Enhanced, rapid, homogeneous cell staining and assays

Abstract

The present invention particularly provides devices, test kits, equipment and methods for rapid homogeneous cell staining and imaging. Specifically, in some embodiments, the present invention can be less than 60 seconds to carry out immunochemical staining of cells or tissues without the need for rinsing. In some embodiments, the present invention stains and observes intracellular analytes (proteins or nucleic acids) within 60 seconds without the need for rinsing.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 722,172, filed on August 23, 2018, by virtue of which the contents of that provisional patent application are incorporated herein by reference in their entirety. The entire disclosure of any publication or patent document mentioned herein is incorporated by reference in its entirety. Technical Field

[0003] In particular, the present invention relates to apparatus and methods for performing rapid cell staining and imaging, such as, but not limited to, immunoassays and staining. Background Art

[0004] There is a need to have rapid cell staining and imaging, and / or to use a cell phone to perform this. Summary of the invention

[0005] The present invention particularly provides devices, test kits, equipment and methods for rapid homogeneous cell staining and imaging. Specifically, in some embodiments, the present invention performs immunochemical staining on cells or tissues in less than 60 seconds without the need for rinsing. In some embodiments, the present invention performs staining and observation of intracellular analytes (proteins or nucleic acids) in 60 seconds without the need for rinsing.

[0006] One aspect of the invention is the use of QMAX cards with dried reagents on the sample contact area of ​​one or both plates to stain samples (eg, cells or tissue).

[0007] Another aspect of the present invention is to use the QMAX card to configure the thickness of the sample into a thin layer to reduce the background signal, so that the stained cells become observable without any washing and / or opening the two plates of the QMAX card. The unbound labeled detection agent in the sample (i.e., the detection agent that is not bound to the target cell) is the main source of the background light signal, wherein the detection agent can be a protein or a nucleic acid.

[0008] Another aspect of the invention is the use of a QMAX card to configure the thickness of the sample into a thin layer so that the stained cells do not substantially overlap in a direction perpendicular to the sample thickness, which can allow the stained cells to be observed from the top of the QMAX card without being substantially blocked by other stained cells.

[0009] Another aspect of the invention is to increase the optical signal (i.e., light signal) from stained cells by non-selectively staining the cells in the sample with other dyes that emit light of the same wavelength as the dye that selectively stains the cells. The addition (i.e., combination) of the optical signal can make the selectively stained cells observable (i.e., distinguishable from the rest of the sample) even without washing out the unbound cells in the sample from the background of the sample.

[0010] Another aspect of the invention is the use of a second labeled antibody to stain target cells, wherein the second labeled antibody selectively attaches to another epitope of the target binding site and has an optical label that emits light at a wavelength overlapping that of the emission of the first labeled antibody.

[0011] Another aspect of the invention is the use of a combination of multiple labeled antibodies, labeled probes, and dyes to stain target cells, wherein the multiple labeled antibodies, labeled probes, and dye labels emit light at wavelengths that overlap with the wavelength of the first labeled antibody.

[0012] Another aspect of the present invention is to provide an apparatus and method for performing rapid RNA fluorescence in situ hybridization (RNA-FISH) staining.

[0013] Another aspect of the invention is to perform Gram-positive and Gram-negative staining.

[0014] Another aspect of the invention is to perform immunostaining.

[0015] In some embodiments, the present invention provides a method for enhancing homogeneous detection of an analyte in cells in a sample, comprising: (a) having a first plate and a second plate, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing cells containing or suspected of containing an analyte, (b) having a detection probe that specifically binds to the analyte and is capable of emitting light of a certain wavelength, wherein the detection probe diffuses in the sample; (c) having an optical enhancer that binds to the cells and is capable of emitting light of a wavelength that overlaps with or is within 30 nm of the wavelength of light emitted by the detection probe; (d) sandwiching the sample, the detection probe, and the optical enhancer between the two sample contact areas of the two plates so as to form a into a thin layer having a thickness of 200 micrometers (μm) or less; and (e) after step (d) and without any washing step, imaging the thin layer using an imager to detect cells having an analyte bound to a detection probe; wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) in the thin layer, positions with bound detection probes are distinguishable from positions without bound detection probes, wherein the bound detection probes are detection probes bound to the analyte in the cells.

[0016] In some embodiments, the present invention provides a method for enhancing homogeneous detection of an analyte in cells in a sample, comprising: (a) having a first plate and a second plate, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing cells containing or suspected of containing an analyte, (b) depositing a detection probe on at least one of the sample contact areas, the detection probe specifically binding to the analyte and emitting light of a certain wavelength, wherein the detection probe diffuses in the sample; (c) having an optical enhancer, the optical enhancer binding to the cells and emitting light in a wavelength range overlapping with or within 30 nm of the wavelength of the light emitted by the detection probe; (d) sandwiching the sample, the detection probe, and the optical enhancer between the two sample contact areas of the two plates to form a thin film. (e) imaging using an imager, after step (d) and without any washing steps, imaging the thin layer using the imager to detect detection probes that have specifically bound to an analyte; wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging; wherein the thickness of the thin layer, the concentration of the detection probes in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that are bound to an analyte in the cell.

[0017] In some embodiments, the present invention provides a method for enhancing homogeneous detection of an analyte in cells in a sample, comprising: (a) having a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample containing cells containing or suspected of containing an analyte, (b) depositing a detection probe on at least one of the sample contact areas, the detection probe specifically binding to the analyte and emitting light of a certain wavelength, wherein the detection probe diffuses in the sample; (c) depositing an optical enhancer on at least one of the sample contact areas, the optical enhancer binding to the cells and emitting light in a wavelength range overlapping with or within 30 nm of the wavelength of light emitted by the detection probe, wherein the optical enhancer diffuses in the sample; (d) combining the sample, the detection probe, and the optical enhancer; The enhancer is sandwiched between two sample contact areas of the two plates to form a thin layer, wherein the thickness of the thin layer is 150 micrometers (μm) or less; and (e) after step (d) and without using any washing steps, using an imager to image the thin layer to detect the detection probe that has specifically bound to the analyte; wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each single cell does not substantially overlap with other cells in the imaging; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) in the thin layer, the position with bound detection probe is distinguishable from the position without bound detection probe, wherein the bound detection probe is a detection probe bound to the analyte in the cell.

[0018] In some embodiments, the present invention provides a kit for enhancing homogeneous detection of an analyte in cells in a sample, comprising: (a) a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample containing cells containing or suspected of containing an analyte, (b) a detection probe that specifically binds to the analyte and is capable of emitting light of a certain wavelength, wherein the detection probe diffuses in the sample; (c) an optical enhancer that binds to the cells and emits light of a wavelength overlapping with or within 30 nm of the wavelength of light emitted by the detection probe; wherein the first plate and the second plate are configured to contact the sample , detection probes and optical enhancers are sandwiched between the two sample contact areas to form a thin layer with a thickness of 200 microns (μm) or less; and wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each single cell does not substantially overlap with other cells during imaging; wherein the thickness of the thin layer, the concentration of the detection probes in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) the positions with bound detection probes are distinguishable from the positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes bound to an analyte in the cell.

[0019] In some embodiments, the present invention provides a device for enhancing homogeneous detection of an analyte in cells in a sample, comprising: (a) a first plate and a second plate, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing cells containing or suspected of containing an analyte, (b) a detection probe that specifically binds to the analyte and emits light of a certain wavelength, wherein the detection probe is coated on at least one of the sample contact areas and diffused in the sample; (c) an optical enhancer that binds to the cells and emits light in a wavelength range that overlaps with or is within 30 nm of the wavelength of light emitted by the detection probe; (d) the sample contact areas in the first and second plates face each other and the sample, the detection probe, and An optical enhancer is sandwiched between two sample contact areas of two plates to form a thin layer, wherein the thickness of the thin layer is 150 micrometers (μm) or less; and (e) an imager that images the thin layer to detect detection probes that have specifically bound to an analyte; wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each single cell does not substantially overlap with other cells in the imaging; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e), positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer. The bound detection probes are detection probes that are bound to the analyte in the cell.

[0020] In some embodiments, the present invention provides a device for enhancing homogeneous detection of an analyte in cells in a sample, comprising: (a) a first plate and a second plate, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing cells containing or suspected of containing an analyte, (b) a detection probe that specifically binds to the analyte and emits light of a certain wavelength, wherein the detection probe is coated on one or both of the sample contact areas and diffused in the sample; (c) an optical enhancer that binds to the cells and emits light in a wavelength range that overlaps with or is within 30 nm of the wavelength of light emitted by the detection probe, wherein the optical enhancer is coated on one or both of the sample contact areas and diffused in the sample; (d) the sample contact areas in the first and second plates face each other, and the sample contact areas are placed in a position adjacent to the sample contact surface. (e) after step (d) and without any washing step, imaging the thin layer using an imager to detect the detection probe that has specifically bound to the analyte; wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each single cell does not substantially overlap with other cells in the imaging; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) in the thin layer, positions with bound detection probes are distinguishable from positions without bound detection probes, wherein the bound detection probes are detection probes bound to the analyte in the cell.

[0021] In some embodiments, the present invention provides a system for enhancing homogeneous detection of an analyte in cells in a sample, comprising: (a) a device or a kit according to any of the above embodiments; and (b) a communication device.

[0022] In some embodiments, the present invention provides a method of using a system for enhancing homogeneous detection of an analyte in cells in a sample, comprising: (a) having a device or a kit according to any of the above embodiments; and (b) having a communication device.

[0023] In some embodiments, the present invention provides a method for rapid homogeneous detection of an analyte in a cell membrane in a sample, comprising: (a) having a first plate and a second plate, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing a cell containing or suspected of containing an analyte inside the cell membrane; (b) having a detection probe that specifically binds to the analyte and can emit light of a certain wavelength, wherein the detection probe diffuses in the sample; (c) having a permeabilizing agent that makes the cell membrane permeable to the detection probe; (d) sandwiching the sample, the detection probe and the permeabilizing agent between the two sample contact areas of the two plates to form a 200 μm thick A thin layer of micrometers (μm) or less; and (e) after step (d) and without using any washing steps, imaging the thin layer to detect cells having analytes bound to detection probes; wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging; wherein the thickness of the thin layer and the concentration of the detection probes in the sample are configured so that locations in the thin layer having detection probes bound to analytes within the cell membrane are distinguishable from locations without cells; and wherein the washing step is a step of removing unbound detection probes, permeabilization reagents, or both from the sample contact area.

[0024] In some embodiments, the present invention provides a method for rapid homogeneous detection of an analyte in a cell membrane in a sample, comprising: (a) having a first plate and a second plate, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing cells containing or suspected of containing an analyte, (b) depositing a detection probe on at least one of the sample contact areas, the detection probe specifically binding to the analyte and emitting light of a certain wavelength, wherein the detection probe diffuses in the sample; (c) having a permeabilizing agent that makes the cell membrane permeable to the detection probe; (d) sandwiching the sample, the detection probe and the optical enhancer between the two sample contact areas of the two plates to form a thin layer, wherein the thin layer has a thickness of 150 micrometers (μm) or less; and (e) ) after step (d) and without any washing steps, imaging the thin layer using an imager to detect detection probes that have specifically bound to the analyte; wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging; wherein the thickness of the thin layer, the concentration of the detection probes in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that are bound to the analyte in the cell; and wherein the washing step is a step of removing unbound detection probes, permeabilization reagents, or both from the sample contact area.

[0025] In some embodiments, the present invention provides a method for rapid homogeneous detection of an analyte in a cell membrane in a sample, comprising: (a) having a first plate and a second plate, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing cells containing or suspected of containing an analyte, (b) depositing a detection probe on at least one of the sample contact areas, the detection probe specifically binding to the analyte and emitting light of a certain wavelength, wherein the detection probe diffuses in the sample; (c) depositing an optical enhancer on at least one of the sample contact areas, the optical enhancer binding to the cells and emitting light in a wavelength range overlapping with or within 30 nm of the wavelength of light emitted by the detection probe, wherein the optical enhancer diffuses in the sample; (d) sandwiching the sample, the detection probe, and the optical enhancer between the two sample contact areas of the two plates to form a thin layer, wherein the thin layer has a thickness of 150 microns (μm) or less; and (e) after step (d) and without using any washing steps, using an imager to image the thin layer to detect the detection probe that has specifically bound to the analyte; wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) in the thin layer, the position with bound detection probe is distinguishable from the position without bound detection probe, wherein the bound detection probe is a detection probe bound to the analyte in the cell; and wherein the washing step is a step of removing unbound detection probe, permeabilization agent, or both from the sample contact area.

[0026] In some embodiments, the present invention provides a method for homogeneously detecting Gram-positive cells or Gram-negative cells in a sample, comprising: (a) having a first plate and a second plate, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing or suspected of containing Gram-positive cells or Gram-negative cells, (b) depositing a Gram-positive stain, a Gram-negative stain, or both on at least one of the sample contact areas, wherein the Gram-positive and Gram-negative stains have two distinguishable colors; wherein the Gram-positive stain only stains Gram-positive cells, while the Gram-negative stain stains both Gram-positive and Gram-negative cells; wherein, in Gram-positive cells stained by both Gram-positive and Gram-negative stains, the Gram-positive stain can be designed by the Gram-negative stain; (c) sandwiching the sample between the two sample contact areas of the two plates so that the sample Forming a thin layer having a thickness of 150 micrometers (μm) or less; and (d) after step (c) and without any washing steps, imaging the thin layer using an imager to detect cells stained by (i) only a Gram-negative stain, (ii) a Gram-positive stain; and (iii); wherein the thin layer sample thickness is configured so that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging; wherein Gram-positive cells appear in a Gram-positive stain color and Gram-negative cells appear only in a Gram-negative stain color; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in the cell.

[0027] In some embodiments, the thickness of the thin layer is configured such that in the imaging step (e) locations in the thin layer with bound detection probes can be distinguished from locations without bound detection probes, wherein the bound detection probes are detection probes that bind to an analyte in the cell.

[0028] In some embodiments, the concentration of the detection probe in the sample is configured such that locations with bound detection probe are distinguishable from locations without bound detection probe in the thin layer in the imaging step (e), wherein the bound detection probe is a detection probe that is bound to an analyte in the cell.

[0029] In some embodiments, the concentration of the optical enhancer in the sample is configured such that in the imaging step (e) locations in the thin layer with bound detection probes are distinguishable from locations without bound detection probes, wherein the bound detection probes are detection probes bound to an analyte in the cell.

[0030] In some embodiments, the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration, wherein: in the open configuration, the first plate and the second plate are partially or completely separated, and the sample is deposited in the sample contact area on one or both of the plates, and the separator is removed from any contact with one or both of the plates; in the closed configuration, at least a portion of the deposited sample is compressed into a thin layer by the two plates.

[0031] In some embodiments, the present invention further includes a plurality of spacers, wherein: (i) the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration, and (ii) one or both of the plates include a spacer fixed to the corresponding plate, wherein in the open configuration, the two plates are completely or partially separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both plates; and wherein in the closed configuration configured after the sample is deposited in the open configuration, at least a portion of the VC sample is between the two plates and in contact with the two plates, and has a very uniform thickness adjusted by the spacer and the two sample surfaces of the plates, and the thickness is equal to or less than 150 μm.

[0032] In some embodiments, imaging includes a step of machine learning.

[0033] In some embodiments, imaging includes a step of machine learning in detecting the analyte.

[0034] In some embodiments, the present invention also includes a non-volatile storage medium having a machine learning algorithm.

[0035] In some embodiments, the imager further comprises a non-volatile storage medium having a machine learning algorithm when detecting the analyte.

[0036] In some embodiments, the analyte comprises a molecule (eg, a protein, peptide, DNA, RNA, nucleic acid or other molecule), cell, tissue, virus or nanoparticle having a different shape.

[0037] In some embodiments, the analyte comprises molecules, proteins, peptides, DNA, RNA, and nucleic acids.

[0038] In some embodiments, analytes within the cell membrane comprise molecules, proteins, peptides, DNA, RNA, and nucleic acids.

[0039] In some embodiments, the sample is a vapor from a biological sample, an environmental sample, a chemical sample, or a clinical sample.

[0040] In some embodiments, detection of the labeled staining reagent is fluorescence-based (referring to other contingencies), chemiluminescence-based (referring to other contingencies), or colorimetric-based (referring to other contingencies) or plasmon-based (referring to other contingencies).

[0041] In some embodiments, the target cells are prokaryotes, such as bacteria and archaea, or eukaryotes, such as animal cells and plant cells.

