5%, 7%, 10%, 15%, 20%, 30%, 40%, or a range between two values. A preferred flat top flatness has a ratio of the column top surface variation to the spacer height of less than 2%, 5%, or 10%. In some embodiments, the spacers are configured to have a fill factor in the range of 1% to 5%. In some embodiments, the spacers are configured to have a fill factor in the range of 5% to 10%. In some embodiments, the spacers are configured to have a fill factor in the range of 10% to 20%. In some embodiments, the spacers are configured to have a fill factor in the range of 20% to 30%. In some embodiments, the spacers are configured to have a fill factor of 5%, 10%, 20%, 30%, 40%, 50%, or a range between any two values. In some embodiments, the spacers are configured such that the fill factor is 50%, 60%, 70%, 80%, or within a range of any two values.

[0200] In some embodiments, the spacer is configured such that the filling factor multiplied by the Young's modulus of the spacer is in the range of 2 MPa to 10 MPa. In some embodiments, the spacer is configured such that the filling factor multiplied by the Young's modulus of the spacer is in the range of 10 MPa and 20 MPa. In some embodiments, the spacer is configured such that the filling factor multiplied by the Young's modulus of the spacer is in the range of 20 MPa to 40 MPa. In some embodiments, the spacer is configured such that the filling factor multiplied by the Young's modulus of the spacer is in the range of 40 MPa to 80 MPa. In some embodiments, the spacer is configured such that the filling factor multiplied by the Young's modulus of the spacer is in the range of 80 MPa to 120 MPa. In some embodiments, the spacer is configured such that the filling factor multiplied by the Young's modulus of the spacer is in the range of 120 MPa to 150 MPa.

[0201] In certain embodiments, the device also includes a dry reagent coated on one or two plates. In certain embodiments, the device also includes a dry binding site with a predetermined area on one or two plates, wherein the dry binding site is combined with and fixed on the analyte in the sample. In certain embodiments, the device also includes a releasable dry reagent and a release time control material on one or two plates, which delays the time when the releasable dry reagent is released into the sample. In certain embodiments, the release time control material delays the time when the dry reagent starts to be released into the sample by at least 3 seconds. In certain embodiments, the reagent includes an anticoagulant and / or a staining agent. In certain embodiments, the reagent includes a cell lysis reagent. In certain embodiments, the device also includes one or more dry binding sites and / or one or more reagent sites on one or two plates. In certain embodiments, the analyte includes molecules (such as proteins, peptides, DNA, RNA, nucleic acids or other molecules), cells, tissues, viruses and nanoparticles with different shapes. In certain embodiments, the analyte includes white blood cells, red blood cells and platelets. In certain embodiments, the analyte is stained.

[0202] In certain embodiments, the spacers that modulate the uniform thickness layer have a fill factor of at least 1%, where the fill factor is the ratio of the area of ​​the spacer in contact with the uniform thickness layer to the total board area in contact with the uniform thickness layer. The layer thickness is uniform. In certain embodiments, for the spacers that modulate the uniform thickness layer, the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 10 MPa, where the fill factor is the ratio of the area of ​​the spacer in contact with the layer. Effect of uniform thickness on the total board area in contact with the uniform thickness layer. In certain embodiments, for the flexible board, 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-μm. In certain embodiments, for the flexible board, the fourth power of the inter-spacer distance (ISD) divided by the thickness (h) of the flexible board and the Young's modulus (E) of the flexible board, ISD4 / (hE), is equal to or less than 106 μm3 / GPa.

[0203] III. Forming a uniform thin fluid layer by imprecise pressure

[0204] In certain embodiments of the present invention, a uniform thin layer of a fluid sample is formed by using compression with an imprecise force. The term "imprecise pressing force" adds a definition of an imprecise pressing force without adding details. As used herein, the term "imprecise" in the context of a force (e.g., "imprecise pressing force") refers to a force that:

[0205] (a) The magnitude of the force is not known or cannot be accurately predicted at the time the force is applied; (b) The pressure is in the range of 0.01 kg / cm2 (square centimeter) to 100 kg / cm2; (c) The magnitude of the force varies from one application of the force to the next; (d) The uncertainty (i.e., variation) of the force in (a) and (c) is at least 20% of the total force actually applied.

[0206] Imprecise forces may be applied by a person's hand, for example, by pinching the subject together between the thumb and index finger, or by pinching and rubbing the subject together between the thumb and index finger.

[0207] In some embodiments, the imprecise force generated by hand pressure has a pressure of 0.01kg / cm2, 0.1kg / cm2, 0.5kg / cm2, 1kg / cm2, 2kg / cm2, kg / cm2, 5kg / cm2, 10kg / cm2, 20kg / cm2, 30kg / cm2, 40kg / cm2, 50kg / cm2, 60kg / cm2, 100kg / cm2, 150kg / cm2, 200kg / cm2 or a range value between any two; the preferred range is 0.1kg / cm2 to 0.5kg / cm2, 0.5kg / cm2 to 1kg / cm2, 1kg / cm2 to 5kg / cm2, 5kg / cm2 to 10kg / cm2 (pressure).

[0208] B) Adapter

[0209] Details of the adapter are described in detail in various publications, including International Application No. PCT / US2018 / 017504 (Essenlix Case No. ESXPCT18F04), which is incorporated herein by reference for all purposes.

[0210] The invention described herein addresses this problem by providing a system comprising an optical adapter and a smartphone. The optical adapter device attaches to the smartphone and transforms it into a microscope, enabling the capture of both fluorescence and brightfield images of samples. This system can be conveniently and reliably operated by the average person from any location. The optical adapter utilizes existing smartphone resources, including the camera, light source, processor, and display, providing a low-cost solution that enables users to perform both brightfield and fluorescence microscopy.

[0211] In the present invention, the optical adapter device includes a holder frame mounted on the upper part of a smartphone and an optical box attached to the holder having a sample container slot and an illumination optical device. In some references (U.S. Patent No. 2016 / 029091 and U.S. Patent No. 2011 / 0292198), the optical adapter design is a whole, including both clip-on mechanical parts that can be mounted on a smartphone and functional optical elements. The problem with this design is that they need to redesign the entire optical adapter for each specific model of smartphone. However, in the present invention, the optical adapter is divided into a bracket frame that is only used to mount the smartphone and a universal optical box that contains all functional components. For smartphones of different sizes, as long as the relative positions of the camera and the light source are the same, only the bracket frame needs to be redesigned, which can save a lot of design and manufacturing costs.

[0212] The optical adapter's optical box includes: a receiving slot that receives the sample and places it in a specimen slide within the field of view and focal length of the smartphone camera; brightfield illumination optics for capturing brightfield microscopy images of the sample; fluorescence illumination optics for capturing fluorescence microscopy images of the sample; and a lever that switches between brightfield illumination optics and fluorescence illumination optics by sliding inward and outward in the optical box.

[0213] The receptacle slot has a rubber door that completely covers the slot, preventing ambient light from entering the optical box and being collected by the camera. In U.S. Patent 2016 / 0290916, the sample slot is always exposed to ambient light, which is not a major problem because it only performs brightfield microscopy. However, the present invention can take advantage of this rubber door when performing fluorescence microscopy, as ambient light can introduce significant noise to the camera's image sensor.

[0214] To capture good fluorescence microscopy images, it is ideal that almost no excitation light enters the camera and the camera only collects the fluorescence emitted by the sample. However, for all common smartphones, due to the large divergence angle of the light beam emitted by the light source and infrared light source, the filter placed in front of the camera is not very effective in blocking the undesirable wavelength range emitted by the smartphone light source. Optical filters are not very effective for uncollimated light beams. Collimating optics can be designed to collimate the light beam emitted by the smartphone light source to solve this problem, but this approach increases the size and cost of the adapter. In contrast, in the present invention, the fluorescence illumination optics enable the excitation light to illuminate the sample at a large oblique incidence angle, partly from the waveguide inside the sample slide and partly from the back side of the sample side, so that the excitation light is almost not collected by the camera to reduce the noise signal entering the camera.

[0215] Brightfield illumination optics in the adapter receive and redirect the light beam from the light source to back-illuminate the sample at normal incidence.

[0216] Typically, the optical box also includes a lens mounted inside it that aligns with the smartphone's camera and magnifies the image captured by the camera. The image captured by the camera can be further processed by the smartphone's processor and the analysis results can be output on the smartphone's screen.

[0217] To enable both brightfield and fluorescence illumination optics in the same optical adapter, the present invention utilizes a slidable lever. The fluorescence illumination optics are mounted on the lever. When the lever is fully inserted into the optical cartridge, the fluorescence illumination optics block the optical path of the brightfield illumination optics, switching the illumination optics to the fluorescence optics. Furthermore, when the lever is removed, the fluorescence illumination optics mounted on the lever move out of the optical path, switching the illumination optics to the brightfield illumination optics. This lever design enables the optical adapter to operate in both brightfield and fluorescence illumination modes without requiring the design of two separate single-mode optical cartridges.

[0218] The lever includes two flat surfaces at different planes at different heights.

[0219] In some embodiments, the two planes can be connected together with a vertical rod and moved into or out of the optical box together. In some embodiments, the two planes can be separated and each plane can be moved into or out of the optical box independently.

[0220] The upper lever plane includes at least one optical element, which may be, but is not limited to, an optical filter. The upper lever plane moves below the light source, with a preferred distance between the upper lever plane and the light source being 0 to 5 mm.

[0221] A portion of the bottom lever plane is nonparallel to the image plane. The surface of the nonparallel portion of the bottom lever plane has a mirror finish with a reflectivity greater than 95%. The nonparallel portion of the bottom lever plane moves under the light source and deflects light emitted from the light source, thereby back-illuminating the sample area directly below the camera. The preferred tilt angle of the nonparallel portion of the bottom lever plane is in the range of 45 to 65 degrees, and the tilt angle is defined as the angle between the nonparallel bottom plane and the vertical plane.

[0222] A portion of the bottom lever plane is parallel to the image plane and positioned below the sample at a distance of 1 to 10 mm. The surface of this portion of the bottom lever plane has a high light absorption rate, exceeding 95%. This absorptive surface is designed to eliminate reflections from the sample at low incident angles.

[0223] To slide the lever in and out to switch the illumination optics, a plug design consisting of a spherical plunger and a groove in the lever is used to stop the lever in a predetermined position when pulled outward from the adapter. This allows the user to pull the lever with any force, but the lever stops in a fixed position, in which the optical adapter's operating mode is switched to bright illumination.

[0224] The sample slider fits into the receptacle slot to accommodate the QMAX device and place the sample within the field of view and focus of the smartphone camera in the QMAX device.

[0225] The sample slide consists of a fixed track frame and a movable arm:

[0226] The frame rails are fixedly mounted in slots in the optical cassette. The rail frame features sliding track slots tailored to the width and thickness of the QMAX device, allowing the QMAX device to slide along the rails. The width and height of the track slots are carefully configured to ensure that the QMAX device deflects less than 0.5mm perpendicular to the sliding direction in the sliding plane and less than 0.2mm across the thickness of the QMAX device.

[0227] The frame rail features an open window under the field of view of the smartphone camera to allow light to illuminate the sample backward.

[0228] The movable arm is pre-built into the sliding track slot of the track frame and moves with the QMAX device to guide the movement of the QMAX device in the track frame.

[0229] The movable arm is equipped with a stop mechanism with two predefined stop positions. In one position, the arm stops the QMAX device so that the fixed sample area on the QMAX device is directly under the smartphone camera. In the other position, the arm stops the QMAX device so that the sample area on the QMAX device is not within the smartphone's field of view, allowing the QMAX device to be easily removed from the track slot.

[0230] By pressing the QMAX device and the movable arm together to the end of the track groove and then releasing it, the movable arm switches between the two stop positions.

[0231] The movable arm indicates whether the QMAX device is inserted in the correct orientation. One corner of the QMAX device is shaped differently than the other three right-angled corners. The movable arm is shaped to match the shape of the specially shaped corner, so the QMAX device will slide into the correct position in the track slot only when it is in the correct orientation.

[0232] C) Smartphone / Detection System

[0233] Details of the smartphone / detection system are described in detail in various publications, including International Application (IA) PCT / US2016 / 046437 filed on August 10, 2016, IA PCT / US2016 / 051775 filed on September 14, 2016, U.S. Provisional Application 62 / 256065 filed on February 7, 2017, U.S. Provisional Application 62 / 456287 and 62 / 456590 filed on February 8, 2017, U.S. Provisional Application 62 / 456504 filed on February 8, 2017, U.S. Provisional Application 62 / 459,544 filed on February 15, 2017, and U.S. Provisional Application 62 / 460075 and 62 / 459920 filed on February 16, 2017, which are incorporated herein by reference in their entireties for all purposes.

[0234] The devices / apparatuses, systems and methods disclosed herein may include or use a Q-Card for sample detection, analysis and quantification. In certain embodiments, the Q-Card is used with an adapter that can connect the Q-Card to a smartphone detection system. In certain embodiments, the smartphone includes a camera and / or an illumination source. In certain embodiments, the smartphone includes a camera that can be used to capture an image or sample when the sample is placed in the camera's field of view (e.g., via an adapter). In certain embodiments, the camera includes a set of lenses (e.g., as in an iPhone TM 6). In some embodiments, the camera includes at least two sets of lenses (e.g., as in the iPhone TM 7). In some embodiments, the smartphone includes a camera, but the camera is not used for image capture.

[0235] In some embodiments, the smartphone includes a light source, such as, but not limited to, an LED (light emitting diode). In some embodiments, the light source is used to illuminate the sample when the sample is in the camera's field of view (e.g., via an adapter). In some embodiments, the light from the light source is enhanced, amplified, modified, and / or optimized by optical components of the adapter.

[0236] In certain embodiments, the smartphone includes a processor configured to process information from the sample. The smartphone includes software instructions that, when executed by the processor, can enhance, amplify, and / or optimize the signal (e.g., image) from the sample. The processor can include one or more hardware components, such as a central processing unit (CPU), an application-specific integrated circuit (ASIC), an application-specific instruction set processor (ASIP), a graphics processing unit (GPU), a physical processing unit (PPU), a digital signal processor (DSP), a field programmable gate array (FPGA), a programmable logic device (PLD), a controller, a microcontroller unit, a reduced instruction set computer (RISC), a microprocessor, the like, or any combination thereof.

[0237] In some embodiments, the smartphone includes a communication unit that is configured and / or used to transmit data and / or images related to the sample to another device. By way of example only, the communication unit may use a cable network, a wired network, a fiber optic network, a telecommunications network, an intranet, the Internet, a local area network (LAN), a wide area network (WAN), a wireless local area network (WLAN), a metropolitan area network (MAN), a wide area network (WAN), a public switched telephone network (PSTN), a Bluetooth network, a ZigBee network, a near field communication (NFC) network, or the like, or any combination thereof. In some embodiments, the smartphone is an iPhone™, an Android TM Phone or Windows TM Telephone.

[0238] D) Manufacturing method

[0239] Details of the manufacturing methods are described in detail in various publications, including International Application PCT / US2018 / 057873, filed October 26, 2018, which is incorporated herein by reference for all purposes.

[0240] The device of the present invention can be manufactured using techniques well known in the art. The choice of manufacturing technology will depend on the material used for the device and the size of the spacer array and / or the size of the spacer. Exemplary materials for manufacturing the device of the present invention include glass, silicon, steel, nickel, polymers such as polymethyl methacrylate (PMMA), polycarbonate, polystyrene, polyethylene, polyolefins, silicones (such as poly(dimethylsiloxane)), polypropylene, cis-polyisoprene (rubber), polyvinyl chloride (PVC), polyvinyl acetate (PVAc), polychloroprene (chloroprene), polytetrafluoroethylene (Teflon), polyvinylidene chloride (SaranA) and cyclic olefin polymer (COP)) and cyclic olefin copolymer (COC) and combinations thereof. Other materials are known in the art. For example, deep reactive ion etching (DRIE) is used to manufacture silicon-based devices with small gaps, small spacers and large aspect ratios (the ratio of spacer height to lateral dimension). For example, thermoforming of plastic parts (embossing, injection molding) can also be used when the smallest lateral features are >20 microns and the aspect ratio of these features is ≤10.

[0241] Other methods include photolithography (e.g., stereolithography or X-ray lithography), molding, embossing, silicon micromachining, wet or dry chemical etching, grinding, diamond cutting, photolithography and electroforming (LIGA), and electroplating. For example, for glass, traditional silicon manufacturing techniques of photolithography can be used, followed by wet (KOH) or dry etching (reactive ion etching with fluorine or other reactive gases). For plastic materials with high photon absorption efficiency, techniques such as laser micromachining can be used. Due to the continuous nature of the process, this technology is suitable for lower-volume manufacturing. For mass-produced plastic devices, thermoplastic injection molding and compression molding may be suitable. Conventional thermoplastic injection molding used for large-scale manufacturing of optical discs (which maintains the fidelity of submicron features) can also be used to manufacture the devices of the present invention. For example, device features are copied onto a glass master by conventional photolithography. The glass master is electroformed to produce a tough, thermal shock resistant, thermally conductive, hard mold. The mold is used as a master template for injection or compression molding the features into the plastic device. Depending on the plastic material used to manufacture the device and the requirements for optical quality and yield of the finished product, compression molding or injection molding can be selected as the manufacturing method. Compression molding (also known as hot embossing or relief embossing) has the advantage of being compatible with high molecular weight polymers, which are very good for small structures and can replicate high aspect ratio structures, but has longer cycle times. Injection molding is suitable for low aspect ratio structures and is best suited for low molecular weight polymers.

[0242] A device can be fabricated in one or more pieces and then assembled. The layers of the device can be bonded together using clamps, adhesives, heating, anodic bonding, or reactions between surface groups (e.g., wafer bonding). Alternatively, a device with channels or gaps in more than one plane can be fabricated as a single piece, for example using stereolithography or other three-dimensional fabrication techniques.

[0243] In order to reduce the nonspecific adsorption of compounds released by cells or lysed cells on the device surface, one or more surfaces of the device can be chemically modified to make it non-adhesive or repellent. The surface can be coated with a thin film coating (e.g., a monolayer) of a commercially available non-stick reagent, such as those used to form hydrogels. Other examples of chemicals that can be used to modify the surface of the device include oligoethylene glycols, fluorinated polymers, organosilanes, thiols, polyethylene glycol, hyaluronic acid, bovine serum albumin, polyvinyl alcohol, mucin, polyHEMA, methacrylate PEG and agarose. Charged polymers can also be used to repel substances with opposite charges. The type of chemical used for repulsion and the method of attachment to the device surface will depend on the nature of the substance being repelled and the nature of the surface and substance to which it is attached. Such surface modification techniques are well known in the art. The surface can be functionalized before or after assembly of the device. The surface of the device can also be coated to capture materials in the sample, such as membrane fragments or proteins.