[0042] In some embodiments, the number of target cells in a sample can be one or more than one.

[0043] In some embodiments, the reagents coated on the device include, but are not limited to, Triton X-100, surfactants, Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tris(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (lla), llb, llc, lld, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium laureth sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, CTAB, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0044] In some embodiments, the reagents coated on the device include agents that render the cell membrane or nucleus permeable by means of osmotic pressure differences between cell membranes during dilution or by adjusting salt concentration.

[0045] In some embodiments, the reagents coated on the device include reagents that cross-link proteins, including but not limited to formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, and potassium permanganate.

[0046] In some embodiments, a dye that stains the WBCs is applied to the first plate or the second plate, or both.

[0047] In some embodiments, a dye that stains WBCs and PLTs is applied to the first plate or the second plate or both.

[0048] In some embodiments, a dye that stains the PLT is applied to the first plate or the second plate, or both.

[0049] In some embodiments, the reagents are applied to the array by droplet printing.

[0050] In some embodiments, the reagents are applied to the plate using the open flow-directing properties of the structure.

[0051] In some embodiments, the reagent is applied by spraying.

[0052] In some embodiments, the reagents are applied by contact printing.

[0053] In some embodiments, the reagents are applied by transfer.

[0054] In some embodiments, a dye that stains the RBCs is applied to the first plate or the second plate, or both.

[0055] In some embodiments, a surfactant for separating and rounding the RBCs is coated onto the first plate or the second plate or both.

[0056] In some embodiments, a chemical that lyses the RBCs is coated onto the first plate or the second plate or both.

[0057] In some embodiments, acridine orange is coated onto the first plate or the second plate or both.

[0058] In some embodiments, the Zwittergent is coated onto the first plate or the second plate or both.

[0059] In some embodiments, methylene blue and Zwittergent are coated onto the first plate or the second plate or both.

[0060] In some embodiments, Acridine Orange and Zwittergent are coated onto the first plate or the second plate or both.

[0061] In some embodiments, the YOYO dye and Zwittergent are applied to the first plate or the second plate or both.

[0062] In some embodiments, the device further comprises a plurality of reagent layers on one or both plates, the plurality of reagent layers comprising anti-adhesion, cell lysis, cell staining, release time control material layers, and combinations thereof;

[0063] In some embodiments, each layer coated on the board has a thickness of 10 nm, 100 nm, 200 nm, 500 nm, 1 um, or a range between any two of the values. .

[0064] In some embodiments, the anti-adhesive agent comprises ethylenediaminetetraacetic acid (EDTA), disodium EDTA, K2EDTA, or K3EDTA.

[0065] In some embodiments, the cell stain comprises Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue and azure B), May-grünwald stain, Leishman's solution ("polychromatic" methylene blue (i.e., demethylated to azure) and eosin), Erythrosine B stain (Erythrosine B), and other fluorescent stains including but not limited to acridine orange dye, 3,3-dihexyloxycarbocyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dyes, ethidium bromide, leucine sulfonamide and diaminostilbene disulfonic acid derivatives, Erythrosine B or Trypan blue, Hoechst 33342, trihydrochloride, Trihydrate, DAPI (4',6-diamidinoflavour-2-phenylindole, dihydrochloride), YOYO.

[0066] In some embodiments, the cell stain comprises Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue and azure B), May-grünwald stain, Leishman's solution ("polychromatic" methylene blue (i.e., demethylated to azure) and eosin), Erythrosine B stain (Erythrosine B), and other fluorescent stains including, but not limited to, acridine orange dye, 3,3-dihexyloxycarbocyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dyes, ethidium bromide, leucine sulfamethoxazole and diaminostilbene disulfonic acid derivatives, Erythrosine B or Trypan blue, Hoechst 33342, trihydrochloride, Trihydrate, DAPI (4',6-diamidino yellow-2-phenylindole, dihydrochloride), YOYO, acid fuchsin, hematoxylin, Hoechst stains including Hoechst 33258 and Hoechst 33342, methyl green, methylene blue, Nile blue, Nile red, osmium tetroxide, rhodamine, safranin, meosic-AAPV-AMC, CFSE, BCECF / AM, silver nitrate, neutral red, pyronin Y, calcein-AM, dihydroethidium, dicrenilide FF, rhodamine 123, 4-methylumbelliferyl palmitate, fast blue B salt, fluorescein yellow, CH dipotassium salt, DAPI double lactate, propidium iodide.

[0067] In some embodiments, the cell lysis agent comprises ammonium chloride, sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), acetic acid, citric acid, or other acids and bases.

[0068] In some embodiments, the release time controlling material comprises albumin, carbomer, carboxymethylcellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinyl pyrrolidone, or polyvinyl alcohol.

[0069] In some embodiments, chemicals with a certain concentration are coated on the plate and dissolved into the blood to achieve uniform distribution of red blood cells in the device.

[0070] In some embodiments, a concentration is coated on a plate and lysed into the blood to lyse red blood cells in the device, where the coating can be on the first plate or the second plate, or both.

[0071] In some embodiments, the chemicals coated in the device include, but are not limited to, surfactants, Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tri(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (lla), llb, llc, lld, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium laureth sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, CTAB, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0072] In some embodiments, the reagents that cause red blood cell lysis coated in the device include but are not limited to Pluronic F-127, polyoxyethylene castor oil, Pluronic F-68, Myrj 52, Brij 35, sodium oleate, sodium dodecyl sulfate, Tween 20, Tween 40, Tween 60, Tween 80, SLS, CTAB, CTAC, tamoxifen, saponin, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, ABS-14, ABS-16, antimalarial drugs (quinine compounds), arsenic, dapsone, metals (chromium / chromates, platinum salts, nickel compounds, copper, lead, cisplatin), nitrites, nitrofurantoin, penicillin, phenazopyridine (marofen), rho immunoglobulin, ribavirin, sulfonamides, and sulfones.

[0073] In some embodiments, the anticoagulant coated in the device includes but is not limited to EDTA, such as dipotassium ethylenediaminetetraacetate (K2EDTA), tripotassium ethylenediaminetetraacetate (K3EDTA), coumarin (vitamin K antagonist), warfarin (comedin), acenaphthenocoumarol, phenprocoumon, atomycin, phenindione, heparin, fondaparinux sodium, and edaparinux, dabigatran etexilate, rivaroxaban, apixaban, edoxaban, betrixaban, NOAC, hirudin, recombinant hirudin, bivalirudin, agratroban, dabigatran etexilate, batroxobin, hirudin, vitamin E, sodium citrate, citrate dextrose, oxalates such as fluoride oxalate, deltaparin, desirudin, enoxaparin.

[0074] In some embodiments, in order to achieve uniform distribution of red blood cells in the device, Zwittergent was added at 3 ng / mm 2 , 5ng / mm 2 , 8ng / mm 2 , 12ng / mm 2 , 15ng / mm 2 , 25ng / mm 2 , 35ng / mm 2 , 50ng / mm 2 , 80ng / mm 2 , 100ng / mm 2 , or a preferred area concentration within a range between any two values ​​is coated on the plate.

[0075] In some embodiments, Zwittergent is added at 100 ng / mm 2 , 120ng / mm 2 , 150ng / mm 2 , 180ng / mm 2 , 200ng / mm2 , 300ng / mm 2 , 400ng / mm 2 , 500ng / mm 2 , 800ng / mm 2 ,1000ng / mm 2 , or a preferred area concentration within a range between any two values ​​is coated on the plate.

[0076] In some embodiments, to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration in the blood of 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 1.0 mg / mL, 2 mg / mL, or in a range between any two of the values.

[0077] In some embodiments, to lyse red blood cells in the device, Zwittergent is coated on the plate at a preferred final concentration in the blood of 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 25 mg / mL, 50 mg / mL, or in a range between any two of the values.

[0078] In some embodiments, in order to achieve uniform distribution of red blood cells in the device, Zwittergent was added at 3 ng / mm 2 , 5ng / mm 2 , 8ng / mm 2 , 12ng / mm 2 , 15ng / mm 2 , 25ng / mm 2 , 35ng / mm 2 , 50ng / mm 2 , 80ng / mm 2 , 100ng / mm 2 , or a preferred area concentration within a range between any two values ​​is coated on the plate.

[0079] In some embodiments, Zwittergent is added at 100 ng / mm 2 , 120ng / mm 2 , 150ng / mm 2 , 180ng / mm 2 , 200ng / mm 2 , 300ng / mm 2 , 400ng / mm 2, 500ng / mm 2 , 800ng / mm 2 , 1000ng / mm 2 , or a preferred area concentration within a range between any two values ​​is coated on the plate.

[0080] In some embodiments, to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration in the blood of 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 1.0 mg / mL, 2 mg / mL, or in a range between any two of the values.

[0081] In some embodiments, to lyse red blood cells in the device, Zwittergent is coated on the plate at a preferred final concentration in the blood of 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 25 mg / mL, 50 mg / mL, or in a range between any two of the values.

[0082] In some embodiments, acridine orange is added at 0.5 ng / mm 2 , 1ng / mm 2 , 2ng / mm 2 , 3ng / mm 2 , 5ng / mm 2 , 8ng / mm 2 , 10ng / mm 2 , 15ng / mm 2 , 20ng / mm 2 , 30ng / mm 2 , or an area concentration within a range between any two values ​​is coated on the plate.

[0083] In some embodiments, acridine orange is added at 3-10 ng / mm 2 The Zwittergent was coated on the plate at an area concentration of 3-10 ng / mm 2 The area concentration is coated on the plate.

[0084] Acridine orange was added at 5-20 ng / mm 2 The Zwittergent was coated on the plate at an area concentration of 10-30 ng / mm 2 The area concentration is coated on the plate.

[0085] In some embodiments, the label is an immunoassay antibody marker, an RNA marker, a staining dye, which is coated on the plate at a preferred final concentration in the sample of 0.1 nM / mL, 0.5 nM / mL, 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL, 50 nM / mL, or in a range between any two of the values.

[0086] In some embodiments, the label is an immunoassay antibody label, an RNA label, a staining dye, which is coated on the plate at a preferred final concentration in the sample of 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL, or in a range between any two values.

[0087] In some embodiments, the label is an immunoassay antibody label, an RNA label, a staining dye, which is coated on the plate at a preferred final concentration in the sample of 0.1 μM / mL, 0.5 μM / mL, 1 μM / mL, 5 μM / mL, 10 μM / mL, 15 μM / mL, 20 μM / mL, 50 μM / mL, or in a range between any two of the values.

[0088] In some embodiments, the label is an immunoassay antibody label, an RNA label, a staining dye, which is coated on the plate at a preferred final concentration in the sample of 0.1 nM / mL, 0.5 nM / mL, 1.0 nM / mL, 1.5 nM / mL, 2.0 nM / mL, 5 nM / mL, 10 nM / mL, 50 nM / mL, or in a range between any two of the values.

[0089] In some embodiments, the label is an immunoassay antibody label, an RNA label, a staining dye, which is coated on the plate at a preferred final concentration in the sample of 0.05 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1.0 mg / mL, or in a range between any two of the values.

[0090] In some embodiments, the label is an immunoassay antibody label, an RNA label, a staining dye, which is coated on the plate at a preferred final concentration in the sample of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL, or in a range between any two of the values.

[0091] In some embodiments, the label is an immunoassay antibody label, an RNA label, a staining dye, which is coated on the plate at a preferred final concentration in the sample of 100 mg / mL, 200 mg / mL, 300 mg / mL, or in a range between any two of the values.

[0092] In some embodiments, the thickness of the sample is 1 um, 2 um, 3 um, 5 um, 10 um, 20 um, 30 um, 50 um, 100 um, 150 um, or in a range between any two of the values.

[0093] In some embodiments, the preferred thickness of the sample is 2 μm, 3 μm, 5 μm, 10 μm, 30 μm, or a range between any two of the values.

[0094] In some embodiments, the concentration of the probe in the sample is 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 10 nM, 50 nM, 100 nM, or a range between any two of these values.

[0095] In some embodiments, the preferred concentration of the probe in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, or a range between any two of the values.

[0096] In some embodiments, the concentration of the enhancer in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 uM, 2 uM, 5 uM, 10 uM, 20 uM, 50 uM, 100 uM, 500 uM, or a range between any two of said values.

[0097] In some embodiments, the preferred concentration of the enhancer in the sample is 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, or a range between any two of the values.

[0098] In some embodiments, the wavelength of light of the optical enhancer and the wavelength of light of the probe are 1 nm, 5 nm, 10 nm, 20 nm, 50 nm, 100 nm, 150 nm, or a range between any two of the values.

[0099] In some embodiments, the light wavelength of the optical enhancer and the light wavelength of the probe are preferably within 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, or within a range between any two of the values.

[0100] In some embodiments, the spacers are secured to the board by directly stamping the board or injection molding the board.

[0101] In some embodiments, the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP, and other plastics.

[0102] In some embodiments, the spacer pitch is in a range of 1 μm to 200 μm.

[0103] In some embodiments, the spacer pitch is in the range of 200 μm to 1000 μm.

[0104] In some embodiments, the spacers that condition the uniform thickness layer have a fill factor of at least 1%, where the fill factor is the ratio of the spacer area in contact with the uniform thickness layer to the total plate area in contact with the uniform thickness layer.

[0105] In some embodiments, for a spacer that adjusts a uniform thickness layer, the Young's modulus of the spacer multiplied by the filling factor of the spacer is equal to or greater than 10 MPa, where the filling factor is the ratio of the spacer area in contact with the uniform thickness layer to the total board area in contact with the uniform thickness layer.

[0106] In some embodiments, for the flexible sheet, the thickness of the flexible sheet multiplied by the Young's modulus of the flexible sheet is in a range of 60 to 750 GPa-μm.

[0107] In some embodiments, for a flexible board, the fourth power of the spacer spacing (ISD) divided by the thickness (h) of the flexible board and the Young's modulus (E) of the flexible board, ISD 4 / (hE), equal to or less than 10 6 um 3 / GPa.

[0108] In some embodiments, one or both plates contain position markers located on the surface or inside the plate, the position markers providing information on the position of the plate.

[0109] In some embodiments, one or both plates contain scale markings on the surface or within the plate that provide information about the lateral dimensions of the structure of the sample and / or the plate.

[0110] In some embodiments, one or both plates contain imaging markers located on the surface or within the plate that aid in imaging of the sample.

[0111] In some embodiments, the spacers act as position markers, scale markers, imaging markers, or any combination thereof.

[0112] In some embodiments, the uniform thickness layer has an average thickness approximately equal to the smallest dimension of the analytes in the sample.

[0113] In some embodiments, the spacer pitch is in a range of 1 μm to 50 μm.

[0114] In some embodiments, the spacer pitch is in a range of 50 μm to 120 μm.

[0115] In some embodiments, the spacer pitch is in a range of 120 μm to 200 μm.

[0116] In some embodiments, the spacer spacing is substantially fixed spacing.

[0117] In some embodiments, the spacers are posts having a cross-sectional shape selected from the group consisting of circular, polygonal, perfect circular, square, rectangular, oval, elliptical, and any combination thereof.

[0118] In some embodiments, the spacers have a columnar shape and have a substantially flat top surface, wherein for each spacer, a ratio of a lateral dimension of the spacer to its height is at least 1.

[0119] In some embodiments, for each spacer, a ratio of a lateral dimension of the spacer to its height is at least 1.

[0120] In some embodiments, the smallest lateral dimension of the spacer is less than or substantially equal to the smallest dimension of the analyte in the sample.

[0121] In some embodiments, the minimum lateral dimension of the spacer is in the range of 0.5 um to 100 um.

[0122] In some embodiments, the minimum lateral dimension of the spacer is in the range of 0.5 um to 10 um.

[0123] In some embodiments, the spacer has a diameter of at least 100 mm. 2 density.

[0124] In some embodiments, the spacer has a thickness of at least 1000 / mm 2 density.

[0125] In some embodiments, at least one of the plates is transparent.

[0126] In some embodiments, at least one of the plates is made of a flexible polymer.

[0127] In some embodiments, the spacers are incompressible with respect to the pressure compressing the plates and / or, independently, only one of the plates is flexible.

[0128] In some embodiments, the flexible sheet has a thickness in a range of 10 μm to 200 μm.

[0129] In some embodiments, the variation is less than 30%.

[0130] In some embodiments, the variation is less than 10%.

[0131] In some embodiments, the variation is less than 5%.

[0132] In some embodiments, the collection plate and the cover plate are connected and are configured to change from an open configuration to a closed configuration by folding the plates.