[0244] In certain embodiments of the present disclosure, the method for manufacturing any Q-card of the present disclosure may include injection molding of a first plate. In certain embodiments of the present disclosure, the method for manufacturing any Q-card of the present disclosure may include nanoimprinting or extrusion printing of a second plate.

[0245] In certain embodiments of the present disclosure, a method for manufacturing any Q-card of the present disclosure may include laser cutting a first plate. In certain embodiments of the present disclosure, a method for manufacturing any Q-card of the present disclosure may include nanoimprinting or extrusion printing of a second plate. In certain embodiments of the present disclosure, a method for manufacturing any Q-card of the present disclosure may include injection molding and laser cutting a first plate. In certain embodiments of the present disclosure, a method for manufacturing any Q-card of the present disclosure may include nanoimprinting or extrusion printing of a second plate. In certain embodiments of the present disclosure, a method for manufacturing any Q-card of the present disclosure may include nanoimprinting or extrusion printing to manufacture a first plate and a second plate. In certain embodiments of the present disclosure, a method for manufacturing any Q-card of the present disclosure may include: manufacturing a first plate or a second plate using injection molding, laser cutting a first plate, nanoimprinting, extrusion printing, or a combination of the above. In certain embodiments of the present disclosure, a method for manufacturing any Q-card of the present disclosure may include the step of attaching hinges to the first plate and the second plate after manufacturing the first plate and the second plate.

[0246] E) Sample type and subjects

[0247] Details of the samples and subjects are described in detail in various publications, including International Application (IA) PCT / US2016 / 046437 filed on August 10, 2016, IA PCT / US2016 / 051775 filed on September 14, 2016, IA No. PCT / US201 / 017307 filed on February 7, 2018, and IA No. PCT / US2017 / 065440 filed on December 8, 2017, which are hereby incorporated by reference for all purposes.

[0248] The sample can be obtained from a subject. The subjects described herein can be of any age and can be adults, infants, or children. In some cases, the age of the subject is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 9697, 98 or 99 years old, or within a certain range (e.g., between 2 and 20 years old, between 20 and 40 years old, or between 40 and 90 years old). A specific category of subjects that may benefit is subjects who have or are suspected of having an infection (e.g., a bacterial and / or viral infection). Another special category of subjects that may benefit is subjects who are at higher risk of infection. In addition, the subjects treated by any of the methods or compositions described herein can be male or female. Any of the methods, devices or kits disclosed herein can also be performed on non-human subjects, such as laboratory or farm animals. Non-limiting examples of non-human subjects include dog, goat, guinea pig, hamster, mouse, pig, non-human primate (eg, gorilla, ape, orangutan, lemur, or baboon), rat, sheep, cow, or zebrafish.

[0249] The devices, apparatus, systems and methods disclosed herein can be used with samples such as, but not limited to, diagnostic samples, clinical samples, environmental samples and food samples.

[0250] For example, in certain embodiments, the devices, apparatus, systems, and methods disclosed herein are used to sample cells, tissues, body fluids, and / or mixtures thereof. In certain embodiments, the sample comprises a human body fluid. In certain embodiments, the sample comprises cells, tissues, body fluids, feces, amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, uterus, chyle, cerumen, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomitus, urine, and at least one of exhaled breath condensate.

[0251] In certain embodiments, the apparatuses, devices, systems, and methods disclosed herein are used with environmental samples obtained from any suitable source, such as, but not limited to, rivers, lakes, ponds, oceans, glaciers, icebergs, rainwater, snow, sewage, reservoirs, tap water, drinking water, and the like; solid samples from soil, compost, sand, rock, concrete, wood, brick, sewage, and the like; in certain embodiments, environmental samples freshly obtained from a source; and gaseous samples from air, underwater vents, industrial exhaust, vehicle exhaust, and the like. In certain embodiments, environmental samples are processed, for example, by converting a non-liquid sample into a liquid form prior to application of the apparatuses, devices, systems, and methods of the present invention.

[0252] In certain embodiments, the devices, apparatuses, systems, and methods disclosed herein are used with food samples that are suitable or have the potential to become suitable for animal consumption, such as human consumption. In certain embodiments, food samples include raw, cooked or processed foods, plant and animal food sources, pre-processed foods, and partially or fully processed foods. In certain embodiments, samples in non-liquid form are converted to liquid form prior to application of the devices, apparatuses, systems, and methods of the present invention.

[0253] The subject devices, apparatus, systems and methods may be used to analyze samples of any volume. Examples of volumes include, but are not limited to, 10 mL or less, 5 mL or less, 3 mL or less, 1 microliter (uL, also referred to herein as "uL") or less, 500 uL or less, 300 uL or less, 250 uL or less, 200 uL or less, 170 uL or less, 150 uL or less, 125 uL or less, 100 uL or less, 75 uL or less, 50 uL or less, Equal to 25uL, less than or equal to 20uL, less than, less than or equal to 15uL, less than or equal to 10uL, less than or equal to 5uL, less than or equal to 3uL, less than or equal to 1uL, less than or equal to 0.5uL, less than or equal to 0.1uL, less than or equal to 0.05uL, less than or equal to 0.001uL, less than or equal to 0.0005uL, less than or equal to 0.0001uL, less than or equal to 10pL, less than or equal to 1pL, or a range between two values.

[0254] In certain embodiments, the volume of the sample includes, but is not limited to, less than or equal to 100uL, less than or equal to 75uL, less than or equal to 50uL, less than or equal to 25uL, less than or equal to 20uL, less than or equal to 15uL, less than or equal to 10uL, less than or equal to 5uL, less than or equal to 3uL, less than or equal to 1uL, less than or equal to 0.5uL, less than or equal to 0.1uL, less than or equal to 0.05uL, less than or equal to 0.001uL, less than or equal to 0.0005uL, less than or equal to 0.0001uL, less than or equal to 10pL, less than or equal to 1pL, or a range between two values. In certain embodiments, the volume of the sample includes but is not limited to less than or equal to 10uL, less than or equal to 5uL, less than or equal to 3uL, less than or equal to 1uL, less than or equal to 0.5uL, less than or equal to 0.1uL, less than or equal to 0.05uL, less than or equal to 0.001uL, less than or equal to 0.0005uL, less than or equal to 0.0001uL, less than or equal to 10pL, less than or equal to 1pL, or a range between two values.

[0255] In some embodiments, the sample is an amount of about one drop of liquid. In some embodiments, the sample is an amount collected from a pricked finger or fingertip. In some embodiments, the sample is an amount collected from a microneedle, micropipette, or intravenous aspiration.

[0256] F) Machine Learning

[0257] Details of the network are described in detail in various publications, including International Application (IA) PCT / US2018 / 017504, filed February 8, 2018, and PCT / US2018 / 057877, filed October 26, 2018, which are incorporated herein by reference for all purposes.

[0258] One aspect of the present invention provides a framework for machine learning and deep learning for analyte detection and localization. A machine learning algorithm is an algorithm that can learn from data. A more rigorous definition of machine learning is: "A computer program is said to learn about some class of tasks T and performance measure P from experience E if its performance in task T (measured by P) improves with experience E." It explores the study and construction of algorithms that can learn from data and make predictions - such algorithms overcome static program instructions by building models based on sample inputs to make data-driven predictions or decisions.

[0259] Deep learning is a special type of machine learning based on a set of algorithms that attempt to model high-level abstractions in data. In a simple case, there might be two groups of neurons: one that receives input signals and one that sends output signals. When the input layer receives input, it passes a modified version of the input to the next layer. In a deep network, there are many layers between the input and output (these layers are not made up of neurons, but they help us think that way), allowing the algorithm to use multiple processing layers composed of multiple linear and non-linear transformations.

[0260] One aspect of the present invention is to provide two methods for analyte detection and localization. The first method is a deep learning method, and the second method is a combination of deep learning and computer vision methods.

[0261] (i) Deep Learning Approach. In the first approach, the publicly available analyte detection and localization workflow consists of two phases: training and prediction. We describe the training and prediction phases in the following paragraphs.

[0262] (a) Training phase

[0263] During the training phase, annotated training data is fed into a convolutional neural network. Convolutional neural networks are specialized neural networks designed for processing data, with a grid-like, feedforward, layered network topology. Examples of such data include time series data, which can be viewed as a 1D grid sampled at regular intervals, and image data, which can be viewed as a 2D grid of pixels. Convolutional networks have proven successful in practical applications. The name "convolutional neural network" indicates that they employ a mathematical operation called convolution, a specialized linear operation. Convolutional networks are simply neural networks that use convolution in at least one layer instead of a typical matrix multiplication.

[0264] The machine learning model receives one or more sample images containing analytes acquired by an imager on a sample-mounted QMAX device as training data. The training data is annotated with the analytes to be analyzed, where the annotations indicate whether the analytes are present in the training data and their locations within the image. Annotations can take the form of tight bounding boxes that completely encompass the analyte or the center location of the analyte. In the latter case, the center location is further converted to a circle that covers the analyte or a Gaussian kernel in the dot map.

[0265] Training machine learning models faces two challenges when the amount of training data is large: annotation (usually done by humans) is time-consuming, and training is computationally expensive. To overcome these challenges, the training data can be divided into smaller patches, and then these patches or parts of patches are annotated and trained. The term "machine learning" can refer to algorithms, systems, and devices in the field of artificial intelligence that typically use statistical techniques and artificial neural networks that are trained from data without being explicitly programmed.

[0266] The annotated images are fed into the machine learning (ML) training module, and the model trainer in the ML module trains an ML model from the training data (annotated sample images). The input data is fed into the model trainer over multiple iterations until a specific stopping condition is met. The output of the ML training module is the ML model—a computational model built from the machine learning training process based on data that enables a computer to perform certain tasks (e.g., detect and classify subjects) on its own.

[0267] The trained machine learning model is applied by a computer during the prediction (or inference) phase. Examples of machine learning models include ResNet and DenseNet, which are also referred to as "deep learning models" due to the depth of the connected layers in their network structures. In certain embodiments, the Caffe library with a fully convolutional network (FCN) is used for model training and prediction, although other convolutional neural network architectures and libraries, such as TensorFlow, can also be used.

[0268] The training phase generates a model that is used during the prediction phase. This model can be reused during the prediction phase to analyze inputs. Therefore, the compute unit only needs access to the generated model. It does not need access to the training data, nor does the training phase need to be run again on the compute unit.

[0269] (b) Prediction stage

[0270] In the prediction / inference phase, the detection component is applied to the input image, which is then fed into the prediction (inference) module pre-loaded with the trained model generated from the training phase. The output of the prediction phase can be a bounding box containing the detected analytes and their center locations, a dot map indicating the location of each analyte, or a heat map containing information about the detected analytes.

[0271] When the output of the prediction phase is a list of bounding boxes, the number of analytes detected in the sample image is represented by the number of bounding boxes detected. When the output of the prediction phase is a dot map, the number of analytes in the sample image used for analysis is represented by the integral of the dot map. When the output of the prediction phase is a heat map, the localization component is used to identify the locations, and the number of detected analytes is represented by the entries in the heat map.

[0272] One embodiment of a localization algorithm is to sort the heatmap values ​​into a one-dimensional ordered list from highest to lowest. The pixel with the highest value is then selected and the pixel and its neighbors are removed from the list. This process is repeated to select the pixel with the highest value in the list until all pixels are removed from the list.

[0273] In the detection component using heatmaps, the input image is fed into a convolutional neural network along with the model generated from the training phase. The output of the detection phase is a pixel-level prediction in the form of a heatmap. The heatmap can be the same size as the input image or a scaled-down version of the input image, and it serves as the input to the localization component. We disclose an algorithm for locating the center of the analyte. The main idea is to iteratively detect local peaks from the heatmap. After locating the peak, we calculate the local area around the peak, but with a smaller value. We remove this area from the heatmap and find the next peak from the remaining pixels. This process is repeated, only to remove all pixels from the heatmap.

[0274] In certain embodiments, the present invention provides a localization algorithm that sorts the heatmap values ​​from highest to lowest into a one-dimensional ordered list. The pixel with the highest value is then selected, and the pixel and its neighboring pixels are removed from the list. This process is repeated to select the pixel with the highest value in the list until all pixels are removed from the list.

[0275]

[0276]

[0277] After sorting, the heatmap is a one-dimensional sorted list, with heatmap values ​​ordered from highest to lowest. Each heatmap value is associated with its corresponding pixel coordinate. The first item in the heatmap is the item with the highest value, which is the output of the pop(heatmap) function. A disk is created, centered at the pixel coordinate of the disk with the highest heatmap value. All heatmap values ​​whose pixel coordinates fall within the disk are then removed from the heatmap. The algorithm repeatedly pops the maximum value in the current heatmap and removes the disks surrounding it until all items are removed from the heatmap.

[0278] In an ordered list heatmap, each item has knowledge of the preceding and following items. When an item is removed from an ordered list, we make the following changes:

[0279] ··Assume that the deleted item is x r , whose continuation term is x p , and its subsequent term is x f .

[0280] ··For ongoing projects x p , redefines its next item to be the item after the deleted item. Therefore, x p The following term is now x f .

[0281] ●·For the deleted item x r , undefine its continuation and successor to remove it from the ordering list.

[0282] ●·For the following items x f , redefines its processing item as the processing item of the deleted item. Therefore, x f The progressive term is now x p .

[0283] After all items are removed from the ordered list, the localization algorithm is complete. The number of elements in the set locus will be the number of analytes, and the position information is the pixel coordinates of each s in the set locus.

[0284] Another embodiment searches for local peaks, which are not the local peaks with the highest heatmap values. To detect each local peak, we start from a random starting point and search for the local maximum. Once a peak is found, we calculate the local area around the peak where the value is smaller. We remove this area from the heatmap and find the next peak from the remaining pixels. This process is repeated until all pixels are removed from the heatmap.

[0285]

[0286] This is a breadth-first search algorithm starting from s, where the conditions for visiting points are changed: the neighbor p of the current position q is added only if heatmap[p]>0 and heatmap[p]<=heatmap[q]. Therefore, every pixel in the cover has a non-descending path to the local peak s.

[0287]

[0288]

[0289] (ii) A hybrid of deep learning and computer vision approaches. In this second approach, detection and localization are achieved using computer vision algorithms, while classification is achieved using a deep learning algorithm. The computer vision algorithm detects and localizes possible candidates for the analyte, while the deep learning algorithm classifies each possible candidate as a true analyte or a false analyte. The locations of all true analytes (along with the total number of true analytes) are recorded as output.

[0290] (a) Detection. Computer vision algorithms detect possible candidates based on the characteristics of the analyte, including but not limited to intensity, color, size, shape, distribution, etc. Preprocessing schemes can improve the detection effect. Preprocessing schemes include contrast enhancement, histogram adjustment, color enhancement, noise reduction, smoothing, defocusing, etc. After preprocessing, the input image is sent to the detector. The detector notifies the presence of possible analytes and gives an estimate of their location. Detection can be based on the structure of the analyte (e.g. edge detection, line detection, circle detection, etc.), connectivity (e.g. blob detection, connected components, contour detection, etc.), intensity, color, shape using schemes such as adaptive thresholding, etc.

[0291] (b) Localization. After detection, computer vision algorithms localize each possible analyte candidate by providing its boundaries or a tight bounding box containing those boundaries. This can be achieved through subject segmentation algorithms such as adaptive thresholding, background subtraction, flood filling, mean shift, and thresholding. Often, localization can be combined with detection to produce a detection result and the location of each analyte candidate subject.

[0292] (c) Classification. Deep learning algorithms such as convolutional neural networks achieve state-of-the-art visual classification. We employ deep learning algorithms to classify each possible analyte. Various convolutional neural networks can be used for analyte classification, such as VGGNet, ResNet, MobileNet, and DenseNet.

[0293] Given each possible analyte candidate, a deep learning algorithm computes layers of neurons through convolutional and nonlinear filters to extract high-level features that distinguish analytes from non-analytes. Fully convolutional network layers combine these high-level features into a classification result, indicating whether it is a true analyte or a likelihood of being an analyte.

[0294] G) Applications, biological / chemical biomarkers and health conditions

[0295] Applications of the present invention include, but are not limited to, (a) detection, purification and quantification of compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental illness, neuropsychiatric diseases and organic diseases, such as lung disease, kidney disease, (b) detection, purification and quantification of microorganisms (such as viruses, fungi and bacteria) in the environment (such as water, soil or biological samples), tissues, body fluids, (c) detection of compounds or biological samples that pose a threat to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitors, such as glucose, blood oxygen levels, total blood cell count; (e) detection and quantification of specific DNA or RNA from biological samples (such as cells, viruses); (f) sequencing and comparison of gene sequences in chromosomal and mitochondrial DNA for genomic analysis; or (g) detection of reaction products during, for example, drug synthesis or purification.

[0296] Detection can be performed in various sample matrices, such as cells, tissues, body fluids and feces. Target body fluids include, but are not limited to, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), uterus (ear wax), chyle, chi, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomitus, urine and exhaled condensate. In some embodiments, the sample comprises a human body fluid. In some embodiments, the sample comprises at least one of cells, tissue, body fluid, stool, amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, uterine, chyle, cerumen, endolymph, peripheral lymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomitus, urine, and exhaled condensate.

[0297] In embodiments, the sample is at least one of a biological sample, an environmental sample, and a biochemical sample.

[0298] The devices, systems, and methods of the present invention can be used in a variety of applications across a wide range of fields where it is desirable to determine the presence and / or quantification of one or more analytes in a sample. For example, the present methods can be used to detect proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds, and the like. These fields include, but are not limited to, human, veterinary, agricultural, food, environmental, and pharmaceutical testing.

[0299] In certain embodiments, the methods of the present invention can be used to detect nucleic acids, proteins, or other biomolecules in a sample. The methods may include detecting a panel of biomarkers in a sample, such as two or more different protein or nucleic acid biomarkers. For example, the methods can be used to rapidly and clinically detect two or more disease biomarkers in a biological sample, such as can be used to diagnose a disease condition in a subject or in ongoing treatment or therapy. As described above, communication with a physician or other healthcare provider can better ensure that the physician or other healthcare provider is aware of and is aware of potential hazards, and therefore more likely to take appropriate action.