[0133] In some embodiments, the collection panel and the cover panel are connected by a hinge and are configured to change from an open configuration to a closed configuration by folding the panels along the hinge.

[0134] In some embodiments, the collection plate and the cover plate are connected by a hinge and are configured to change from an open configuration to a closed configuration by folding the plate along the hinge, the hinge being a separate material from the plate.

[0135] In some embodiments, the collection plate and cover plate are made from a single sheet of material and are configured to change from an open configuration to a closed configuration by folding the plates.

[0136] In some embodiments, the device is configured to analyze a sample in 60 seconds or less.

[0137] In some embodiments, in the closed configuration, the final sample thickness device is configured to analyze the sample in 60 seconds or less.

[0138] In some embodiments, the collection plate or cover plate further comprises a first predetermined assay site and a second predetermined assay site on its surface, wherein the distance between the edges of the assay sites is substantially greater than the thickness of the uniform thickness layer when the plate is in a closed position, wherein at least a portion of the uniform thickness layer is above the predetermined assay sites, and wherein the sample has one or more analytes capable of diffusing in the sample.

[0139] In some embodiments, the collection plate or cover plate has at least three analyte assay sites on its surface, and the distance between the edges of any two adjacent assay sites is substantially greater than the thickness of the uniform thickness layer when the plate is in a closed position, wherein at least a portion of the uniform thickness layer is above the assay sites, and wherein the sample has one or more analytes capable of diffusing in the sample.

[0140] In some embodiments, the collection plate or cover plate has at least two adjacent analyte assay sites on its surface, and the at least two adjacent analyte assay sites are not separated by a distance substantially greater than the thickness of the uniform thickness layer when the plate is in a closed position, wherein at least a portion of the uniform thickness layer is above the assay sites, and wherein the sample has one or more analytes capable of diffusing in the sample.

[0141] In some embodiments, the present invention provides a system for quickly analyzing steam condensation samples using a mobile phone, comprising: (a) a device according to claim 1; and (b) a mobile communication device, which comprises: i. one or more cameras for detecting signals and / or imaging steam condensate samples; and ii. electronic equipment, signal processors, and hardware and software for receiving and / or processing detected signals and / or images of steam condensate samples and for remote communication.

[0142] In some embodiments, the system may further include a light source from a mobile communication device or an external source.

[0143] In some embodiments, one of the plates has a binding site for binding an analyte, wherein at least a portion of the uniform thickness sample layer is above the binding site and is substantially smaller than an average lateral linear dimension of the binding site.

[0144] In some embodiments, the present invention further comprises: (d) a housing configured to hold a sample and to be mounted to a mobile communication device.

[0145] In some embodiments, the housing includes optics for facilitating imaging and / or signal processing of a sample by a mobile communication device, and a mount configured to hold the optics on the mobile communication device.

[0146] In some embodiments, an element of the optical device in the housing is movable relative to the housing.

[0147] In some embodiments, the mobile communication device is configured to transmit the test results to medical personnel, a medical institution, or an insurance company.

[0148] In some embodiments, the mobile communication device is further configured to communicate information about the test and the subject to medical personnel, a medical facility, or an insurance company.

[0149] In some embodiments, the mobile communication device is further configured to transmit the tested information to a cloud network, and the cloud network is configured to process the information to improve the test results.

[0150] In some embodiments, the mobile communication device is further configured to transmit the test and subject information to a cloud network, the cloud network is configured to process the information to improve the test results, and the improved test results are sent back to the subject.

[0151] In some embodiments, the mobile communication device is configured to receive a prescription, diagnosis, or advice from a medical professional.

[0152] In some embodiments, the spacers have a predetermined substantially uniform height and a predetermined constant spacer spacing, and wherein at least one of the spacers is within the sample contacting region.

[0153] In some embodiments, the optical signal from the optical enhancer is equal to or less than the optical signal from areas of the sample layer that do not contain target cells.

[0154] In some embodiments, the detection agent and optical enhancement agent are coated on the inner surface of the plates before the sample is deposited between the two plates.

[0155] In some embodiments, the detection agent and optical enhancement agent are coated on the inner surface of the plates before the sample is deposited between the two plates.

[0156] In some embodiments, the layer thickness of at least a portion of the sample is configured such that the time for the detection agent and the optical enhancement agent is 30 seconds or less.

[0157] In some embodiments, the layer thickness of at least a portion of the sample is configured such that the time for the detection agent and the optical enhancement agent is 60 seconds or less.

[0158] In some embodiments, the layer thickness of at least a portion of the sample is configured such that the time for the detection agent and the optical enhancement agent is 120 seconds or less.

[0159] In some embodiments, attachment of the optical enhancer to the target cells is specific.

[0160] In some embodiments, attachment of the optical enhancer to the target cells is non-specific.

[0161] In some embodiments, attachment of the optical enhancer to the target cell is non-specific, wherein the non-specific attachment is binding to nucleic acid of the target cell.

[0162] In some embodiments, the thin layer sample thickness is configured such that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging;

[0163] In some embodiments, an optical enhancer is further included that binds to the cells and is capable of emitting light at a wavelength overlapping with or within 30 nm of the wavelength of light emitted by the detection probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present invention in any way. In the drawings, some drawings are not drawn to scale. In the drawings where experimental data points are given, the lines connecting the data points are only used to guide the observation of the data and have no other purpose.

[0165] Figure 1 An embodiment of QMAX (Q: quantification); M: amplification; A: addition of reagents; X: acceleration; also known as a compression regulated open flow (CROF) device, which includes a first plate and a second plate. Figure (a) shows a cross-sectional view of the device without a sample, with a certain gap between the two plates, and two reagents (1 and 2) coated on the two plates; Figure (b) shows a cross-sectional view of the QMAX device, where the sample is located between the two plates.

[0166] Figure 2An embodiment of a QMAX device is shown, comprising a first plate and a second plate. Figure (a) shows a cross-sectional view of the plates in an open configuration when the plates are separated, with two reagents (1 and 2) applied to the two plates; Figure (b) shows a cross-sectional view of a sample deposited on one of the plates in an open configuration; Figure (c) shows a perspective view of the QMAX device in a closed configuration, with the sample located between the two plates.

[0167] Figure 3 An exemplary effect of using a combination of a labeled antibody and a cell enhancer (e.g., a non-specific cell dye or other specific antibody) on the detection of a signal from a cell or non-cell analyte within a sample is shown. Graph (a) shows that cells without an antibody-labeled enhancer have a signal below the detection threshold; Graph (b) shows that cells with an antibody-labeled enhancer have a signal above the detection threshold.

[0168] Figure 4 An exemplary effect of changing the spacer height on the signal detection of cells or non-cellular analytes in the sample is shown. Figure (a) shows that cells labeled with antibodies at large spacer heights have signals covered by high background from non-cellular analytes in the sample; Figure (b) shows that cells labeled with antibodies at small spacer heights have signals on the background from non-cellular analytes in the sample.

[0169] Figure 5 The effect of different Zwittergent concentrations on leukocyte permeabilization during rapid FISH using a GAPDH probe in QMAX is shown. Panel (a) GAPDH probe was observed using a smartphone-based microscope, and 0.625 mg / mL of Zwittergent in blood did not produce a FISH signal. Panel (b) Zwittergent gave a strong fluorescent signal from GAPDH-labeled white blood cells observed by a smartphone-based microscope, and panel (c) used a fluorescent microscope in (c) (DAPI WBC nuclear counterstain).

[0170] Figure 6 Exemplary photographs of Chlamydia staining using a QMAX device without an optional rinse step are shown. Figure (a) Chlamydia-positive cells were stained with strong fluorescence; Figure (b) Chlamydia-negative cells were not stained with fluorescence. In the experiment, slides fixed with Chlamydia-infected human tissue cells were incubated with 40ug / mL of 1μL DyLight633-labeled anti-Chlamydia antibodies for 2min. X-plates with a column height of 30μm were used in QMAX immunostaining. Images were taken by microscope without rinsing.

[0171] Figure 7Exemplary photographs showing the use of different DNA dyes to differentiate between Gram-positive and Gram-negative bacteria. The three images were taken from different bacterial mixtures that were analyzed using the same rapid staining assay. Bacterial mixtures analyzed: A) mixture of bacterial species, B) only Escherichia coli, C) only Staphylococcus epidermidis.

[0172] Figure 8 An exemplary photograph of CD4 T cell enumeration using the QMAX device taken by an iPhone-based microscope is shown. Cells were stained with SYTO62 non-specific nuclear stain along with detection antibodies. DETAILED DESCRIPTION

[0173] The following detailed description illustrates some embodiments of the present invention by way of example and not limitation. The section headings and any subtitles used herein, if any, are for organizational purposes only and should not be construed as limiting the subject matter described in any way. The content under a section heading and / or subheading is not limited to the section heading and / or subheading, but applies to the entire description of the present invention.

[0174] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0175] It should be noted that the drawings are not intended to show these elements in exact proportion. For the sake of clarity, some elements are shown enlarged in the drawings. The dimensions of the elements should be depicted by the description provided herein and incorporated by reference.

[0176] The terms "optical enhancer" and "enhancer" are interchangeable.

[0177] The terms "cell" and "target cell" are used interchangeably.

[0178] Typically, staining an analyte on or in a cell membrane for imaging is a multi-step process (about 10 steps) that typically requires hours to perform. One aspect of the invention is that cells or tissues are immunochemically stained in less than 60 seconds without the need for rinsing. In certain embodiments, the present invention stains and observes intracellular analytes (proteins or nucleic acids) in 60 seconds without the need for rinsing.

[0179] Example of using optical enhancers to enhance detection of specifically stained cells

[0180] Figures 1 to 4 Several embodiments of the present invention are shown that rapidly stain cells or tissues without washing.

[0181] In some embodiments, a method of enhancing homogeneous detection of an analyte in cells in a sample comprises:

[0182] (a) having a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample comprising cells containing or suspected of containing an analyte,

[0183] (b) a detection probe that specifically binds to the analyte and can emit light of a certain wavelength, wherein the detection probe diffuses in the sample;

[0184] (c) having an optical enhancer that binds to the cell and is capable of emitting light at a wavelength overlapping or within 30 nm of the wavelength of light emitted by the detection probe;

[0185] (d) sandwiching the sample, the detection probe, and the optical enhancer between two sample contacting areas of two plates so as to form a thin layer having a thickness of 200 micrometers (μm) or less; and

[0186] (e) after step (d) and without any washing step, imaging the thin layer using an imager to detect cells having analyte bound to the detection probe;

[0187] wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging;

[0188] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in a cell.

[0189] In some embodiments, a method of enhancing homogeneous detection of an analyte in cells in a sample comprises:

[0190] (a) having a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample comprising cells containing or suspected of containing an analyte,

[0191] (b) depositing a detection probe on at least one of the sample contact regions, the detection probe specifically binding to the analyte and emitting light of a certain wavelength, wherein the detection probe diffuses in the sample;

[0192] (c) having an optical enhancer that binds to the cell and emits light in a wavelength range that overlaps with or is within 30 nm of the wavelength of light emitted by the detection probe;

[0193] (d) sandwiching the sample, the detection probe and the optical enhancer between two sample contact areas of two plates, wherein the thickness of the thin layer is 150 μm or less; and

[0194] (e) after step (d) and without any washing step, imaging the thin layer using an imager to detect the detection probes that have specifically bound to the analyte;

[0195] wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging;

[0196] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in a cell.

[0197] In some embodiments, a method of enhancing homogeneous detection of an analyte in cells in a sample comprises:

[0198] (a) having a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample comprising cells containing or suspected of containing an analyte,

[0199] (b) depositing a detection probe on at least one of the sample contact regions, the detection probe specifically binding to the analyte and emitting light of a certain wavelength, wherein the detection probe diffuses in the sample;

[0200] (c) depositing an optical enhancer on at least one of the sample contact regions, the optical enhancer binding to the cells and emitting light in a wavelength range overlapping or within 30 nm of the wavelength of light emitted by the detection probe, wherein the optical enhancer diffuses in the sample;

[0201] (d) sandwiching the sample, the detection probe and the optical enhancer between two sample contact areas of two plates, wherein the thickness of the thin layer is 150 μm or less; and

[0202] (e) after step (d) and without any washing step, imaging the thin layer using an imager to detect the detection probes that have specifically bound to the analyte;

[0203] wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging;

[0204] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in a cell.

[0205] In some embodiments, a kit for enhancing homogeneous detection of an analyte in cells in a sample comprises:

[0206] (a) a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample comprising cells containing or suspected of containing an analyte,

[0207] (b) a detection probe that specifically binds to the analyte and is capable of emitting light of a certain wavelength, wherein the detection probe diffuses in the sample;

[0208] (c) an optical enhancer that binds to cells and emits light at a wavelength that overlaps with or is within 30 nm of the wavelength of light emitted by the detection probe;

[0209] wherein the first plate and the second plate are configured to sandwich the sample, the detection probe, and the optical enhancer between two sample contact areas so as to form a thin layer having a thickness of 200 micrometers (μm) or less; and

[0210] wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging;

[0211] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in a cell.

[0212] In some embodiments, a device for enhancing homogeneous detection of an analyte in cells of a sample comprises:

[0213] (a) a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample comprising cells containing or suspected of containing an analyte,

[0214] (b) a detection probe that specifically binds to the analyte and emits light of a certain wavelength, wherein the detection probe is coated on at least one of the sample contact regions and diffuses in the sample;

[0215] (c) an optical enhancer that binds to cells and emits light in a wavelength range that overlaps with or is within 30 nm of the wavelength of light emitted by the detection probe;

[0216] (d) the sample contact areas in the first and second plates face each other, and the sample, the detection probe, and the optical enhancer are sandwiched between the two sample contact areas of the two plates to form a thin layer, wherein the thickness of the thin layer is 150 micrometers (μm) or less; and

[0217] (e) an imager that images the thin layer to detect detection probes that have specifically bound to the analyte;

[0218] wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging;

[0219] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in a cell.

[0220] In some embodiments, a device for enhancing homogeneous detection of an analyte in cells of a sample comprises:

[0221] (a) a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample comprising cells containing or suspected of containing an analyte,

[0222] (b) a detection probe that specifically binds to the analyte and emits light of a certain wavelength, wherein the detection probe is coated on one or both of the sample contact regions and diffuses in the sample;

[0223] (c) an optical enhancer that binds to the cells and emits light in a wavelength range that overlaps with or is within 30 nm of the wavelength of light emitted by the detection probe, wherein the optical enhancer is coated on one or both of the sample contact regions and diffuses in the sample;

[0224] (d) the sample contact areas in the first and second plates face each other, and the sample, the detection probe, and the optical enhancer are sandwiched between the two sample contact areas of the two plates to form a thin layer, wherein the thickness of the thin layer is 150 micrometers (μm) or less; and

[0225] (e) after step (d) and without any washing step, imaging the thin layer using an imager to detect the detection probes that have specifically bound to the analyte;

[0226] wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging;

[0227] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in the imaging step (e) positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in a cell.

[0228] In some embodiments, a system for enhancing homogeneous detection of an analyte in cells in a sample comprises:

[0229] (a) a device or a kit according to any of the above embodiments; and

[0230] (b) Communication device.

[0231] In some embodiments, a method for rapid homogeneous detection of an analyte in a cell membrane in a sample comprises:

[0232] (a) having a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample containing cells containing or suspected of containing an analyte inside a cell membrane,

[0233] (b) a detection probe that specifically binds to the analyte and can emit light of a certain wavelength, wherein the detection probe diffuses in the sample;

[0234] (c) having a permeabilizing agent that makes the cell membrane permeable to the detection probe;

[0235] (d) sandwiching the sample, the detection probe, and the permeabilizing agent between two sample contact areas of two plates so as to form a thin layer having a thickness of 200 micrometers (μm) or less; and

[0236] (e) after step (d) and without any washing steps, imaging the thin layer to detect cells having analyte bound to the detection probe;

[0237] wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging;

[0238] wherein the thickness of the thin layer and the concentration of the detection probe in the sample are configured so that locations in the thin layer having the detection probe bound to the analyte within the cell membrane are distinguishable from locations having no cells; and

[0239] The washing step is a step of removing unbound detection probes, permeabilization reagents, or both from the sample contact area.

[0240] In some embodiments, a method for rapid homogeneous detection of an analyte in a cell membrane in a sample comprises:

[0241] (a) having a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact surface contacts a sample comprising cells containing or suspected of containing an analyte,

[0242] (b) depositing a detection probe on at least one of the sample contact regions, the detection probe specifically binding to the analyte and emitting light of a certain wavelength, wherein the detection probe diffuses in the sample;

[0243] (c) having a permeabilizing agent that makes the cell membrane permeable to the detection probe;

[0244] (d) sandwiching the sample, the detection probe and the optical enhancer between two sample contact areas of two plates, wherein the thickness of the thin layer is 150 μm or less; and

[0245] (e) after step (d) and without any washing step, imaging the thin layer using an imager to detect the detection probes that have specifically bound to the analyte;

[0246] wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell does not substantially overlap with other cells in the imaging;

[0247] wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured to make locations in the thin layer with bound detection probe distinguishable from locations without bound detection probe in the imaging step (e), wherein the bound detection probe is a detection probe that binds to an analyte in the cell; and

[0248] The washing step is a step of removing unbound detection probes, permeabilization reagents, or both from the sample contact area.

[0249] A method and apparatus according to any of the above embodiments, wherein the optical signal from the optical enhancer is equal to or less than the optical signal from a region of the sample layer that does not contain target cells.

[0250] The method and apparatus according to any of the above embodiments, wherein the detection agent and optical enhancement agent are coated on the inner surface of the plates before the sample is deposited between the two plates.

[0251] The method and apparatus according to any of the above embodiments, wherein the detection agent and optical enhancement agent are coated on the inner surface of the plates before the sample is deposited between the two plates.

[0252] A method and apparatus according to any of the above embodiments, wherein the layer thickness of at least a portion of the sample is configured so that the time for the detection agent and the optical enhancement agent is 30 seconds or less.

[0253] A method and apparatus according to any of the above embodiments, wherein the layer thickness of at least a portion of the sample is configured so that the time for the detection agent and the optical enhancement agent is 60 seconds or less.

[0254] A method and apparatus according to any of the above embodiments, wherein the layer thickness of at least a portion of the sample is configured such that the time for the detection agent and the optical enhancement agent is 120 seconds or less.

[0255] The method and device according to any of the above embodiments, wherein the attachment of the optical enhancer to the target cells is specific.

[0256] The method and apparatus according to any of the above embodiments, wherein the attachment of the optical enhancer to the target cells is non-specific.

[0257] In some embodiments, the reagent is coated with a slow-release layer that releases the reagent (ie, stain) at least 3 seconds, and in some embodiments at least 10 seconds, or at least 60 seconds after the sample contacts the reagent.

[0258] Immunostaining

[0259] In some embodiments, immunostaining can be used to detect cells or non-cellular analytes in a sample. In some embodiments, immunostaining can be used to detect cells or non-cellular analytes by enhancing the signal from the cells or non-cellular analytes relative to the background signal. Immunostaining generally refers to any method of using one or more antibodies to detect cells or non-cellular analytes in a sample. Naturally occurring antibodies can be proteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain can be composed of a heavy chain variable region (V H ) and a heavy chain constant region. The heavy chain constant region can be composed of three domains C H1 , C H2 and C H3 Each light chain can be composed of a light chain variable region (V L ) and a light chain constant region. The light chain constant region may consist of a domain C L Composition. H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). H and V LIt may be composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1 q) of the classical complement system. The antibody may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), subclass or a modified form thereof.

[0260] Antibodies can include intact immunoglobulins or fragments thereof. Antibody fragments can refer to one or more fragments of an antibody that retain the ability to specifically bind to a cell or non-cell analyte (e.g., an antigen). In addition, aggregates, polymers, and conjugates of immunoglobulins or fragments thereof can be used as appropriate, as long as the binding affinity for a specific molecule is maintained. Examples of antibody fragments include Fab fragments, which consist of V L 、V H , C L and C H1 A monovalent fragment consisting of a V domain; a F(ab)2 fragment, a bivalent fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region; H and C H1 The Fd fragment consists of a single-arm V L and V H Fv fragment composed of V H Single domain antibody (dAb) fragments composed of domains; and isolated CDRs and single chain fragments (scFv), wherein V L and V H The two V domains are paired to form a monovalent molecule (collectively referred to as single-chain Fv (scFv). Therefore, antibody fragments include Fab, F(ab)2, scFv, Fv, dAb, etc. L and V H The domains are encoded by separate genes, but they can be connected using recombinant methods by artificial peptide linkers that enable them to be made into single protein chains. Such single-chain antibodies include one or more antigen-binding portions. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of the fragments can be screened in the same manner as complete antibodies. The antibody can be a human antibody, a humanized antibody, a chimeric antibody, a separated antibody, a dog antibody, a cat antibody, a donkey antibody, a sheep antibody, any plant antibody, an animal antibody, or a mammalian antibody.

[0261] In any embodiment of the present disclosure, any number and / or type of antibodies can be used. Specifically, about 1 antibody, about 2 antibodies, about 3 antibodies, about 4 antibodies, about 5 antibodies, about 10 antibodies, about 25 antibodies, or more than about 25 antibodies can be used. For example, Figure 3 As shown, labeled antibodies can be used to detect cellular analytes within a sample. Figure 3 As shown, 2 kinds of different labeled antibodies can be used for detecting cells or non-cellular analytes (for example, antigens) in samples, thereby enhancing the signal detected from cells or non-cellular analytes relative to background signals. In certain embodiments, two or more antibodies can be used for detecting cells or non-cellular analytes, and the two or more antibodies can be combined with the same molecule. In certain embodiments, two or more antibodies can be used for detecting cells or non-cellular analytes, and the two or more antibodies can be combined with the same epitope. In certain embodiments, two or more antibodies can be used for detecting cells or non-cellular analytes, and the two or more antibodies can be combined with different epitopes (for example, polyclonal antibodies, etc.) on the same antigen. In certain embodiments, two or more antibodies can be used for detecting cells or non-cellular analytes, and the two or more antibodies can be combined with the same epitope on different molecules. In certain embodiments, two or more antibodies can be used for detecting cells or non-cellular analytes, and the two or more antibodies can be combined with different molecules.

[0262] Any antibody used in the embodiments of the present disclosure may be labeled or unlabeled. A label may refer to a molecule that, when connected to another molecule (directly or indirectly, such as by an antibody connection), provides or enhances the means for detecting another molecule. The signal emitted from the label may allow detection of the molecule or complex to which it is attached, and / or the label itself. A label may be a molecular species that causes a physical or chemical response that can be observed or detected by the naked eye or by an instrument, such as but not limited to a camera (e.g., a mobile phone camera), a scintillation counter, a colorimeter, a UV spectrophotometer, etc. Labels include but are not limited to radioisotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (e.g., biotin, avidin, streptavidin or haptens), etc. When excited at different wavelengths, a fluorescent or fluorescent marker or label emits detectable light at a specific wavelength. Radioactive labels or radioactive markers emit radioactive particles that are detectable with instruments such as but not limited to scintillation counters. Other signal generation detection methods include chemiluminescence detection, electrochemiluminescence detection, Raman energy detection, colorimetric detection, hybridization protection assay and mass spectrometry. The non-limiting examples of labeling include fluorophores, chromophores, FITC, TRITC, DTAF, Texas Red, phycoerythrin, allophycocyanin, green fluorescent protein (GFP), blue fluorescent protein (BFP), rhodamine, FAM, TET, HEX, JOE, TAMRA, ROX, aromatic substituted xanthene dyes, 4,7-dichloro-fluorescein, 4,7-dichloro-rhodamine, cyanine dyes, enzymes, horseradish peroxidase (HRP enzyme), alkaline phosphatase, beta-galactosidase, luciferase, chloramphenicol acetyltransferase (CAT enzyme), avidin, streptavidin, biotin, biotinylated protein, or any combination thereof, fragments or derivatives. In any embodiment of the present disclosure, one or more labels can be used to detect cell or non-cellular analytes. In any embodiment of the present disclosure, two or more labels can be used for detecting cells or non-cellular analytes. In certain embodiments, when using two or more labels to detect cells or non-cellular analytes, the excitation and / or emission spectra of two or more labels can be the same. For example, a sample can be contacted with two antibodies (for example, each antibody binds to the same epitope on the same antigen), and each antibody has different labels excited at the same wavelength. In another example, a sample can be contacted with two antibodies (for example, each antibody binds to different epitopes on the same antigen), and each antibody has the same label excited at a predetermined wavelength. In certain embodiments, when using two or more labels to detect cells or non-cellular analytes, the excitation and / or emission spectra of two or more labels can be different.In some embodiments, when three or more markers are used to detect cells or non-cellular analytes, the excitation and / or emission spectra of at least two or more markers may be the same. In some embodiments, when three or more markers are used to detect cells or non-cellular analytes, the excitation and / or emission spectra of at least two or more markers may be different.

[0263] If the antibody contains a label, the detection method may include fluorescence, luminescence, radioactivity, etc. If the antibody is unlabeled, the detection of binding can be based on changes in some physical properties of the target analyte. These physical properties may include, for example, refractive index or electrical impedance. Detecting the binding of unlabeled antibodies may include, for example, mass spectrometry. In a competitive approach, the binding site occupancy may be determined indirectly. In this method, a target cell or non-cellular analyte may be exposed to a solution containing an antibody with an associated label and an unlabeled antibody. The labeled homologous antibody and the unlabeled antibody compete for binding sites on the target analyte. The affinity of the unlabeled antibody for the target analyte relative to the labeled homologous antibody is determined by the reduction in the amount of binding of the labeled antibody.

[0264] Dyeing

[0265] In certain embodiments, dye staining can be used for detecting cells or non-cellular analytes in samples. In certain embodiments, dye staining can be used for detecting cells or non-cellular analytes by enhancing the signal from cells or non-cellular analytes relative to background signal. Dye staining can refer to the technology for enhancing the contrast in the image, and specifically enhancing the detectable signal of the target cell or non-cellular analyte from the sample. Staining agents and dyes can be used for highlighting the structure in biological tissue or cells for observation. Staining agents can be used for determining and checking cell populations (for example, different blood cells or bacteria are classified, such as gram-positive bacteria and gram-negative bacteria), or organelles in individual cells. Dye staining can involve contacting class-specific (for example, DNA, protein, lipid or carbohydrate) dyes with samples to qualitatively or quantitatively determine the presence of specific cells or non-cellular analytes in samples.

[0266] In any embodiment of the present disclosure, any number and / or type of dye can be used. In particular, about 1 dye, about 2 dyes, about 3 dyes, about 4 dyes, about 5 dyes, about 10 dyes, about 25 dyes, or more than about 25 dyes can be used. In certain embodiments, two or more dyes can be used to detect cells or non-cellular analytes, and two or more dyes can be combined with the same molecule. In certain embodiments, two or more dyes can be used to detect cells or non-cellular analytes, and two or more dyes can dye the same region (e.g., the same organelle) of the same target cell or non-cellular analyte. In certain embodiments, two or more dyes can be used to detect cells or non-cellular analytes, and two or more dyes can dye different regions (e.g., cytoplasm and nucleus) of the same target cell or non-cellular analyte.

[0267] The signal emitted from the dye can allow detection of the molecule or complex to which it is attached. A dye can be a molecular species that causes a physical or chemical response that can be observed or detected by the naked eye or by an instrument such as, but not limited to, a camera (e.g., a mobile phone camera), a scintillation counter, a colorimeter, a UV spectrophotometer, and the like. The dyes may include, but are not limited to, 5-ethynyl-2'-deoxyuridine, 7-aminoactinomycin D, acid fuchsin, acridine orange, acridine yellow, Alcian blue stain, aniline blue WS, aniline yellow, auramine O, Bismarck brown Y, brilliant green (dye), bromodeoxyuridine, Calcofluor fluorescent whitening agent, carbol fuchsin, succinimidyl acetate, carmine, Congo red, Coomassie brilliant blue, crystal violet, DAPI, DiI, DiOC6, eosin, ethidium bromide, ethyl green, fast green FCF, Feulgen stain, fluorescein, fluorescein isothiocyanate, fuchsin, gel green, gel red, Giemsa stain, food green S, H&E stain, hematoxylin, hematoxylin, hematin, Hoechst stain, Janus Green B, Jaswant Singh–Bhattacharji (JSB) stain, Light Green SF, Lugol's iodine, Malachite Green, Mallory's trichrome stain, Methyl Blue, Methyl Violet, Methylene Blue, Neutral Red, New Methylene Blue, Aniline Black, Nile Blue, Nile Red, Oil Blue 35, Oil Red O, Orange G, Lichen Red, Osmium Tetroxide, β-Dimethylaminocinnamaldehyde, Phyloxin, Ponceau 2R, Ponceau 6R, Propidium Iodide, Pyranine, Quinoline Yellow SS, Red 2G, Rhodamine, Rhodamine 123, Rhodamine 6G, Rhodamine B, RiboGreen, Ruthenium Red, Safranin, Silver Nitrate, Stain, Sudan Black B, Sudan III, Sudan IV, Sudan Red B, SYBR Green I, SYBR Safe, SYTOX, Template: Stain, Texas Red, Toluidine Blue Stain, Trypan Blue, User: Kyle MoJo / sandbox, Victoria Blue BO, Aqua Blue, Wayson stain, and Ziehl-Neelsen stain.

[0268] In some embodiments, the dye selected is a metachromatic dye. The term "metachromatic dye" may refer to a fluorescent dye that contains two or more peaks in its emission spectrum when bound to a cell or a cell component. When combined with different types of cells or molecules, such as when combined with RNA, DNA or other cell components, the metachromatic dye may fluoresce at different wavelengths. For example, when bound to double-stranded DNA, single-stranded DNA or single-stranded RNA, the metachromatic dye used in any embodiment of the present disclosure fluoresces at different wavelengths.

[0269] Various metachromatic dyes are known in the art, including but not limited to xanthene dyes, carbocyanine dyes, polymethine dyes (including Astra Violet FR), thiofalvine T, pseudocyanine, oxacarbocyanine dyes, acridine dyes, azine dyes, diphenylmethane dyes, methine dyes, oxazine dyes, cyanine dyes, styryl dyes, nonylacridine orange dye (3,6-bis(dimethylamino)-10-nonylacridine bromide), Molecular Probes, Eugene, Oreg., rhodamine, acridine red dye, toluidine blue dye (2-amino-7-dimethylamino-3-methylphenothiazine chloride), hydrosystilbamidine, and cyanine dyes including SYTO dyes, TOTO dyes, YOYO dyes, BOBO dyes, and combinations or derivatives thereof.

[0270] In some embodiments, the dye can be a non-metachromatic dye. The term "non-metachromatic dye" can refer to a fluorescent dye that provides a single wavelength excitation when irradiated with a predetermined wavelength. Such dyes can be used in methods for distinguishing multiple cell types, or can be used in situations where there may be a second fluorescent dye or antibody with a metachromatic wavelength that interferes with sample analysis. Such dyes can be used to stain cellular components of cells, including eosinophilic granules, basophilic granules, and cell membranes. Non-limiting examples of non-metachromatic dyes include, but are not limited to, neutral red dye (3-amino-7-dimethylamino-2-methylphenazine hydrochloride, basic orange 21 dye, DiOC dye (1,1'-dimethyloxacarbocyanine), pyronine Y dye, methylene blue dye (3-bis(dimethylamino)-phenothiazine-5-onium chloride), auramine O dye (4,4'-di(N,N-dimethylaniline) monohydrochloride, LDS 751 dye (quinolinium, 6-(dimethylamino)-2-[4-(dimethylamino)phenyl]-1,3-butadienyl)-2-perchlorate ethyl ester, red series dyes, and combinations or derivatives thereof. Other dyes also suitable for use include ethidium bromide, propidium iodide (3,8-diamino-5-(3-diethylaminopropyl)-6-phenyl-phenanthridinium iodide, hexidium iodide (hexanediol iodide), dihydroethidium, ethidium monoazide, thiazole orange dye, and combinations and derivatives thereof.

[0271] In some embodiments, the reagents coated on the device include, but are not limited to, Triton X-100, surfactants, Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tris(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (lla), llb, llc, lld, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium laureth sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, CTAB, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0272] In some embodiments, the reagents coated on the device include agents that render the cell membrane or nucleus permeable by means of osmotic pressure differences between cell membranes during dilution or by adjusting salt concentration.

[0273] In some embodiments, the reagents coated on the device include reagents that cross-link proteins, including but not limited to formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, and potassium permanganate.