[0300] Applications of the devices, systems, and methods of the present invention using CROF devices include, but are not limited to, (a) detection, purification, and quantification of compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, psychiatric diseases, neuropsychiatric diseases, and organic diseases, such as lung disease and kidney disease; (b) detection, purification, and quantification of microorganisms (such as viruses, fungi, and bacteria) in environments such as water, soil, or biological samples such as tissues and body fluids; (c) detection of compounds or biological samples that pose a threat to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitoring, such as glucose, blood oxygen levels, and total blood cell counts; (e) detection and quantification of specific DNA or RNA from biological samples (such as cells, viruses); (f) sequencing and comparison of gene sequences in chromosomal and mitochondrial DNA for genomic analysis; or (g) detection of reaction products during, for example, drug synthesis or purification. Some specific applications of the devices, systems, and methods of the present invention are now further described in detail.

[0301] Applications of the present invention include, but are not limited to, (a) detection, purification and quantification of compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental illness, neuropsychiatric diseases and organic diseases, such as lung disease, kidney disease, (b) detection, purification and quantification of microorganisms (such as viruses, fungi and bacteria) in the environment (such as water, soil or biological samples), tissues, body fluids, (c) detection of compounds or biological samples that pose a threat to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitors, such as glucose, blood oxygen levels, total blood cell count; (e) detection and quantification of specific DNA or RNA from biological samples (such as cells, viruses); (f) sequencing and comparison of gene sequences in chromosomal and mitochondrial DNA for genomic analysis; or (g) detection of reaction products during, for example, drug synthesis or purification.

[0302] Implementation of the devices, systems, and methods of the present invention may include: a) obtaining a sample, b) applying the sample to a CROF device containing a capture agent that binds to a target analyte under conditions suitable for analyte binding; placing the sample in the capture agent; c) cleaning the CROF device; and d) reading the CROF device to obtain a measurement of the amount of analyte in the sample. In some embodiments, the analyte can be a biomarker, an environmental marker, or a food marker. In some cases, the sample is a liquid sample and can be a diagnostic sample (e.g., saliva, serum, blood, sputum, urine, sweat, tears, semen, or mucus); an environmental sample obtained from a river, ocean, lake, rainwater, snow, sewage, sewage treatment runoff, agricultural runoff, industrial runoff, tap water, or drinking water; or a food sample obtained from tap water, drinking water, prepared food, processed food, or raw food.

[0303] In any embodiment, the CROF device can be placed in a microfluidic device, and applying step b) can include applying the sample to the microfluidic device including the CROF device.

[0304] In any embodiment, reading step d) may include detecting a fluorescent or luminescent signal from the CROF device.

[0305] In any embodiment, the reading step d) may comprise reading the CROF device using a handheld device configured to read the CROF device. The handheld device may be a mobile phone, such as a smartphone.

[0306] In any embodiment, the CROF device can include a labeling agent that can bind to the analyte-capture agent complex on the CROF device.

[0307] In any embodiment, the devices, systems and methods of the present invention may further comprise the step of applying a labeling agent of the analyte-capture complex bound to the CROF device to the CROF device between steps c) and d), and then washing the CROF device.

[0308] In any embodiment, the reading step d) may include reading an identifier of the CROF device. The identifier may be an optical barcode, a radio frequency ID tag, or a combination thereof.

[0309] In any embodiment, the devices, systems and methods of the present invention can further comprise applying a control sample to a CROF control device comprising a capture agent that binds to the analyte, wherein the control sample comprises a known detectable amount of the analyte, and reading the control CROF device to obtain a control measurement of the known detectable amount of the analyte in the sample.

[0310] In any embodiment, the sample can be a diagnostic sample obtained from a subject, the analyte can be a biomarker, and the measured amount of the analyte in the sample can be diagnostic of a disease or condition.

[0311] In any embodiment, the devices, systems, and methods of the present invention may further comprise receiving or providing a report to the subject indicating that the measured amount of the biomarker is absent or low and the range of measured values ​​for the biomarker in the individual is at risk for a disease or condition, wherein the measured amount of the biomarker relative to the range of measured values ​​is diagnostic of the disease or condition.

[0312] In any embodiment, the devices, systems, and methods of the present invention may further comprise diagnosing the subject based on information including the measured amounts of biomarkers in the sample. In some cases, the diagnosing step comprises transmitting data including the measured amounts of biomarkers to a remote location and receiving a diagnosis based on the information including the measured values ​​from the remote location.

[0313] In any embodiment, applying step b) can include isolating miRNA from the sample to produce an isolated miRNA sample, and applying the isolated miRNA sample to a disk-coupled post-on-point antenna (CROF device) array.

[0314] In any embodiment, the method can include receiving or providing a report indicating the safety or harmfulness of the subject's exposure to the environment from which the sample was obtained.

[0315] In any embodiment, the method can include: transmitting data comprising the measured amounts of the environmental markers to a remote location; and receiving a report indicating the safety or harmfulness of the subject's exposure to the environment from which the sample was obtained.

[0316] In any embodiment, the CROF device array can include a plurality of capture agents that each bind to an environmental marker, and wherein reading step d) can include obtaining a measure of the amount of the plurality of environmental markers in the sample.

[0317] In any embodiment, the sample can be a food sample, wherein the analyte can be a food marker, and wherein the amount of the food marker in the sample can be correlated with the safety of the food for consumption.

[0318] In any embodiment, the method can include receiving or providing a report indicating the safety or harmfulness of consumption by the subject of the food from which the sample was obtained.

[0319] In any embodiment, the method may include transmitting data including the measured amounts of the food markers to a remote location and receiving a report indicating the safety or harmfulness of the subject consuming the food from which the sample was obtained.

[0320] In any embodiment, the CROF device array can include a plurality of capture agents each bound to a food marker, wherein said obtaining can include obtaining a measure of the amount of the plurality of food markers in the sample, and wherein the plurality of food markers in the sample can be correlated with the safety of the consumed food.

[0321] Also provided herein are kits useful in practicing the devices, systems, and methods of the present invention.

[0322] The sample size can be about one drop of sample. The sample size can be the amount collected from a finger prick or fingertip. The sample size can be the amount collected from a microneedle or intravenous aspiration.

[0323] After obtaining the sample from the source, it can be used without further processing, or it can be processed to, for example, enrich the target analyte, remove large particulate matter, dissolve or resuspend solid samples, etc.

[0324] Any suitable method for applying the sample to the CROF device can be used. Suitable methods may include using a pipette, dropper, syringe, etc. In certain embodiments, when the CROF device is in the form of a dipstick on a support, as described below, the sample can be applied to the CROF device by dipping the sample into the receiving area of ​​the dipstick into the sample.

[0325] Samples can be collected one or more times. Samples collected over time can be aggregated and / or processed separately (by applying to a CROF device and obtaining measurements of the amount of analyte in the sample, as described herein). In some cases, measurements obtained over time can be aggregated and used for longitudinal analysis over time to facilitate screening, diagnosis, treatment, and / or disease prevention.

[0326] As described above, the CROF device can be washed in any convenient manner to remove unbound sample components. In certain embodiments, the surface of the CROF device is washed with a binding buffer to remove unbound sample components.

[0327] The detectable labeling of the analyte can be accomplished by any convenient method. The analyte can be labeled directly or indirectly. In direct labeling, the analyte in the sample is labeled before the sample is applied to the CROF device. In indirect labeling, as described below, the unlabeled analyte in the sample is labeled after the sample is applied to the CROF device to capture the unlabeled analyte.

[0328] Samples from subjects, health conditions of subjects, and other applications of the present invention are further described below. Exemplary samples, health conditions, and applications are also disclosed in, for example, U.S. Publication Nos. 2014 / 0154668 and 2014 / 0045209, which are incorporated herein by reference.

[0329] The present invention can be used in a variety of applications, among which these applications are generally analyte detection applications, in which the presence of a specific analyte in a given sample is detected qualitatively, if not quantitatively. Protocols for performing analyte detection assays are well known to those skilled in the art and need not be described in detail here. Typically, a sample suspected of containing the target analyte is contacted with the surface of a subject nanosensor under conditions sufficient to allow the analyte to bind to its respective capture agent bound to the sensor. The capture agent has a highly specific affinity for the target targeting molecule. This affinity can be an antigen-antibody reaction where an antibody binds to a specific epitope on an antigen, or a DNA / RNA or DNA / RNA hybridization reaction with sequence specificity between two or more complementary chains. Therefore, if the target analyte is present in the sample, it is likely to bind to the sensor at the location of the capture agent and form a complex on the sensor surface. That is, the captured analyte is immobilized on the sensor surface. After removing unbound analyte, the presence of the bound complex (i.e., the immobilized target analyte) on the sensor surface is then detected, for example, using a labeled second capture agent.

[0330] Target specific analyte detection applications include hybridization assays using nucleic acid capture agents and protein binding assays using polypeptides such as antibodies. In these assays, a sample is first prepared and, after sample preparation, is contacted with a subject nanosensor under specific binding conditions, thereby forming a complex between the target nucleic acid or polypeptide (or other molecule) that is complementary to the capture agent bound to the sensor surface.

[0331] In one embodiment, the capture oligonucleotide is a synthetic single-stranded DNA of 20-100 bases in length, one end of which is thiolated. These molecules are immobilized on the surface of the nanodevice to capture target single-stranded DNA (at least 50 bp in length) with a complementary sequence to the immobilized capture DNA. After the hybridization reaction, a detection single-stranded DNA (20–100 bp in length) is added, whose sequence is complementary to the unoccupied nucleic acid of the target DNA, thereby hybridizing with the target. One end of the detection DNA is coupled to a fluorescent label whose emission wavelength is within the plasma resonance range of the nanodevice. Therefore, by detecting the fluorescent emission emitted from the surface of the nanodevice, the targeted single-stranded DNA can be accurately detected and quantified. The length of the capture and detection DNA determines the melting temperature (the nucleotide chains will separate above the melting temperature) and the degree of mismatch (the longer the chain, the lower the degree of mismatch).