[0274] In any embodiment of the present disclosure, for Gram-positive and Gram-negative stains, acridine orange and other SYTO green dyes (such as SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 16, SYTO 21 and SYTO 24) can stain both Gram-positive and Gram-negative bacteria. Fluorescently labeled wheat germ agglutinin can specifically stain Gram-positive bacteria. Iodinated hexanediol can specifically stain Gram-positive bacteria. Crystal violet and iodine stain Gram-positive and negative. Decolorization using ethanol, acetone. Safranin / carbol fuchsin counterstaining

[0275] Any dye used in the embodiments of the present disclosure can be a cell permeable dye or a cell impermeable dye. The term "cell permeable agent" can refer to a dye that easily penetrates the cell wall and stains its components without the need for the additional presence of a permeabilizing agent. Typically, cell permeable dyes are used to stain living cells or unlysed cell components. In some embodiments, cell permeable dyes are used to analyze samples containing whole blood. In some embodiments of the present disclosure, if a cell impermeable dye is used, a cell permeabilizing agent can be used to increase the permeability of the cell to the dye.

[0276] In some embodiments, dyes can be used in combination with labeled antibodies. Figure 3 As shown, labeled antibodies can be used to detect cellular analytes within a sample. Figure 3 As shown, the combination of labeled antibodies and cell dyes can be used to detect cells or non-cellular analytes (e.g., antigens) in a sample, thereby enhancing the signal detected from cells or non-cellular analytes relative to background signals. Simple staining can refer to staining with only one staining agent / dye. There are various multiple stainings, many of which are examples of counterstaining, differential staining or both, including double staining and triple staining. In any embodiment of the present disclosure, one or more dyes can be used to detect cells or non-cellular analytes. In any embodiment of the present disclosure, two or more dyes can be used to detect cells or non-cellular analytes. In certain embodiments, when two or more dyes are used to detect cells or non-cellular analytes, the excitation and / or emission spectra of the two or more dyes can be the same. For example, a sample can be contacted with two antibodies (e.g., each antibody binds to the same epitope on the same antigen), and each antibody has different dyes excited at the same wavelength. In another example, a sample can be contacted with two antibodies (e.g., each antibody binds to different epitopes on the same antigen), and each antibody has the same dye excited at a predetermined wavelength. In some embodiments, when two or more dyes are used to detect cells or non-cellular analytes, the excitation and / or emission spectra of the two or more dyes may be different. In some embodiments, when three or more dyes are used to detect cells or non-cellular analytes, the excitation and / or emission spectra of at least two or more dyes may be the same. In some embodiments, when three or more dyes are used to detect cells or non-cellular analytes, the excitation and / or emission spectra of at least two or more dyes may be different.

[0277] In any embodiment disclosed herein, the labeled antibody, stain and / or dye can be applied to one or more surfaces of the first plate and / or the second plate. It is expected that the labeled antibody, stain and / or dye applied to one or more surfaces of the first plate or the second plate can be used to reduce the number of steps performed by the user in order to analyze the sample. For example, if the first plate is coated with a cell staining reagent, the user will simply need to deposit the sample on the QMAX card or in the QMAX card (for example, in the sample contact area). When contacting the cell staining reagent, the cells in the sample can be automatically (for example, without user intervention) stained. The reduction of the number of steps performed by the user can reduce errors (for example, human errors), increase the accuracy of data analysis, and reduce the amount of time required for analyzing the sample. In some embodiments, stains or dyes can be applied to one or more surfaces of the first plate, and the labeled antibody can be applied to one or more surfaces of the second plate. In another embodiment, the labeled antibody and stain or dye can be applied to one or more surfaces of the first plate or the second plate. In another embodiment, the labeled antibody and stain or dye can be applied to one or more surfaces of both the first plate and the second plate.

[0278] It is also contemplated that a blocking agent competing with a stain or dye can be used to enhance the signal from a target cell or non-cellular analyte. For example, a blocking agent can be used to bind to an unbound antibody to quench the reporter molecule on the antibody and reduce the background signal that may otherwise be produced by the unbound antibody. Blocking agents can block the non-specific interaction of dyes with cells or cellular components. Blocking agents can compete with other dyes present in the binding site on the cell to be analyzed or the non-cellular analyte. Blocking agents can compete with low-affinity dyes for specific binding sites on cells or non-cellular analytes. Blocking agents themselves can be dyes; in this case, dyes are selected so that they can be detected from dyes or stains that are combined with target cells or non-cellular analytes. Alternatively, blocking agents can be any compound that blocks the non-specific interaction of dyes or stains without affecting the specific binding of dyes or stains to their targets.

[0279] The blocking agent is non-fluorescent or is selected from a dye that fluoresces at a wavelength different from the wavelength of the dye used to detect the target cell or non-cellular analyte. The blocking agent may be non-fluorescent at a wavelength that activates the fluorescence of the dye or stain. Many suitable blocking agents are known in the art and can be readily used in the embodiments of the present disclosure, including but not limited to, bisbenzamide (N,N'-(dithiodi-2,1-phenyl)-bisbenzamide, Hoechst TM 33258 dye (bisbenzamide, 2'-p-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5-bis-1H-benzimidazole trihydrochloride pentahydrate, Hoechst 34580 dye, Hoechst 33342 dye (bisbenzimide, 2'-(ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5-bi-1H-benzimidazole trihydrochloride trihydrate), 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), 4',6-bis-[2-imidazoline-4H,5H]-2-phenyl-indole (DIPI), eosin Y dye, orcein dye, fluorescein B dye (2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein disodium salt), pentoxifylline dye, quinacrine dye (6-chloro-9-(4-diethyl-1)-methylbutylamino)-2-methoxyacridine dihydrochloride), combinations thereof, and derivatives thereof.

[0280] RNA fluorescence in situ hybridization

[0281] RNA fluorescence in situ hybridization (RNA-FISH) is a molecular cytogenetic technique used to detect and localize specific RNA targets (mRNA, lncRNA, and miRNA) in single cells by fluorescence microscopy. Traditional RNA-FISH methods usually include multiple steps, such as: fixation, permeabilization, hybridization, and imaging. Although FISH has a wide range of medical applications, the complexity of the technique limits its potential for rapid diagnosis. Therefore, there is a need to develop rapid, accurate, portable, and / or inexpensive RNA-FISH assays that can be performed by non-professionals.

[0282] In some embodiments, fluorescence in situ hybridization (FISH) can be used to detect cellular or non-cellular analytes within a sample. In some embodiments, FISH can be used to detect cellular or non-cellular analytes by enhancing the signal from the cellular or non-cellular analyte relative to the background signal. FISH can generally refer to any method of detecting cellular or non-cellular analytes within a sample using one or more probes, including DNA probes that detect and locate the presence or absence of specific DNA sequences on chromosomes and RNA probes that detect or locate specific RNA targets (mRNA, lncRNA, and miRNA). Therefore, FISH is often used to discover specific features in DNA for genetic counseling, medicine, and species identification, as well as to define the spatiotemporal patterns of gene expression within cells and tissues. In some embodiments, the probe can be a short strand (usually 10-25 nucleotides) of DNA or RNA that is complementary to a given target sequence that is typically used to locate the target. In some embodiments, FISH can be used RNA FISH probes, an RNA visualization method that allows for the simultaneous detection, localization, and quantification of individual mRNA molecules.

[0283] In one embodiment, the surfactant used to make the probe accessible includes, but is not limited to, surfactants, Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tri(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (lla), llb, llc, lld, CTAC, Tween 20, Tween 40, Tween 60, Tween80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium lauryl ether sulfate (SLES), sodium myristyl alcohol polyether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, CTAB, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0284] In some embodiments, the reagents that cause red blood cell lysis coated in the device include but are not limited to Pluronic F-127, polyoxyethylene castor oil, Pluronic F-68, Myrj 52, Brij 35, sodium oleate, sodium dodecyl sulfate, Tween 20, Tween 40, Tween 60, Tween 80, SLS, CTAB, CTAC, tamoxifen, saponin, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, ABS-14, ABS-16, antimalarial drugs (quinine compounds), arsenic, dapsone, metals (chromium / chromates, platinum salts, nickel compounds, copper, lead, cisplatin), nitrites, nitrofurantoin, penicillin, phenazopyridine (marofen), rho immunoglobulin, ribavirin, sulfonamides, and sulfones.

[0285] Any RNA / DNA probe used in the embodiments of the present disclosure may be labeled. A label may refer to a molecule that, when attached (directly or indirectly) to another molecule, provides or enhances the means for detecting another molecule. The signal emitted from the label may allow detection of the molecule or complex to which it is attached, and / or the label itself. A label may be a molecular species that causes a physical or chemical response that can be observed or detected by the naked eye or by an instrument, such as but not limited to a camera (e.g., a mobile phone camera), a scintillation counter, a colorimeter, a UV spectrophotometer, etc. Labels include but are not limited to radioisotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (e.g., biotin, avidin, streptavidin or haptens), etc. When excited at different wavelengths, a fluorescent or fluorescent marker or label emits detectable light at a specific wavelength. A radioactive label or radioactive marker emits radioactive particles detectable by an instrument such as but not limited to a scintillation counter. Other signal generation detection methods include chemiluminescence detection, electrochemiluminescence detection, Raman energy detection, colorimetric detection, hybridization protection assay and mass spectrometry. Non-limiting examples of labeling include fluorophores, chromophores, FITC, TRITC, DTAF, Texas Red, phycoerythrin, allophycocyanin, green fluorescent protein (GFP), blue fluorescent protein (BFP), rhodamine, FAM, TET, HEX, JOE, TAMRA, ROX, aromatic substituted xanthene dyes, 4,7-dichloro-fluorescein, 4,7-dichloro-rhodamine, cyanine dyes, enzymes, horseradish peroxidase (HRP enzyme), alkaline phosphatase, beta-galactosidase, luciferase, chloramphenicol acetyltransferase (CAT enzyme), avidin, streptavidin, biotin, biotinylated protein, or any combination thereof, fragments or derivatives. In any embodiment of the present disclosure, one or more labels can be used to detect cells or non-cellular analytes. In any embodiment of the present disclosure, two or more labels can be used for detecting cells or non-cellular analytes. In certain embodiments, when using two or more labels to detect cells or non-cellular analytes, the excitation and / or emission spectra of two or more labels can be the same. For example, a sample can be contacted with two antibodies (for example, each antibody binds to the same epitope on the same antigen), and each antibody has different labels excited at the same wavelength. In another example, a sample can be contacted with two antibodies (for example, each antibody binds to different epitopes on the same antigen), and each antibody has the same label excited at a predetermined wavelength. In certain embodiments, when using two or more labels to detect cells or non-cellular analytes, the excitation and / or emission spectra of two or more labels can be different.In some embodiments, when three or more markers are used to detect cells or non-cellular analytes, the excitation and / or emission spectra of at least two or more markers may be the same. In some embodiments, when three or more markers are used to detect cells or non-cellular analytes, the excitation and / or emission spectra of at least two or more markers may be different.

[0286] protocol

[0287] 1. Design, synthesis and purification of target RNA fluorescent probes.

[0288] 2. Use Biodot to print probes (1 nM) and surfactant (eg Zwittergent) on X-plate.

[0289] 3. Apply 1 drop of blood on the substrate, close the wafer, press, and incubate at room temperature for 1 minute.

[0290] 4. Insert the chip into the device, take pictures, and analyze the data.

[0291] Spacer height & sample thickness

[0292] In some embodiments of the present disclosure, the height of the spacer can be changed to adjust the thickness of the sample to be analyzed, thereby enhancing the detection of signals from target cells or non-cellular analytes. Figure 4 As shown, the signal detected from cells or non-cellular analytes relative to the background signal can be detected by Figure 4 A) to smaller thickness ( Figure 4B) changing the thickness of the sample to enhance. In some embodiments, the spacer height and / or thickness of the sample can be 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, at most about 0.1 microns, or at most about 0.05 microns. For example, the spacer height can be at most about 30 microns. In some embodiments, the spacer height and / or sample thickness can be about 25 microns to about 35 microns. In some embodiments, the spacer height and / or sample thickness can be about 20 microns to about 40 microns. In some embodiments, the spacer height and / or sample thickness may be about 10 microns to about 30 microns. In some embodiments, the spacer height and / or sample thickness may be about 30 microns to about 50 microns. In some embodiments, the spacer height and / or sample thickness may be about 50 microns to about 75 microns. In some embodiments, the spacer height and / or sample thickness may be about 1 micron to about 25 microns. For example, the spacer height and / or sample thickness may be about 25 microns to about 35 microns. In some embodiments, the spacer height and / or thickness of the sample may be determined based on the size or shape of the target cell or non-cellular analyte. For example, lymphocytes may have an average diameter of about 15 microns in a suspension. Therefore, in some embodiments, the spacer height and / or thickness of the sample may be about 15 microns, about 20 microns, about 25 microns, about 30 microns or greater than about 30 microns.

[0293] Examples of the Invention

[0294] A1. A method for analyzing a sample, the method comprising:

[0295] (a) obtaining a device according to embodiments disclosed herein, the device comprising a first plate, a second plate, and a spacer, wherein the spacer has a predetermined substantially uniform height equal to or less than about 200 microns;

[0296] (b) contacting the sample with two or more labeled antibodies to obtain a mixture, wherein the two or more labeled antibodies are capable of binding to one or more epitopes on target cells within the sample;

[0297] (c) depositing a product on one or both of the plates when the plates are configured in an open configuration, wherein in the open configuration the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers;

[0298] (d) After (c), forcing the two plates into a closed configuration, in which: at least a portion of the mixture is compressed by the two plates into a layer of very uniform thickness, wherein the uniform thickness of the layer is limited by the mixture contacting surfaces of the plates and adjusted by the plates and spacers.

[0299] A2. A method for analyzing a sample, the method comprising:

[0300] (a) obtaining a device according to an embodiment disclosed herein, the device comprising a first plate, a second plate, and a spacer, wherein the spacer has a predetermined substantially uniform height equal to or less than about 200 microns, and wherein at least one of the first plate, the second plate, and the spacer comprises at least one surface coated with two or more labeled antibodies;

[0301] (b) depositing the sample on one or both of the plates when the plates are configured in an open configuration, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not regulated by a spacer; wherein when the sample is deposited on one or both plates, the sample is contacted with two or more labeled antibodies, wherein the two or more labeled antibodies are capable of binding to one or more epitopes on a target cell within the sample;

[0302] (d) After (c), forcing the two plates into a closed configuration, in which: at least a portion of the sample is compressed by the 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 adjusted by the plates and spacers.

[0303] A3. A method according to any of the above embodiments, wherein the spacer has a predetermined substantially uniform height equal to or less than about 100 microns, equal to or less than about 75 microns, equal to or less than about 50 microns, equal to or less than about 30 microns, equal to or less than about 25 microns, equal to or less than about 10 microns, equal to or less than about 5 microns, or equal to or less than about 1 micron.

[0304] A4. A method according to any of the above embodiments, wherein the predetermined substantially uniform height of the spacer is determined based on the size or shape of the target cells.

[0305] A5. The method according to any of the above embodiments, wherein two or more labeled antibodies bind to different epitopes on target cell molecules.

[0306] A6. A method according to any of the above embodiments, wherein two or more labeled antibodies bind to the same epitope.

[0307] A7. The method according to any of the above embodiments, wherein two or more labeled antibodies bind to different molecules on the target cell.

[0308] A7.1 A method according to any of the above embodiments, wherein the labels coupled to the two or more labeled antibodies can be excited at the same wavelength.

[0309] A8. A method for analyzing a sample, the method comprising:

[0310] (a) obtaining a device according to an embodiment disclosed herein, the device comprising a first plate, a second plate, and a spacer, wherein the spacer has a predetermined substantially uniform height equal to or less than about 200 microns, wherein the first plate comprises a surface coated with a first antibody, and the second plate comprises a surface coated with a second antibody;

[0311] (b) depositing the sample on one or both of the plates when the plates are configured in an open configuration, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not regulated by a spacer, wherein when the sample is deposited on one or both plates, the sample is contacted with a first antibody and a second antibody, wherein the first antibody and the second antibody are capable of binding to one or more epitopes on a target cell within the sample;

[0312] (d) After (c), forcing the two plates into a closed configuration, in which: at least a portion of the sample is compressed by the 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 adjusted by the plates and spacers.

[0313] A9. A method for analyzing a sample, the method comprising:

[0314] (a) obtaining a device according to embodiments disclosed herein, the device comprising a first plate, a second plate, and a spacer, wherein the spacer has a predetermined substantially uniform height equal to or less than about 200 microns;

[0315] (b) contacting the sample with two or more dyes to obtain a mixture, wherein the two or more labeled dyes are capable of binding to target cells within the sample;

[0316] (c) depositing a product on one or both of the plates when the plates are configured in an open configuration, wherein in the open configuration the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers;

[0317] (d) After (c), forcing the two plates into a closed configuration, in which: at least a portion of the mixture is compressed by the two plates into a layer of very uniform thickness, wherein the uniform thickness of the layer is limited by the mixture contacting surfaces of the plates and adjusted by the plates and spacers.

[0318] A10. A method for analyzing a sample, the method comprising:

[0319] (a) obtaining a device according to an embodiment disclosed herein, the device comprising a first plate, a second plate, and a spacer, wherein the spacer has a predetermined substantially uniform height equal to or less than about 200 microns, and wherein at least one of the first plate, the second plate, and the spacer comprises at least one surface coated with two or more dyes;

[0320] (b) depositing the sample on one or both of the plates when the plates are configured in an open configuration, wherein in the open configuration the two plates are partially or completely separated and the spacing between the plates is not regulated by a spacer; wherein when the sample is deposited on one or both plates, the sample is contacted with two or more dyes, wherein the two or more dyes are capable of binding to target cells within the sample;

[0321] (d) After (c), forcing the two plates into a closed configuration, in which: at least a portion of the sample is compressed by the 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 adjusted by the plates and spacers.

[0322] A11. A method according to any of the above embodiments, wherein the spacer has a predetermined substantially uniform height equal to or less than about 100 microns, equal to or less than about 75 microns, equal to or less than about 50 microns, equal to or less than about 30 microns, equal to or less than about 25 microns, equal to or less than about 10 microns, equal to or less than about 5 microns, or equal to or less than about 1 micron.

[0323] A12. A method according to any of the above embodiments, wherein the predetermined substantially uniform height of the spacer is determined based on the size or shape of the target cells.

[0324] A13. A method according to any of the above embodiments, wherein two or more dyes bind non-specifically to target cells.

[0325] A14. The method according to any of the above embodiments, wherein two or more dyes can be excited at the same wavelength.

[0326] A15. A method for analyzing a sample, the method comprising:

[0327] (d) obtaining a device according to embodiments disclosed herein, the device comprising a first plate, a second plate, and a spacer, wherein the spacer has a predetermined substantially uniform height equal to or less than about 200 microns, wherein the first plate comprises a surface coated with an antibody, and the second plate comprises a surface coated with a dye;

[0328] (e) depositing a sample on one or both of the plates when the plates are configured in an open configuration, wherein in the open configuration the two plates are partially or completely separated and the spacing between the plates is not regulated by a spacer; wherein when the sample is deposited on one or both plates, the sample is contacted with an antibody and a dye, wherein the antibody and the dye are capable of binding to target cells within the sample;

[0329] (f) After (b), forcing the two plates into a closed configuration, in which: at least a portion of the sample is compressed by the 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 adjusted by the plates and spacers.

[0330] A16. A method for analyzing a sample, the method comprising:

[0331] (e) obtaining a device according to embodiments disclosed herein, the device comprising a first plate, a second plate, and a spacer, wherein the spacer has a predetermined substantially uniform height equal to or less than about 200 microns;

[0332] (f) contacting the sample with a labeled antibody and a dye to obtain a mixture, wherein the labeled antibody and the dye are capable of binding to target cells in the sample;

[0333] (c) depositing the mixture on one or both of the plates when the plates are in an open configuration, wherein in the open configuration the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers;

[0334] (h) after (c), forcing the two plates into a closed configuration, in which: at least a portion of the mixture is compressed by the two plates into a layer of substantially uniform thickness, wherein the uniform thickness of the layer is limited by the mixture contacting surfaces of the plates and adjusted by the plates and spacers.

[0335] A17. A method for analyzing a sample, the method comprising:

[0336] (d) obtaining a device according to an embodiment disclosed herein, the device comprising a first plate, a second plate, and a spacer, wherein the spacer has a predetermined substantially uniform height equal to or less than about 200 microns, and wherein at least one of the first plate, the second plate, and the spacer comprises at least one surface coated with a labeled antibody and a dye;

[0337] (e) depositing a sample on one or both of the plates when the plates are configured in an open configuration, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not regulated by a spacer; wherein when the sample is deposited on one or both plates, the sample is contacted with a labeled antibody and a dye, wherein the labeled antibody and the dye are capable of binding to target cells within the sample;

[0338] (f) After (b), forcing the two plates into a closed configuration, in which: at least a portion of the sample is compressed by the 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 adjusted by the plates and spacers.

[0339] A18. A method according to any of the above embodiments, wherein the spacer has a predetermined substantially uniform height equal to or less than about 100 microns, equal to or less than about 75 microns, equal to or less than about 50 microns, equal to or less than about 30 microns, equal to or less than about 25 microns, equal to or less than about 10 microns, equal to or less than about 5 microns, or equal to or less than about 1 micron.

[0340] A19. A method according to any of the above embodiments, wherein the predetermined substantially uniform height of the spacer is determined based on the size or shape of the target cells.

[0341] A20. A method according to any of the above embodiments, wherein the dye non-specifically binds to the target cells.

[0342] A21. A method according to any of the above embodiments, wherein the label on the labeled antibody and the dye can be excited at the same wavelength.

[0343] In one embodiment, the reagents and additives are coated on the same plate (either the first plate or the second plate), or on separate plates (the first plate and the second plate).

[0344] In one embodiment, the additive is a chemical that has a physical or chemical or physiological effect on the target cells. Examples are surfactants (referring to other transients) and ions (referring to other transients).

[0345] In one embodiment, the staining reagent is a labeled antibody, peptide, oligonucleotide, aptamer, small molecule, or any other substance that has binding affinity for one or more specific components inside or outside the target cell.

[0346] In one embodiment, detection of the labeled staining reagent is fluorescence-based (referring to other contingencies), chemiluminescence-based (referring to other contingencies), or colorimetric-based (referring to other contingencies) or plasmon-based (referring to other contingencies).

[0347] In one embodiment, the target cell is a prokaryotic organism, such as bacteria and archaea, or a eukaryotic organism, such as animal cells and plant cells. Common examples include mammalian cells, yeast, and algae, among others.

[0348] In one embodiment, the number of target cells in a sample can be one or more than one.

[0349] In some embodiments, the reagents coated on the device include, but are not limited to, Triton X-100, surfactants, Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tris(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (lla), llb, llc, lld, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium laureth sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, CTAB, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0350] In some embodiments, the reagents coated on the device include agents that render the cell membrane or nucleus permeable by means of osmotic pressure differences between cell membranes during dilution or by adjusting salt concentration.

[0351] In some embodiments, the reagents coated on the device include reagents that cross-link proteins, including but not limited to formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, and potassium permanganate.

[0352] The device, kit, system or method according to any of the above embodiments, wherein a dye for staining WBCs is applied to the first plate or the second plate or both.

[0353] The device, kit, system or method according to any of the above embodiments, wherein a dye for staining WBC and PLT is applied to the first plate or the second plate or both.

[0354] The device, kit, system or method according to any of the above embodiments, wherein the dye for PLT staining is applied to the first plate or the second plate or both.

[0355] The device, kit, system or method according to any of the above embodiments, wherein the reagents are applied into the array by droplet printing.

[0356] The method, device, system and kit according to any of the above embodiments, wherein the reagents are coated on the plate using the open flow-guiding properties of the structure.

[0357] The device, kit, system or method according to any of the above embodiments, wherein the reagent is applied by spraying.

[0358] The device, kit, system or method according to any preceding embodiment, wherein the reagent is applied by contact printing.

[0359] The device, kit, system or method according to any preceding embodiment, wherein the reagent is applied by transfer.

[0360] The device, kit, system or method according to any of the above embodiments, wherein the dye that stains the RBCs is applied to the first plate or the second plate or both.

[0361] The device, kit, system or method according to any of the above embodiments, wherein a surfactant for separating and rounding the RBCs is coated on the first plate or the second plate or both.

[0362]

[00136] The device, kit, system or method of any preceding embodiment, wherein the chemical that lyses the RBCs is coated onto the first plate or the second plate or both.

[0363] The device, kit, system or method according to any preceding embodiment, wherein acridine orange is coated onto the first plate or the second plate or both.

[0364] The device, kit, system or method according to any preceding embodiment, wherein the Zwittergent is coated onto the first plate or the second plate or both.

[0365] The device, kit, system or method according to any of the preceding embodiments, wherein methylene blue and Zwittergent are coated onto the first plate or the second plate or both.

[0366] The device, kit, system or method according to any preceding embodiment, wherein acridine orange and Zwittergent are coated onto the first plate or the second plate or both.

[0367] The device, kit, system or method according to any of the above embodiments, wherein the YOYO dye and Zwittergent are coated on the first plate or the second plate or both.

[0368] The method and device according to any of the above embodiments, wherein the device further comprises a plurality of reagent layers on one or both plates, the plurality of reagent layers comprising anti-adhesion, cell lysis, cell staining, release time control material layers, and combinations thereof;

[0369] The thickness of each layer coated on the board is 10 nm, 100 nm, 200 nm, 500 nm, 1 um, or in a range between any two of the values.

[0370] The anti-adhesive agent includes ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetate, K2EDTA, K3EDTA, etc.;

[0371] Wherein the cell staining agent comprises Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue and azure B), May-grünwald stain, Leishman's solution ("polychromatic" methylene blue (i.e., demethylated to azure) and eosin), Erythrosin B stain (Erythrosin B), and other fluorescent stains, including but not limited to acridine orange dye, 3,3-dihexyloxycarbocyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dyes, ethidium bromide, leucine sulfonamide and diaminostilbene disulfonic acid derivatives, Erythrosin B or Trypan blue, Hoechst 33342, trihydrochloride, Trihydrate, DAPI (4,6-diamidino yellow-2-phenylindole, dihydrochloride), YOYO;

[0372] The cell stains include Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue and azure B), May-grünwald stain, Leishman's solution ("polychromatic" methylene blue (i.e., demethylated to azure) and eosin), erythrosine B stain (erythrosine B), and other fluorescent stains including, but not limited to, acridine orange dye, 3,3-dihexyloxycarbocyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dyes, ethidium bromide, leucine sulfaquinone and diaminostilbene disulfonic acid derivatives, erythrosine B or trypan blue, Hoechst 33342, trihydrochloride, Trihydrate, DAPI (4,6-diamidino yellow-2-phenylindole, dihydrochloride), YOYO, acid fuchsin, hematoxylin, Hoechst stains including Hoechst 33258 and Hoechst 33342, methyl green, methylene blue, Nile blue, Nile red, osmium tetroxide, rhodamine, safranin, meosic-AAPV-AMC, CFSE, BCECF / AM, silver nitrate, neutral red, pyronin Y, calcein-AM, dihydroethidium, ditolyl cyanide FF, rhodamine 123, 4-methylumbelliferyl palmitate, fast blue B salt, fluorescein yellow, CH dipotassium salt, DAPI double lactate, propidium iodide;

[0373] The cell lysis agents include ammonium chloride, sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), acetic acid, citric acid, other acids and bases, etc.

[0374] The release time controlling material includes albumin, carbomer, carboxymethyl cellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinyl pyrrolidone, or polyvinyl alcohol.

[0375] In some embodiments, chemicals with a certain concentration are coated on the plate and dissolved into the blood to achieve uniform distribution of red blood cells in the device.

[0376] In some embodiments, a chemical having a certain concentration is coated on the plate and dissolved into the blood to lyse the red blood cells in the device, wherein the coating can be on the first plate or the second plate, or both.

[0377] In one embodiment, the chemicals coated in the device include, but are not limited to, surfactants, Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tri(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (lla), llb, llc, lld, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium laureth sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, CTAB, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0378] In some embodiments, the reagents that cause red blood cell lysis coated in the device include but are not limited to Pluronic F-127, polyoxyethylene castor oil, Pluronic F-68, Myrj 52, Brij 35, sodium oleate, sodium dodecyl sulfate, Tween 20, Tween 40, Tween 60, Tween 80, SLS, CTAB, CTAC, tamoxifen, saponin, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, ABS-14, ABS-16, antimalarial drugs (quinine compounds), arsenic, dapsone, metals (chromium / chromates, platinum salts, nickel compounds, copper, lead, cisplatin), nitrites, nitrofurantoin, penicillin, phenazopyridine (marofen), rho immunoglobulin, ribavirin, sulfonamides, and sulfones.

[0379] In some embodiments, the anticoagulant coated in the device includes but is not limited to EDTA, such as dipotassium ethylenediaminetetraacetate (K2EDTA), tripotassium ethylenediaminetetraacetate (K3EDTA), coumarin (vitamin K antagonist), warfarin (comedin), acenaphthenocoumarol, phenprocoumon, atomycin, phenindione, heparin, fondaparinux sodium, and edaparinux, dabigatran etexilate, rivaroxaban, apixaban, edoxaban, betrixaban, NOAC, hirudin, recombinant hirudin, bivalirudin, agratroban, dabigatran etexilate, batroxobin, hirudin, vitamin E, sodium citrate, citrate dextrose, oxalates such as fluoride oxalate, deltaparin, desirudin, enoxaparin.

[0380] In some embodiments, in order to achieve uniform distribution of red blood cells in the device, Zwittergent was added at 3 ng / mm 2 , 5ng / mm 2 , 8ng / mm 2 , 12ng / mm 2 , 15ng / mm 2 , 25ng / mm 2 , 35ng / mm 2 , 50ng / mm 2 , 80ng / mm 2 , or a preferred area concentration within a range between any two values ​​is coated on the plate.

[0381] In some embodiments, Zwittergent is added at 100 ng / mm 2 , 120ng / mm 2 , 150ng / mm 2 , 180ng / mm 2 , 200ng / mm 2 , 300ng / mm 2 , 400ng / mm 2 , 500ng / mm 2 , 800ng / mm 2 ,1000ng / mm 2 , or a preferred area concentration within a range between any two values ​​is coated on the plate.

[0382] In some embodiments, to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred final concentration in blood of 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 1.0 mg / mL, 2 mg / mL, or in a range between any two of the values.

[0383] In a certain embodiment, to lyse red blood cells in the device, Zwittergent is coated on the plate at a preferred final concentration of 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 25 mg / mL, 50 mg / mL, or in a range between any two of the values.

[0384] In some embodiments, in order to achieve uniform distribution of red blood cells in the device, Zwittergent was added at 3 ng / mm 2 , 5ng / mm 2 , 8ng / mm 2 , 12ng / mm 2 , 15ng / mm 2 , 25ng / mm 2 , 35ng / mm 2 , 50ng / mm 2 , 80ng / mm 2 , 100ng / mm 2 , or a preferred area concentration within a range between any two values ​​is coated on the plate.

[0385] In some embodiments, Zwittergent is added at 100 ng / mm 2 , 120ng / mm 2 , 150ng / mm 2 , 180ng / mm 2 , 200ng / mm 2 , 300ng / mm 2 , 400ng / mm 2 , 500ng / mm 2 , 800ng / mm 2 , 1000ng / mm 2 , or a preferred area concentration within a range between any two values ​​is coated on the plate.

[0386] In some embodiments, to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred final concentration in blood of 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 1.0 mg / mL, 2 mg / mL, or in a range between any two of the values.

[0387] In a certain embodiment, to lyse red blood cells in the device, Zwittergent is coated on the plate at a preferred final concentration of 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 25 mg / mL, 50 mg / mL, or in a range between any two of the values.

[0388] According to any of the above embodiments of the device, kit, system or method, wherein acridine orange is 0.5 ng / mm 2 , 1ng / mm 2 , 2ng / mm 2 , 3ng / mm 2 , 5ng / mm 2 , 8ng / mm 2 , 10ng / mm 2 , 15ng / mm 2 , 20ng / mm 2 , 30ng / mm 2 , or an area concentration within a range between any two values ​​is coated on the plate.

[0389] According to any of the above embodiments of the device, kit, system or method, wherein acridine orange is 3 to 10 ng / mm 2 The area concentration of Zwittergent was coated on the plate, and Zwittergent was coated on the plate at a concentration of 3 to 10 ng / mm 2 The area concentration is coated on the plate.

[0390] According to any of the above embodiments of the device, kit, system or method, wherein acridine orange is 5 to 20 ng / mm 2 The area concentration of Zwittergent was coated on the plate, and the Zwittergent was 10 to 30 ng / mm 2 The area concentration is coated on the plate.

[0391] In certain embodiments, a marker (e.g., as an immunoassay antibody marker, an RNA marker, a staining dye) is coated on the plate at a preferred final concentration in the sample of 0.1 nM / mL, 0.5 nM / mL, 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL, 50 nM / mL, or in a range between any two of said values.

[0392] In some embodiments, a marker (e.g., as an immunoassay antibody marker, RNA marker, staining dye) is coated on the plate at a preferred final concentration in the sample of 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL, or in a range between any two of the values.