[0332] One consideration in selecting the length of the complementary bond is the need to minimize mismatches while keeping the melting temperature as high as possible. Additionally, the overall length of the hybridization distance is determined to achieve optimal signal amplification.

[0333] The subject sensor can be used in a method for diagnosing a disease or condition, comprising: (a) obtaining a liquid sample from a patient suspected of having a disease or condition, (b) contacting the sample with a subject nanosensor, wherein a capture agent of the nanosensor specifically binds to a biomarker of the disease, and wherein the contacting is performed under conditions suitable for the biomarker to specifically bind to the capture agent; (c) removing any biomarker that is not bound to the capture agent; (d) reading a light signal from the biomarker that is still bound to the nanosensor, wherein the light signal indicates that the patient has the disease or condition, wherein the method further comprises labeling the biomarker with a luminescent tag before or after binding to the capture agent. As will be described in more detail below, the patient may be suspected of having cancer, and the antibody binds to the cancer biomarker. In other embodiments, the patient is suspected of having a neurological disease, and the antibody binds to a biomarker of a neurological disease.

[0334] Applications of subject sensors include, but are not limited to, (a) detection, purification, and quantification of chemical compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular disease, cancer, psychiatric disorders, neuropsychiatric disorders, and organic diseases, such as lung disease, kidney disease, (b) detection, purification, and quantification of microorganisms (e.g., viruses, fungi, and bacteria) in the environment (e.g., water, soil, or biological samples), tissues, body fluids, (c) detection of compounds or biological samples that pose a risk to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitors, such as glucose, blood oxygen levels, total blood cell count; (e) detection and quantification of specific DNA or RNA from biological samples (e.g., cells, viruses); (f) sequencing and comparison of gene sequences in chromosomal and mitochondrial DNA for genomic analysis; or (g) detection of reaction products during, for example, drug synthesis or purification.

[0335] Detection can be performed in various sample matrices (e.g., cells, tissues, body fluids, and feces. Target body fluids include, but are not limited to, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), earwax (ear wax), chyle, cerumen, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomitus, urine, and exhaled condensate.

[0336] In some embodiments, a subject biosensor can be used to diagnose pathogen infection by detecting a target nucleic acid from a pathogen in a sample. The target nucleic acid can be, for example, selected from human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cell leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial virus (RSV), adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human papillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex virus 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8, also known as Kaposi's sarcoma herpesvirus) and flaviviruses, including yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus and Ebola virus. However, the invention is not limited to the detection of nucleic acids, such as DNA or RNA sequences, from the above-mentioned viruses, but can be applied without problems to other pathogens of importance for veterinary and / or human medicine.

[0337] Human papillomavirus (HPV) is further subdivided into over 70 different types based on their DNA sequence homology. These types cause different diseases. HPV types 1, 2, 3, 4, 7, 10, and 26-29 cause benign warts. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25, and 46-50 cause lesions in patients with weakened immune systems. Types 6, 11, 34, 39, 41-44, and 51-55 cause benign condyloma acuminata on the mucous membranes of the genitals and respiratory tract. HPV types 16 and 18 are of particular medical significance because they cause epithelial dysplasia in the genital mucosa and contribute significantly to invasive cancers of the cervix, vagina, vulva, and anal canal. HPV DNA integration is believed to play a decisive role in the carcinogenicity of cervical cancer. HPV can be detected, for example, from the DNA sequences of the capsid proteins L1 and L2. Therefore, the method of the present invention is particularly suitable for detecting DNA sequences of HPV types 16 and / or 18 in tissue samples to assess the risk of cancer.

[0338] In some cases, nanosensors can be used to detect biomarkers present at low concentrations. For example, nanosensors can be used to detect cancer antigens in readily accessible body fluids (e.g., blood, saliva, urine, tears, etc.), to detect biomarkers of tissue-specific diseases in readily accessible body fluids (e.g., biomarkers for neurological diseases such as Alzheimer's disease antigens), to detect infections (especially detection of low-titer latent viruses such as HIV), to detect fetal antigens in maternal blood, and to detect exogenous compounds (e.g., drugs or pollutants).

[0339] The table below provides a list of protein biomarkers and their associated diseases that can be detected using the subject nanosensors (when used in conjunction with appropriate monoclonal antibodies). The table also indicates a potential source of the biomarker (e.g., "CSF"; cerebrospinal fluid). In many cases, the subject biosensors can detect those biomarkers in a different bodily fluid than the indicated bodily fluid. For example, a biomarker present in CSF can be identified in urine, blood, or saliva.

[0340] H) Practicality

[0341] The methods of the present invention can be used in a variety of applications where it is desired to determine the presence and / or quantification of one or more analytes in a sample. For example, the subject methods can be used to detect proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds, and the like.

[0342] In certain embodiments, the methods of the present invention can be used to detect nucleic acids, proteins, or other biomolecules in a sample. The methods may include detecting a panel of biomarkers in a sample, such as two or more different protein or nucleic acid biomarkers. For example, the methods can be used to rapidly and clinically detect two or more disease biomarkers in a biological sample, such as can be used to diagnose a disease condition in a subject or in ongoing treatment or therapy. As described above, communication with a physician or other healthcare provider can better ensure that the physician or other healthcare provider is aware of possible concerns and is therefore more likely to take appropriate action.

[0343] Applications of the devices, systems, and methods of the present invention using CROF devices include, but are not limited to, (a) detection, purification, and quantification of compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular disease, cancer, psychiatric disorders, neuropsychiatric disorders, and organic diseases, such as lung disease and kidney disease; (b) detection, purification, and quantification of microorganisms, such as viruses, fungi, and bacteria, in environmental samples, such as water, soil, or biological samples, such as tissues and body fluids; (c) detection of compounds or biological samples that pose a risk to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitoring, such as glucose, blood oxygen levels, and total blood cell counts; (e) detection and quantification of specific DNA or RNA from biological samples (e.g., cells, viruses); (f) sequencing and comparison of gene sequences in chromosomal and mitochondrial DNA for genomic analysis; or (g) detection of reaction products, such as in drug synthesis or purification processes. Some specific applications of the devices, systems, and methods of the present invention are now described in further detail.

[0344] I) Diagnostic methods

[0345] In certain embodiments, the methods of the present invention can be used to detect biomarkers. In some embodiments, the devices, systems, and methods of the present invention using CROF are used to detect the presence or absence of specific biomarkers, as well as increases or decreases in the concentration of specific biomarkers in blood, plasma, serum, or plasma. Other body fluids or excretions, such as but not limited to urine, blood, serum, plasma, saliva, semen, prostatic fluid, nipple aspirate, tears, sweat, feces, cheek swabs, cerebrospinal fluid, cell lysate samples, amniotic fluid, gastrointestinal fluid, biopsy tissue, etc. Therefore, samples such as diagnostic samples can include various fluids or solid samples.

[0346] In some cases, the sample can be a body fluid sample from the subject to be diagnosed. In some cases, a solid or semi-solid sample can be provided. The sample can include tissue and / or cells collected from the subject. The sample can be a biological sample. Examples of biological samples can include, but are not limited to, blood, serum, plasma, nasal swabs, nasopharyngeal washes, saliva, urine, gastric juice, spinal fluid, tears, feces, mucus, sweat, earwax, oil, glandular secretions, cerebrospinal fluid, tissue, semen, vaginal fluid, tissue fluid derived from tumor tissue, eye fluid, spinal fluid, throat swabs, breath, hair, nails, skin, biopsy, placental fluid, amniotic fluid, umbilical cord blood, lymph, cavity fluid, sputum, pus, microbial flora, meconium, breast milk, exhaled condensate and / or other excreta. The sample can include nasopharyngeal washes. Nasal swabs, throat swabs, stool samples, hair, nails, earwax, breath and other solid, semi-solid or gaseous samples can be processed in an extraction buffer, such as a fixed time or a variable time for their analysis. If desired, the extraction buffer or an aliquot thereof may then be processed similarly to other fluid samples.Examples of tissue samples from a subject may include, but are not limited to, connective tissue, muscle tissue, neural tissue, epithelial tissue, cartilage, cancerous samples, or bone.

[0347] In some cases, the subject from which the diagnostic sample is obtained can be a healthy individual, or can be an individual who is at least suspected of having a disease or health condition. In some cases, the subject can be a patient.

[0348] In certain embodiments, the CROF device includes a capture agent configured to specifically bind to a biomarker in a sample provided by a subject. In certain embodiments, the biomarker can be a protein. In certain embodiments, the biomarker protein is specifically bound by an antibody capture agent present in the CROF device. In certain embodiments, the biomarker is an antibody that specifically binds to an antigen capture agent present in the CROF device. In certain embodiments, the biomarker is a nucleic acid that specifically binds to a nucleic acid capture agent that is complementary to one or both strands of a double-stranded nucleic acid biomarker or to a single-stranded biomarker. In certain embodiments, the biomarker is a nucleic acid that is specifically bound by a nucleic acid binding protein. In certain embodiments, the biomarker is specifically bound by an aptamer.