[0393] In some embodiments, the label (e.g., immunoassay antibody label, RNA label, staining dye) is coated on the plate at a preferred final concentration in the sample of 0.1uM / mL, 0.5uM / mL, 1uM / mL, 5uM / mL, 10uM / mL, 15uM / mL, 20uM / mL, 50uM / mL, or in a range between any two of the values.

[0394] In some embodiments, the label (e.g., immunoassay antibody label, RNA label, staining dye) is coated on the plate at a final concentration in the sample of 0.1 nM / mL, 0.5 nM / mL, 1.0 nM / mL, 1.5 nM / mL, 2.0 nM / mL, 5 nM / mL, 10 nM / mL, 50 nM / mL, or in a range between any two of the values.

[0395] In some embodiments, the marker (e.g., immunoassay antibody marker, RNA marker, staining dye) is coated on the plate at a preferred final concentration in the sample of 0.05 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1.0 mg / mL, or in a range between any two of the values.

[0396] In some embodiments, the label (e.g., immunoassay antibody label, RNA label, staining dye) is coated on the plate at a preferred final concentration in the sample of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL, or in a range between any two of the values.

[0397] In some embodiments, the marker (e.g., immunoassay antibody marker, RNA marker, staining dye) is coated on the plate at a preferred final concentration in the sample of 100 mg / mL, 200 mg / mL, 300 mg / mL, or in a range between any two of the values.

[0398] Example 1:

[0399] Rapid detection of Gram-negative and Gram-positive bacteria

[0400] Material

[0401] 1. Hexanediol iodide: This nucleic acid stain is permeable to mammalian cells and selectively stains most Gram-positive bacteria orange (Thermofisher, H7593).

[0402] 2. Example 9: Staining Gram-positive and Gram-negative bacteria green (Thermofisher, S34854).

[0403] 3.500nm wavelength excitation filter.

[0404] 4. Staphylococcus epidermidis (Gram-positive bacteria), Escherichia coli (Gram-negative bacteria).

[0405] method:

[0406] Bacterial culture preparation:

[0407] 1. Staphylococcus epidermidis and Escherichia coli were used as Gram-positive bacteria and Gram-negative bacteria, respectively. These different bacterial species were cultured in a nutrient liquid medium (R061582, Remel) at 37°C overnight, followed by staining.

[0408] Q card preparation:

[0409] 1. A mixture containing 12 μg / mL iodinated hexanediol and 8 μM SYTO 9 suspended in PBS was added at 3 μL / cm 2 Print on a batch of X plates (containing 2 μm high columns) and air dry.

[0410] 2.2uL of PBS or blood containing Staphylococcus epidermidis and Escherichia coli was added to the substrate. The printed X-plate and the substrate containing bacteria-blood sample were firmly pressed together ( Figure 7 ).

[0411] Imaging:

[0412] Fluorescence and brightfield images of the assay were captured using an iPhone 6s after a 60-second incubation period. Only Gram-positive bacteria are orange, while both Gram-positive and Gram-negative bacteria are green ( Figure 7 ).

[0413] In this experiment, the QMAX card had two plates, a flat PMMA substrate with a thickness of 1 mm, and a 175 μm thick PMMA film with a pillar array on it with pillar dimensions of 30 × 40 μm, 80 μm pitch (pillars), and 2 μm pillar height.

[0414] Example 2:

[0415] Rapid CD4 immunostaining

[0416] Material

[0417] 1. CD4 antibodies: Ab34276 (Abcam) and 10R-CD4KHUP (Fitzgerald industrial international)

[0418] 2. Antibody labeling kit:

[0419] 2.Alexa Fluor 647 NHS ester kit (A37573, Thermofisher)

[0420] 2.2 Zenon Mouse IgG1 Antibody Labeling Kit (Z25008, Thermofisher)

[0421] 3. 5 mM SYTO62 red fluorescent nucleic acid dye (S11344, Thermofisher) in DMSO

[0422] 4. Bioworld Antibody Biostabilizer 10X (22050005-2, Fisher Scientific)

[0423] 5.Triton X-100 (X-100, 100ml, Sigma)

[0424] method:

[0425] 1. Antibody double labeling method:

[0426] 1.1. Primary fluorescent labeling: According to the manufacturer's protocol, 100ug of each CD4 detection antibody (Ab34276 and 10R-CD4KHUP) was labeled using the Alexa Fluor 647 NHS ester labeling kit (Thermofisher). After labeling the antibodies, the residual dye was removed using a Sephadex-G25 column (17-0853-01, GE Health, Switzerland). The purified antibodies were resuspended in a mixture consisting of 30% glycerol and 1% BSA to a final concentration of 1 μg / μL. The antibodies were stored at -20°C before further use.

[0427] 1.2. Secondary fluorescent labels:The Alexa Fluor 647NHS ester-labeled CD4 antibody was secondarily labeled using the Zenon Alexa Fluor 647 Labeling Kit (Fc Domain Labeling) according to the manufacturer's instructions. Briefly, 1ug ​​of each CD4 antibody was mixed with 8ul of mouse IgG1 Alexa Fluor 647 labeling reagent in 15μl PBS. The reaction mixture was incubated at room temperature for 5 minutes and then kept at 4°C before further use. This dual labeling method significantly enhanced the fluorescence signal of the CD4-specific antibody.

[0428] 2. Detection antibody mixture:

[0429] 2D assay: 6ug / ml of each detection antibody that has been dual labeled was mixed with 5uM SYTO 62 (red fluorescent DNA dye from Thermofisher), 0.5X antibody biostabilizer (Bioworld) and 0.1% Triton X-100 (Sigma) in PBS. Thus, the mixture had a final antibody concentration of 1μg / mL mixed with the dye that stains DNA.

[0430] 2A determination: 6ug / ml of each detection antibody that had been dual labeled was mixed with 0.5X Antibody Bio-stabilizer (Bioworld) and 0.1% Triton X-100 (Sigma) in PBS. This mixture also had a final antibody concentration of 12ug / mL, but the DNA dye was omitted.

[0431] 1A Determination: 6ug / ml of one detection antibody that has been dual labeled was mixed with 0.5X Antibody Bio-stabilizer (Bioworld) and 0.1% Triton X-100 (Sigma) in PBS. This mixture contained only a final antibody concentration of 1 μg / mL without DNA dye.

[0432] 1D determination: 6ug / ml of one detection antibody that has been dual labeled was mixed with 0.5X Antibody Biostabilizer (Bioworld) and 0.1% Triton X-100 (Sigma) in PBS. This mixture contains only one antibody with DNA dye.

[0433] 3. Surface treatment of Q card:

[0434] 3.1. Treat both the X-plate (containing 10 μm columns) and the base plate with 1% NaOH at 50° C. for 1 hour. Both plates were then rinsed once with distilled water, PBS, and distilled water for 5 minutes each.

[0435] 3.2. After washing the plates, they were blocked with 4% BSA for 2 hours at room temperature. The plates were washed twice with distilled water to remove residual BSA for 5 minutes and then air-dried.

[0436] 4. Print the antibody mixture onto Q-card:

[0437] 4.1 Prepare the antibody mixture at about 3ul / cm 2 Print on two plates (X plate and base plate). Once the antibodies are printed, the plates are air dried and protected from light before further use.

[0438] 5. Use iPhone 6s to detect CD4:

[0439] 5.1. Add fresh whole blood to the prepared substrate. Then firmly press the prepared X-plate onto the blood sample. Take a picture after incubation for 1 minute using an iPhone 6s.

[0440] 6. CD4 count per micron:

[0441] 6.1.X plate column height: 10uM

[0442] 6.2 fov: Field of view (the total area of ​​the Q card in a photo taken with an iPhone or a fluorescence microscope)

[0443] 6.3.fov sample size:

[0444] 6.3.1. Length (um) x width (um) x height (10um) = volume of fov (um 3 )

[0445] 6.4.1μl=10 9 μm 3

[0446] 6.5. CD4 count per micron:

[0447] 6.5.1.: In FOV X 10 9 um 3 / FOV volume (um 3 )

[0448]

[0449] Total CD4 count

[0450] FOV volumeum 3

[0451] 6.6. Example: In Figure 8 In the iPhone image shown, there are 31 CD4 positive signals. Figure 8An exemplary photograph of CD4 T cell enumeration using the QMAX device taken by an iPhone-based microscope is shown. Cells were stained with SYTO62 non-specific nuclear stain along with detection antibodies.

[0452] 6.6.1. CD4 counts per micron:

[0453] 31x10 9 um 3 / (1680um x 2200um x 10um)=31x10 9 um 3 / 3.7x10 7 um 3 =838 / ul

[0454] Example 3:

[0455] RNA fluorescence in situ hybridization (RNA-FISH)

[0456] RNA fluorescence in situ hybridization (RNA-FISH) is a molecular cytogenetic technique used for fluorescence microscopy detection and localization of specific RNA targets (mRNA, lncRNA and miRNA) within single cells. Traditional RNA-FISH methods usually include multiple steps, such as: fixation, permeabilization, hybridization and imaging. Although FISH has a wide range of medical applications, the complexity of the technology limits its potential in rapid diagnosis. Therefore, it is necessary to develop a fast, accurate, portable and / or inexpensive RNA-FISH assay that can be performed by non-professionals. The present invention meets these needs.

[0457] 1. Materials

[0458] FISH probes, with 670 dye for human GAPDH (BioSearch Technologies, cat# SMF-2019-1).

[0459] Q card with column height of 10 μm.

[0460] 2. Procedure:

[0461] The plate was coated with 0.25uM 670 labeled FISH probes (with different concentrations of Zwittergent (Sigma-Aldrich) as a surfactant in 50% Stellaris RNA FISH hybridization buffer (BioSearch Technologies, cat# SMF-HB1-10) containing 3.5% formamide) were printed on X-plates with a column height of 10 μm and air-dried.

[0462] Add sample. 3 μL of fresh whole blood is dropped onto the center of the base plate and covered by the X-plate.

[0463] Staining. Incubate the card at room temperature for 1 minute.

[0464] Imaging The stained cards were imaged by an iPhone 6S under external laser illumination.

[0465] 3. Results:

[0466] An embodiment of an X-FISH device, comprising a first plate (X plate) and a second plate (substrate). Specific probes and surfactants (e.g., Zwittergent) are printed on the X plate. After a blood sample is dripped onto the substrate, the X plate is covered and pressed onto the substrate. After 1 minute, the device is inserted into a smartphone device for imaging and analysis. (b) is a diagram of detecting specific RNA expressed in leukocytes by X-FISH. The column on the X plate forms a gap between the two plates, where the assay is performed. The printed probes and surfactants (e.g., Zwittergent) are dissolved in the blood, the surfactant (e.g., Zwittergent) lyses the red blood cells, and also permeabilizes the leukocytes to facilitate the probe to enter the cell to bind to the target RNA. It is noteworthy that the probe is designed to bind to the target RNA in series to amplify the signal.

[0467] Flowchart description of X-FISH. Preparation is printing of X-plates with fluorophore-labeled detection probes and surfactant. The assay involves applying the sample to a substrate plate, covering with the X-plate, incubating at room temperature for 1 minute, and inserting into a smartphone device for reading.

[0468] Figure 5 The effect of different Zwittergent concentrations on leukocyte permeabilization during rapid FISH using a GAPDH probe in QMAX is shown. Figure (a) GAPDH probe was observed using a smartphone-based microscope, and 0.625 mg / mL of Zwittergent in blood did not produce a FISH signal. Figure (b) 2.5 mg / mL and above (5 mg / mL) of Zwittergent gave strong fluorescence signals from GAPDH-labeled white blood cells observed by a smartphone-based microscope, and Figure (c) used fluorescence microscopy in (c) (DAPI WBC nuclear counterstaining).

[0469] Example 4:

[0470] Chlamydia staining

[0471] In the experiments, mouse anti-Chlamydia infection (Abcam, cat#ab41196) was used for staining and Chlamydia antigen control slides (MBL Bion, cat#QCHE-4502) were used as a device to fix the samples.

[0472] In one of the experiments, slides fixed with Chlamydia-infected human tissue cells were incubated with 40ug / mL of 1μL DyLight633-labeled anti-Chlamydia antibody for 30 seconds. X-plates with a column height of 30μm were used in QMAX immunostaining. The slides were then rinsed three times with PBST and images were taken by fluorescence microscopy. BF: bright field. DL633: DyLight633 fluorescence.

[0473] Figure 6 An exemplary photograph of Chlamydia staining using the QMAX device without the optional rinse step is shown. In the experiment, slides fixed with Chlamydia-infected human tissue cells were incubated with 40ug / mL of 1μLDyLight633-labeled anti-Chlamydia antibodies for 1 min. X-plates with a column height of 30μm were used in QMAX immunostaining. Images were taken by fluorescence microscopy without rinsing.

[0474] In the conventional staining experiment, the slides fixed with Chlamydia-infected human tissue cells were incubated with 20 μL DyLight633 labeled anti-Chlamydia antibody at 40 ug / mL for 30 min. The slides were then rinsed three times with PBST and images were taken using a fluorescence microscope.

[0475] Examples of the Invention

[0476] One-step staining assay for detecting Chlamydia

[0477] AA1. A method for detecting Chlamydia in a sample, comprising:

[0478] (a) obtaining a first plate, the first plate comprising a sample contact area on an inner surface thereof configured to contact a sample;

[0479] (b) depositing a sample on a sample contact area, wherein the sample is suspected of containing Chlamydia-infected cells; and

[0480] (c) depositing a chlamydia-staining medium on the sample, wherein the staining medium comprises antibodies that bind to chlamydia and the staining medium forms a mixture with the sample;

[0481] (d) covering the mixture of sample and staining medium with a second plate,

[0482] (e) pressing the first plate and the second plate so that at least a portion of the mixture is compressed into a thin layer;

[0483] (f) incubating for a predetermined period of time of about 60 seconds or less; and

[0484] (g) Detection of Chlamydia-associated signals from the mixture.

[0485] AA2. A method according to any of the above embodiments, wherein the predetermined time period is approximately 30 seconds or less.

[0486] AA3. A method according to any of the above embodiments, wherein the predetermined time period is approximately 15 seconds or less.

[0487] AA4. A method according to any of the above embodiments, wherein the Chlamydia antibody is fluorescently labeled.

[0488] AA5. The method of any preceding embodiment, wherein the thin layer has a uniform thickness of less than 100 μm.

[0489] AA6. The method of any preceding embodiment, wherein the thin layer has a uniform thickness of less than 50 μm.

[0490] AA7. The method of any of the preceding embodiments, wherein the thin layer has a uniform thickness of about 30 μm or less.

[0491] AA8. A method according to any of the above embodiments, wherein the Chlamydia-related signal is detected by imaging the sample.

[0492] One-step sandwich assay for detecting Chlamydia

[0493] AB1. A method for detecting Chlamydia in a sample, comprising:

[0494] (a) obtaining a first plate, the first plate comprising a sample contacting area having binding sites on its inner surface, wherein the binding sites comprise immobilized capture antibodies that bind to Chlamydia in a sample suspected of containing Chlamydia;

[0495] (b) obtaining a second plate comprising a sample contacting area having a storage site on its inner surface, wherein the storage site comprises a detection antibody capable of diffusing in the sample upon contact with the sample, and wherein the capture antibody and the detection antibody bind to different sites in the Chlamydia to form a capture antibody-Chlamydia-detection antibody sandwich;

[0496] (c) depositing the sample on one or both sample contact areas of the plate;

[0497] (d) after (c), bringing the two plates into a closed configuration, wherein in the closed configuration, at least a portion of the sample deposited in (c) is confined between sample contact areas of the two plates and has an average thickness in the range of 0.01 μm to 200 μm; and

[0498] (e) Detection of Chlamydia-associated signals captured by the capture antibody.

[0499] AB2. A method according to any of the above AB embodiments, wherein the sample is from a human subject.

[0500] AB3. A method according to any of the above AB embodiments, wherein the capture site further comprises a protein stabilizer.

[0501] AB4. A method according to any of the above AB embodiments, wherein the storage site further comprises a protein stabilizer.

[0502] AB5. A method according to any of the above AB embodiments, wherein the detection antibody comprises a fluorescent label.

[0503] AB6. The method according to any of the above AB embodiments, wherein the sample between the two plates has a uniform thickness in the range of 0.5 to 50 μm.

[0504] AB7. A method according to any of the above AB embodiments, wherein the sample between the two plates has a uniform thickness in the range of 1 to 35 μm.