[0349] The presence or absence of a biomarker or a significant change in the concentration of a biomarker can be used to diagnose disease risk, the presence of a disease in an individual, or to adjust the treatment of a disease in an individual. For example, the presence of a specific biomarker or biomarker group can affect the choice of drug treatment or dosing regimen given to an individual. When evaluating potential drug therapies, biomarkers can be used as surrogate indicators for natural endpoints (such as survival or irreversible morbidity). If a treatment changes a biomarker that is directly related to improved health status, the biomarker can be used as a surrogate endpoint to evaluate the clinical benefit of a specific treatment or dosing regimen. Therefore, personalized diagnosis and treatment based on a specific biomarker or biomarker group detected in an individual is facilitated by this subject method. In addition, as described above, the high sensitivity of the devices, systems and methods of the present invention facilitates early detection of biomarkers associated with the disease. Due to the ability to detect multiple biomarkers using mobile devices (such as smartphones) combined with sensitivity, scalability and ease of use, the methods disclosed in the present invention can be used in portable and point-of-care or near-patient molecular diagnostics.

[0350] In certain embodiments, the methods of the present invention can be used to detect biomarkers of diseases or disease states. In some cases, the methods of the present invention can be used to detect biomarkers to characterize cell signaling pathways and intracellular communication for drug discovery and vaccine development. For example, the subject method can be used to detect and / or quantify the amount of biomarkers in diseased, healthy, or benign samples. In certain embodiments, the methods of the present invention can be used to detect biomarkers of infectious diseases or disease states. In some cases, the biomarkers can be molecular biomarkers, such as, but not limited to, proteins, nucleic acids, carbohydrates, small molecules, and the like.

[0351] The subject methods can be used in diagnostic assays, such as, but not limited to, the following: as described above, detecting and / or quantifying biomarkers; screening assays, in which samples from asymptomatic subjects are examined periodically; prognostic assays, in which the presence and / or amount of biomarkers is used to predict the likely course of a disease; stratified analyses, in which a subject's response to different drug treatments can be predicted; efficacy analyses, in which the efficacy of a drug treatment is monitored; and the like.

[0352] In some embodiments, a subject biosensor can be used to diagnose pathogen infection by detecting a target nucleic acid from a pathogen in a sample. The target nucleic acid can be, for example, selected from human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cell leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial virus (RSV), adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human papillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex virus 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8, also known as Kaposi's sarcoma herpesvirus) and flaviviruses, including yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus and Ebola virus. However, the invention is not limited to the detection of nucleic acids, such as DNA or RNA sequences, from the above-mentioned viruses, but can be applied without problems to other pathogens of importance for veterinary and / or human medicine.

[0353] Human papillomavirus (HPV) is further subdivided into over 70 different types based on their DNA sequence homology. These types cause different diseases. HPV types 1, 2, 3, 4, 7, 10, and 26-29 cause benign warts. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25, and 46-50 cause lesions in patients with weakened immune systems. Types 6, 11, 34, 39, 41-44, and 51-55 cause benign condyloma acuminata on the mucous membranes of the genitals and respiratory tract. HPV types 16 and 18 are of particular medical significance because they cause epithelial dysplasia in the genital mucosa and contribute significantly to invasive cancers of the cervix, vagina, vulva, and anal canal. HPV DNA integration is believed to play a decisive role in the carcinogenicity of cervical cancer. HPV can be detected, for example, from the DNA sequences of the capsid proteins L1 and L2. Therefore, the method of the present invention is particularly suitable for detecting DNA sequences of HPV types 16 and / or 18 in tissue samples to assess the risk of cancer.

[0354] Other pathogens that can be detected in diagnostic samples using the devices, systems and methods of the present invention include, but are not limited to, varicella zoster; Staphylococcus epidermidis, Escherichia coli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcal keratitis, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Klebsiella pneumoniae, gonorrhea (Neisseria gonorrhoeae), syphilis (Treponema pallidum), Chlamydia trachomatis (Chlamydia trachomatis), nongonococcal urethritis (Ureaplasma urealyticum), pulmonitis (Pseudomonas aeruginosa), and chlamydia spp. Meningitis (Haemophilus influenzae), Trichomonas (trichomoniasis); Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MSRA), Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Stenotrophomonas maltophilia, Haemophilus parahaemolyticus, Escherichia coli, Haemophilus enterococci, Enterococci, Enterococci, Enterococci, Citrobacter freundii, Enterococcus faecalis, Klebsiella aerobicus, Pseudomonas fluorescens, Neisseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii, Klebsiella pneumoniae, Legionella pneumoniae, Mycoplasma pneumoniae, Mycoplasma, etc.

[0355] In some cases, CROF devices can be used to detect low concentrations of biomarkers. For example, CROF devices can be used to detect cancer antigens in readily accessible body fluids (e.g., blood, saliva, urine, tears, etc.), to detect tissue-specific disease biomarkers in readily accessible body fluids (e.g., a biomarker for a neurological disease (e.g., an Alzheimer's disease antigen), to detect infection (particularly detecting low titers of latent viruses such as HIV), to detect fetal antigens in maternal blood, and to detect exogenous compounds (e.g., drugs) or pollutants).

[0356] The table also indicates a potential source of the biomarker (e.g., "CSF"; cerebrospinal fluid). In many cases, the subject biosensor can detect those biomarkers in a body fluid different from the indicated body fluid. For example, a biomarker present in CSF can be identified in urine, blood, or saliva. It will also be clear to one of ordinary skill in the art that the CROF device of the present invention can be configured to capture and detect many more biomarkers known in the art for diagnosing diseases or health conditions.

[0357] Unless otherwise specified, a biomarker can be a protein or nucleic acid (e.g., mRNA) biomarker. Unless otherwise specified, a diagnosis can be associated with an increase or decrease in the level of a biomarker in a sample. A list of biomarkers, diseases that can be diagnosed, and samples in which the biomarkers can be detected are described in Tables 1 and 2 of U.S. Patent Application No. 62 / 234,538, filed September 29, 2015, which is incorporated herein by reference.

[0358] In some cases, the devices, systems, and methods of the present invention are used to inform the subject of the sample's health. Health conditions that can be diagnosed or measured using the devices, systems, and methods of the present invention include, but are not limited to: chemical balance; and nutritional health; exercise; fatigue; sleep; stress; prediabetes; allergies; aging; exposure to environmental toxins, pesticides, herbicides, synthetic hormone analogs; pregnancy; menopause; and perimenopause. U.S. Provisional Application Serial No. 3 U.S. Patent Application 62 / 234,538, filed September 29, 2015, provides a list of biomarkers that can be detected using the present CROF device (when used in conjunction with appropriate monoclonal antibodies, nucleic acids, or other capture agents) and their associated health conditions.

[0359] J) Kit

[0360] As described above, aspects of the present disclosure include kits useful in performing the devices, systems, and methods of the present invention. In certain embodiments, the kit includes instructions for practicing the subject method using a handheld device, such as a mobile phone. These instructions can be present in the subject kit in various forms, one or more of which can be present in the kit. One form in which these instructions can appear is information printed on an appropriate medium or substrate, for example, in the packaging of the kit, in a package insert, or on paper with information printed on it. The device will be a computer-readable medium, such as a disk, CD, DVD, Blu-ray, computer-readable memory, etc., on which information has been recorded or stored. Another means that can be present is a website address that can be used via the Internet to access information on a deleted site. The kit may further include software provided on a computer-readable medium for implementing the method for measuring an analyte on a device as described herein. Any convenient method can be used in the kit.

[0361] In some embodiments, the kit includes a detection agent comprising a detectable marker, such as an antioxidant, or a fluorescently labeled antibody or oligonucleotide that specifically binds to the target analyte for labeling the target analyte. The detection agent can be provided in a separate container or within the CROF device.

[0362] In some embodiments, the kit includes a control sample comprising a known detectable amount of an analyte to be detected in the sample. The control sample can be provided in a container and can be present in a solution at a known concentration, or can be provided in a dried form, such as lyophilized or freeze-dried. If provided in a dried form, the kit can also include a buffer for dissolving the control sample.

[0363] Related documents

[0364] The present invention includes various embodiments, which can be combined in various ways as long as the various components do not conflict with each other. The embodiments should be considered as individual invention documents: each document has the other documents as references and is also cited in its entirety for all purposes, rather than as discrete, independent documents. These embodiments include not only the disclosure in the present document, but also documents cited, incorporated, or claiming priority herein.

[0365] (1) definition

[0366] The terms used to describe the devices, systems and methods disclosed herein are defined in the present application or in PCT applications (designating the U.S.) PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application Nos. 62 / 456,065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456,287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456,504, filed on February 8, 2017, all of which are hereby incorporated herein in their entireties for all purposes.

[0367] 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, but in some embodiments, the COF card does not include a spacer. The terms refer to a device that includes a first board and a second board that are movable relative to each other to different configurations (including an open configuration and a closed configuration) and includes a spacer (except in some embodiments of the COF card) to adjust the spacing between the boards. The term "X-board" refers to one of the two boards in the CROF card, with the spacer fixed to the board. Further description of COF cards, CROF cards and X-boards is provided in U.S. patent application 62 / 456,065 filed on February 7, 2017, which is incorporated herein in its entirety for all purposes.

[0368] (2) Q-cards, spacers, and uniform sample thickness

[0369] The devices, systems and methods disclosed herein may include or use Q cards, spacers and uniform sample thickness embodiments for sample detection, analysis and quantification. In some embodiments, the Q card includes a spacer that helps to present at least a portion of the sample as a highly uniform layer. The structure, materials, functions, variations and sizes of the spacers and the uniformity of the spacers and sample layers are disclosed or listed herein, described and summarized in PCT applications (designated U.S.) 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, 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 by reference in their entirety for all purposes.

[0370] (3) Hinges, slots, recessed edges and slides

[0371] 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, recesses and slides that help facilitate the manipulation of the Q-card and the measurement of samples. The structures, materials, functions, variations and sizes of the hinges, notches, notches and slides are disclosed or listed herein, described and summarized in PCT applications (designated U.S.) 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, all of which are incorporated herein in their entirety for all purposes.

[0372] (4) Q-card, slider and smartphone inspection system

[0373] 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 is used with a slider that allows a smartphone detection system to read the card. The structure, materials, functions, variations, dimensions, and connections of the Q-card, slider, and smartphone detection system are disclosed or listed herein, described, and summarized in PCT applications (designating the United States) 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, all of which are incorporated herein in their entirety for all purposes.

[0374] (5) Detection method

[0375] The devices, systems, and methods disclosed herein may include or be used for various types of detection methods. The detection methods are disclosed herein and in PCT applications (designating the United States) 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, all of which are incorporated herein in their entirety for all purposes.

[0376] (6) Labels, capture agents, and detectors

[0377] The devices, systems, and methods disclosed herein can employ various types of labels, capture agents, and detectors for analyte detection. The labels herein are disclosed, listed, described, and summarized in PCT applications (designating the United States) PCT / US2016 / 045437 and PCT / US0216 / 051775, filed August 10, 2016, and September 14, 2016, U.S. Provisional Application No. 62 / 456065, filed February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0378] (7) Analytes

[0379] The devices, systems, and methods disclosed herein can be applied to the manipulation and detection of various types of analytes, including biomarkers. These analytes are disclosed or listed, described, and summarized in PCT applications (designating the United States) 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 hereby incorporated by reference in their entirety for all purposes.

[0380] (8) Application (field and sample)

[0381] The devices, systems, and methods disclosed herein can be used for a variety of applications (field and sample). These applications are disclosed or listed, described, and summarized in PCT applications (designating the United States) PCT / US2016 / 045437 and PCT / US0216 / 051775, filed August 10, 2016, and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0382] (9) Cloud

[0383] The devices, systems, and methods disclosed herein may employ cloud technologies for data transmission, storage, and / or analysis. Relevant cloud technologies are disclosed or listed, described, and summarized in PCT applications (designating the United States) PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016, and September 14, 2016, respectively, and in U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, and 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.

[0384] Other implementations

[0385] Other examples of inventive subjects according to the present disclosure are described in the following enumerated paragraphs.

[0386] 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, for example, when the word "singular" 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 detector and multiple detectors, and the word "agent" includes one agent and multiple agents.

[0387] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about," it will be understood that the particular value forms another embodiment. It will also be understood that the endpoints of each range are significant both relative to the other endpoint and independently of the other endpoint. The term "about" or "approximately" can mean within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limits of the value. For example, according to practice in the art, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold of a value. Where specific values ​​are described in this application and the claims, unless otherwise indicated, the term "about" should be assumed to mean that the specific value is within an acceptable error range. The term "about" has the meaning commonly understood by those of ordinary skill in the art. In some embodiments, the term "about" refers to ±10%. In some embodiments, the term "about" refers to ±5%.

[0388] As used herein, the terms "adapted" and "configured" refer to an element, component, or other object that is designed and / or intended to perform a given function. Thus, the use of the terms "adapted" and "configured" should not be interpreted to mean that a given element, component, or other object is merely "capable" of performing a given function. Similarly, an object described as being configured to perform a particular function may additionally or alternatively be described as being operable to perform that function.

[0389] As used herein, 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 are 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. Therefore, 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 that are structurally and / or functionally similar and / or equivalent are also within the scope of the present disclosure.

[0390] As used herein, the phrases "at least one" and "one or more" when referring 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 entity explicitly 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") can refer to A alone, B alone, or A and B in combination.

[0391] As used herein, the term "and / or" placed between first and second entities means one of (1) the first entity, (2) the second entity, and (3) the first and second entities. Multiple entities listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the entities so connected. In addition to the entities expressly identified by the "and / or" clause, other entities may optionally be present, whether related or unrelated to those specifically identified entities.

[0392] When numerical ranges are mentioned herein, the present 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 is excluded. Unless otherwise indicated, both endpoints should be assumed to be included. Furthermore, unless otherwise indicated or obvious from the context and understanding of a person of ordinary skill in the art.

[0393] If any patent, patent application, or other reference is incorporated herein by reference and (1) defines a term in a manner that is inconsistent with one of the following, and / or (2) is inconsistent with the unincorporated portion of the following: in this disclosure or any other incorporated reference, the unincorporated portion of this disclosure shall control, and a term therein or in the incorporated disclosure shall control solely in the reference and / or incorporated disclosure in which the term was defined.

Claims

1. A device for analyzing target cells and non-cellular analytes in a sample, comprising: a first plate, a second plate, a plurality of spacers, a plurality of particles, and a capture agent, wherein: a) the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration; b) each plate having a sample contact area on its respective surface for contacting a sample containing or suspected of containing one or more target cells and non-cellular analytes; c) the spacer comprises an array of posts having a uniform height of 150 μm or less fixed to the first plate; d) the particles have a size of 0.2 μm to 100 μm and are distributed in one of the sample contact areas of the plate when the device is in an open state; and e) attaching a capture agent to the particle, wherein the capture agent binds to the non-cellular analyte, wherein the non-cellular analyte is capable of binding to the luminescent label to form a labeled non-cellular analyte; One of the open configurations is the configuration where the two plates are separated and the sample is deposited on one or both plates. wherein the closed configuration is a configuration configured after depositing the sample in the open configuration; the associated sample volume is compressed into a layer of substantially uniform thickness by the two plates and substantially stagnant relative to the plates, and the uniform thickness of the layer is defined by the plates and adjusted by the plates and the spacers; wherein the relevant sample volume is the portion of the sample analyzed for both non-cellular analytes and target cells; and wherein the height of the spacer and the geometry of the particles are configured such that in the closed configuration there is no significant overlap between the one or more target cells, between the particles, and between the target cells and the particles; and wherein the capture agent or the detection agent or both the capture agent and the detection agent specifically bind to the non-cellular analyte.

2. A method for determining target cells and non-cellular analytes in a sample, comprising: a) A device according to claim 1; b) depositing a sample containing or suspected of containing target cells and non-cellular analytes; c) after step (b), the two plates are placed in a closed state, and the sample is provided with a labeled detection agent; and d) using an imager and in a closed configuration of the plate, imaging the target cells and non-cellular analytes captured by the capture agent and bound to the labeled detection agent; Wherein the capture agent or the detection agent or both the capture agent and the detection agent specifically bind to the non-cellular analyte.

3. A device for measuring target cells and non-cellular analytes in a sample, comprising: The device according to claim 1; The imager takes at least one image of the relevant sample size; and The processor is configured to process the at least one image to (i) detect the non-cellular analyte by analyzing the image of the particle, and (ii) detect the target cellular analyte.

4. A kit for determining target cell and non-cellular analytes in a sample, comprising: The device according to claim 1; and a labeled detection agent; wherein the capture agent or the detection agent or both the capture agent and the detection agent specifically bind to the non-cellular analyte.

5. A device comprising: a. First board; b. a second plate having a sample contact area on its surface; wherein the first plate and the second plate are movable relative to each other: i. an open structure in which the first plate and the second plate are at least partially separated so that a sample comprising one or more target cells and acellular analytes can be deposited therebetween, and ii. a closed configuration wherein the first plate is placed on top of the second plate, thereby compressing at least a portion of the sample between the first plate and the second plate into a layer having a uniform thickness; c. one or more spacers comprising a periodic array of pillars; and wherein the uniform thickness of the layer is adjusted by one or more spacers, d. a plurality of particles having one or more capture agents on their surface, wherein, in the closed configuration, the first plate and the second plate compress at least a portion of the sample such that there is substantially no overlap between (i) one or more target cells, (ii) the plurality of particles, and (b) iii) the one or more target cells and the plurality of particles.

6. A method for determining cellular and non-cellular analytes in a liquid sample, comprising: a) A device according to claim 1; b) having a sample containing or suspected of containing one or more target cells and non-cellular analytes; c) having an optical tag capable of binding to a non-cellular analyte to form a labeled non-cellular analyte; d) storing samples in the sample contact area of ​​the device in an open configuration, e) after step (d), closing the apparatus to compress at least a portion of the sample into a layer having a uniform thickness; f) capturing at least one image of the sample volume associated with the sample using an imager, wherein the imager is capable of imaging the optical label and the target cell. and g) analyzing at least one image to detect the presence or amount of (i) target cells and / or (ii) non-cellular analytes.

7. A method for determining cellular and non-cellular analytes in a liquid sample, comprising: a) A device according to claim 1; b) having a sample containing or suspected of containing one or more target cells and non-cellular analytes; c) having an optical tag capable of binding to a non-cellular analyte to form a labeled non-cellular analyte; d) storing samples in the sample contact area of ​​the device in an open configuration, e) after step (d), closing the apparatus to compress at least a portion of the sample into a layer having a uniform thickness; f) capturing at least one image of the sample volume associated with the sample using an imager, wherein the imager is capable of imaging the optical label and the target cell. and g) analyzing at least one image using a processor to detect the presence or amount of (i) target cells and / or (ii) non-cellular analytes; wherein the processor is configured to process the at least one image to (i) detect the non-cellular analyte by analyzing images of at least 10 particles, and (ii) detect the target cellular analyte.

Images