[0505] AB8. The method according to any of the above AB embodiments, further comprising determining the presence or absence of Chlamydia.

[0506] AB9. A method according to any of the above AB embodiments, wherein the total time of steps (a) to (e) is less than 10 minutes.

[0507] AB10. A method according to any of the above AB embodiments, wherein the total time of steps (e) to (e) is less than 3 minutes.

[0508] AB11. A method according to any of the above AB embodiments, wherein the total time of steps (a) to (e) is less than 2 minutes.

[0509] Additional Features:

[0510] AC1. A method according to any of the above embodiments, wherein one or both of the sample contact areas include a spacer, wherein the spacer adjusts the spacing between the sample contact areas of the plate when the plate is in a closed configuration.

[0511] AC2. A method according to any of the above embodiments, wherein the first plate comprises a plurality of binding sites and the second plate comprises a plurality of corresponding storage sites, wherein each binding site faces a corresponding storage site when the plates are in a closed configuration.

[0512] AC3. A method according to any of the above embodiments, wherein the detection antibody is dried on the storage site.

[0513] AC4. A method according to any of the above embodiments, wherein the capture antibody at the binding site is on an amplification surface that amplifies the optical signal of the analyte or captured detection agent in any of the previous embodiments.

[0514] AC5. A method according to any of the above embodiments, wherein the capture agent at the binding site is on an amplification surface, and the amplification surface amplifies the optical signal of the analyte or captured detection agent in any of the previous embodiments, wherein the amplification is proximity-dependent in that the amplification decreases significantly as the distance between the capture agent and the analyte or detection agent increases.

[0515] AC6. A method according to any of the above embodiments, wherein the detection of the signal is electrical, optical, or both. (Including but not limited to Fluorescence, SPR, etc.

[0516] Quantification of CD4 expressing cells

[0517] In a method for staining cells expressing CD4 according to some embodiments of the present invention, a first plate (referred to as an "X plate") and a second plate (e.g., made of glass or acrylic) are obtained, wherein the first plate and the second plate are movable relative to each other. In certain embodiments, the first plate and the second plate are not connected. In certain embodiments, the first plate and the second plate are connected by a rotating structure (e.g., a hinge). Each plate has two surfaces: an inner surface and an outer surface, wherein the inner surfaces face each other when the plates are pressed against each other. On the inner surface, each plate includes a sample contact area for contacting a liquid sample.

[0518] In some embodiments, a detection agent (e.g., a labeled anti-CD4 antibody) is fixed to the sample contact area of ​​one or both plates. In certain embodiments, the detection agent comprises an anti-CD4 antibody. In certain embodiments, the detection agent is labeled with a fluorophore. In certain embodiments, the anti-CD4 antibody is labeled with Alex 647.

[0519] In step 2, a liquid sample is deposited on a sample contact area of ​​one or both of the plates while the plates are in an open configuration in which the plates are separated. In certain embodiments, the sample is blood.

[0520] In step 3, the plates are pressed against each other in a closed configuration. In some embodiments, the imprecise force is applied by human hands. In the closed configuration, the plates are pressed against each other with a gap therebetween, and the sample is compressed into a thin layer. In some embodiments, the thin layer has a uniform thickness. In some embodiments, one of the two plates includes a spacer fixed in one or both sample contact areas. When the plates are pressed into the closed configuration, the spacer adjusts the thickness of the sample layer. In some embodiments, the spacer has a column shape.

[0521] In step 4, the sample layer is imaged and the number of cells expressing CD4 is quantified.

[0522] In the experiment, the QMAX device has two plates. The first plate is an X-plate with 2μm or 10μm column height, 30×40μm column size, 80μm spacing, and is made of 175μm thick PMMA. The second plate is 1mm thick glass or acrylic resin. AlexaFluor 647-labeled anti-CD4 antibodies are positioned on the second plate in liquid form or dry form. In its liquid form, the anti-CD4 antibody is 5 to 50ug / mL and the volume is 0.5 to 1uL. The anti-CD4 antibody is printed into an array with a fixed spacing of 300μm and dried, and the surface concentration after drying is 1-100ng / cm 2 .

[0523] In the experiment, for step 2, the sample was fresh whole blood with a volume of about 1 μL.

[0524] In this experiment, for step 3, the sample layer was incubated with the detection agent for approximately 60 seconds after pressing the plates against each other.

[0525] In the experiments, for step 4, the stained whole blood sample layer was imaged with a laboratory microscope or with a mobile device-adapter system.

[0526] Below is a device for capturing an image of a sample according to some embodiments of the present invention. Taking the iPhone as an example, the iPhone / reader setting uses a laser diode as the light source. The laser diode has a central wavelength of 638nm and a power of 10-20mW. The short pass of the excitation filter in front of the light source is 650nm. The light is reflected by the aluminum mirror to the back of the QMAX device, and the typical illumination area is 1mm×4mm. The observation system is located in front of the QMAX device, and the iPhone is equipped with an emission filter and lens. The long pass of the emission filter is 670nm. The lens has a focal length of about 4mm and an NA of 0.2.

[0527] Fluorescence photographs of CD-4 stained whole blood in a 2 μm thick QMAX card under a phone / reader system. The left photo used a relatively high antibody concentration of 50 ug / mL, and the right photo used an antibody concentration of 10 ug / mL. The fluorescence photographs show clear fluorescence of stained CD-4 T cells. (b) Fluorescence photographs of CD-4 stained whole blood in a 10 μm thick QMAX card under a phone / reader system. The left photo used a relatively high antibody concentration of 50 ug / L, and the right photo used an antibody concentration of 10 ug / mL. No CD-4 T cells were observed under the 10 μm QMAX, which may be due to the lower sensitivity and dynamic range of the iPhone reader compared to the inverted microscope system.

[0528] The number of CD4 expressing T cells counted using the QMAX device and the iPhone-laser device is listed below. The CD4 T cells counted by the QMAX system were 900 μL, and the samples had known T cell values ​​between 500-1600 μL.

[0529] In some embodiments, staining comprises the following steps:

[0530] (a) obtaining a first plate and a second plate, wherein each plate comprises a sample contact area on a respective inner surface thereof configured to contact a liquid sample,

[0531] wherein the detection agent is located on the sample contact area of ​​one or both plates, and

[0532] wherein the detection agent is configured to specifically bind to the biomarker,

[0533] (b) depositing a sample in the sample contact area, wherein the sample comprises cells expressing the biomarker;

[0534] (c) pressing the first plate and the second plate to compress the sample into a thin layer, the thin layer being at least partially defined by two sample contact areas facing each other;

[0535] (d) incubating for a predetermined period of time of about 60 seconds or less; and

[0536] (e) Quantifying cells expressing the biomarker by imaging the sample layer and counting the cells expressing the biomarker.

[0537] An exemplary flow chart demonstrating a process for performing a staining assay for CD4 expressing cells comprises the following steps:

[0538] (a) obtaining a first plate and a second plate, wherein each plate comprises a sample contact area on a respective inner surface thereof configured to contact a liquid sample,

[0539] wherein the detection agent is located on the sample contact area of ​​one or both plates, and

[0540] wherein the detection agent is configured to specifically bind to the biomarker,

[0541] (b) depositing a sample in the sample contact area, wherein the sample comprises cells expressing the biomarker;

[0542] (c) pressing the first plate and the second plate to compress the sample into a thin layer, the thin layer being at least partially defined by two sample contact areas facing each other;

[0543] (d) incubating for a predetermined period of time of about 60 seconds or less; and

[0544] (e) Quantifying cells expressing the biomarker by imaging the sample layer and counting the cells expressing the biomarker.

[0545] Examples of the Invention

[0546] One-step staining assay for CD4 T cells in whole blood

[0547] BA1.1 A method for quantifying cells expressing a biomarker in a sample, comprising:

[0548] (a) obtaining a sample holder configured to hold a liquid sample containing an analyte, wherein a detection agent is positioned in the sample holder and configured to specifically bind to a biomarker;

[0549] (b) depositing a sample in a sample contact area, wherein the sample comprises cells expressing a biomarker; and contacting the sample with a detection reagent in a sample holder;

[0550] (c) adjusting the sample holder so as to compress the sample into a thin layer,

[0551] (d) incubating for a predetermined period of time; and

[0552] (e) Quantifying cells expressing the biomarker by imaging the sample layer and counting the cells expressing the biomarker.

[0553] BA1.2 A method for quantifying cells expressing a biomarker in a sample, comprising:

[0554] (a) obtaining a first plate and a second plate, wherein each plate comprises a sample contact area on a respective inner surface thereof configured to contact a liquid sample,

[0555] wherein the detection agent is located on the sample contact area of ​​one or both plates, and

[0556] wherein the detection agent is configured to specifically bind to the biomarker,

[0557] (b) depositing a sample in the sample contact area, wherein the sample comprises cells expressing the biomarker;

[0558] (c) pressing the first plate and the second plate to compress the sample into a thin layer, the thin layer being at least partially defined by two sample contact areas facing each other;

[0559] (d) incubating for a predetermined period of time of about 60 seconds or less; and

[0560] (e) Quantifying cells expressing the biomarker by imaging the sample layer and counting the cells expressing the biomarker.

[0561] BA1.3 A method for quantifying cells expressing CD4 (cluster of differentiation 4) in a blood sample, comprising:

[0562] (a) obtaining a first plate and a second plate, wherein each plate comprises a sample contacting area on a respective inner surface thereof configured to contact a blood sample,

[0563] wherein the detection agent is located on the sample contact area of ​​one or both plates, and

[0564] wherein the detection agent is configured to specifically bind to CD4,

[0565] (b) depositing a blood sample in the sample contacting area, wherein the blood sample comprises cells expressing CD4;

[0566] (c) pressing the first plate and the second plate to compress the blood sample into a thin layer, the thin layer being at least partially defined by two sample contact areas facing each other;

[0567] (d) incubating for a predetermined period of time of about 60 seconds or less; and

[0568] (e) Quantification of cells expressing CD4 by imaging the sample layer and counting cells expressing CD4.

[0569] BA2.1 An apparatus for quantifying cells expressing a biomarker in a sample, comprising:

[0570] a sample holder configured to hold a liquid sample containing cells expressing the biomarker, wherein the detection agent is positioned in the sample holder and configured to specifically bind to the biomarker; and

[0571] An adapter configured to receive a sample holder and attachable to a mobile device, wherein:

[0572] i. The mobile device includes an imager,

[0573] ii. the adapter is configured to position the sample in the field of view (FOV) of the imager when the adapter is attached to the mobile device, and

[0574] iii. The imager is configured to capture an image of the sample, thereby detecting / measuring a signal resulting from the binding of the biomarker to the detection agent after the sample is incubated with the detection agent for a predetermined period of about 60 seconds or less.

[0575] BB1.1 A method or apparatus according to any of the above embodiments, wherein the predetermined time period is approximately 30 seconds or less.

[0576] BB1.2 A method or apparatus according to any of the above embodiments, with the proviso that the sample contact area is not rinsed after step (d).

[0577] BB1.3 A method or device according to any of the above embodiments, wherein the detection agent is an antibody.

[0578] BB1.4 A method or device according to any of the above embodiments, wherein the antibody is labeled with a fluorophore.

[0579] BB1.5 A method or device according to any of the above embodiments, wherein the detection agent is labeled with a signal molecule that emits a signal when excited.

[0580] BBl.6 A method or apparatus according to any of the above embodiments, wherein the thin layer has a uniform thickness approximately equal to or less than 10 μm.

[0581] BBl.7 A method or apparatus according to any of the above embodiments, wherein the thin layer has a uniform thickness approximately equal to or less than 2 μm.

[0582] BB1.8. A method or apparatus according to any of the above embodiments, wherein the sample is whole blood.

[0583] BB1.9 A method or apparatus according to any of the above embodiments, wherein the biomarker is CD4 (cluster of differentiation 4).

[0584] BB1.10 A method or apparatus according to any of the above embodiments, wherein the cell is a T cell.

[0585] BB1.11 A method or device according to any of the above embodiments, wherein the detection agent is immobilized on the sample contact area.

[0586] Machine Learning (ML) for Image-Based Assays

[0587] In the present invention, a machine learning model for detecting objects in image-based determinations is established based on training data. In some embodiments, machine learning-based detection is performed on images for determination, and in some embodiments, machine learning-based detection is performed on transformed images for determination with higher contrast and lower signal-to-noise ratio. It starts with the action of collecting a set of training images DBO for determination taken by an imager. These images are collected by taking multiple images of a sample in a sample holding device such as a QMAX card. Then, it obtains each training image from DB0 and marks the object of interest in the image for training. The marked images are saved in a separate training set DB 1 for establishing a machine learning model.

[0588] In the machine learning model training, a labeled training database DBI is used, a machine learning model structure is selected in the form of a deep neural network, and the model is trained for the training database DBI. In some embodiments, the machine learning model of RetinaNet is used, and in some other embodiments, the machine learning model of Fast-RCNN is selected. The machine learning model is trained using Tensorflow and PyTourch, and the training database DB1 is used for detection and segmentation. When the loss function on the training set DBI or a separate evaluation image data set meets the preset stopping criteria, the machine learning model training process ends after a certain iteration of the training data in DBI. The machine learning model obtained from the machine learning training process is saved for the determination application.

[0589] In this assay process, the image of the sample is the input to the machine learning based reasoning module. The machine learning based reasoning module performs machine learning based detection and segmentation based on the model obtained in the previous training process based on the training set DB1. The detected objects and their locations are saved in a database, which can be searched and retrieved for image-based assay applications.

[0590] In some embodiments, machine learning model training is performed on a local computing server, and in some embodiments, for scalability and efficiency, machine learning model training is performed in a hybrid cloud. In image-based assays, machine learning-based detection and segmentation are either performed on the device or using distributed computing resources, such as a hybrid cloud using both local and cloud resources, depending on computational complexity, resources on the device, network connectivity, assay latency requirements, etc.

[0591] Other embodiments

[0592] The present disclosure includes various embodiments that can be combined in various ways as long as various components are not contradictory to each other. These embodiments include not only the disclosure in the current document, but also the documents cited, incorporated or claiming priority here.

[0593] (1) Q-Card, spacers, and uniform sample thickness

[0594] The devices, systems and methods disclosed herein may include or use QMAX cards, spacers and uniform sample thickness embodiments for sample detection, analysis and quantification. In some embodiments, the Q card contains a spacer that helps to make at least a portion of the sample a very uniform layer.

[0595] The devices, systems, and methods disclosed herein may include or use QMAX 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 make at least a portion of the sample a very uniform layer. The above-mentioned PCT applications (designated US) PCT / US2016 / 046437 and PCT / US2016 / 051775, and the corresponding US provisional applications disclose the structure, materials, functions, variations, and dimensions of the spacers, as well as the uniformity of the spacers and the sample layer.

[0596] Other Notes

[0597] Other embodiments according to the inventive subject matter of the present disclosure are described in the following enumerated paragraphs.

[0598] As used herein, the terms "adapted" and "configured" mean that an element, component, or other subject matter is designed and / or intended to perform a given function. Thus, the use of the terms "adapted" and "configured" should not be interpreted as meaning that a given element, component, or other subject matter is simply "capable of" performing a given function. Similarly, a subject matter stated as being configured to perform a particular function may additionally or alternatively be described as being operable to perform that function.

[0599] As used herein, the phrase "for example", the phrase "as an example" and / or simply the terms "example" and "exemplary", when used with reference 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, including structurally and / or functionally similar and / or equivalent components, features, details, structures, embodiments and / or methods, are also within the scope of the present disclosure.

[0600] As used herein, the phrases "at least one" and "one or more" with respect to a list of more than one entity refer to any one or more entities in the list of entities, and are not limited to at least one of each and every entity specifically listed in the list of entities. For example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") may refer to A alone, B alone, or A and B in combination.

[0601] As used herein, the term "and / or" placed between a first entity and a second entity means one of (1) the first entity, (2) the second entity, and (3) the first entity and the second entity. Multiple entities listed using "and / or" should be interpreted in the same manner, i.e., "one or more" of the entities so conjoined. In addition to the entities specifically identified by the "and / or" clause, other entities may optionally be present, whether related or unrelated to those entities specifically identified.

[0602] 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 endpoint is excluded. Both endpoints should be assumed to be included unless otherwise stated. Furthermore, unless otherwise stated or apparent to one of ordinary skill in the art from context and understanding.

Claims

1. A method for enhancing homogeneous detection of an analyte in cells of a sample, comprising: (a) providing a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact area contacts a sample containing or suspected of containing cells of an analyte, (b) providing a detection probe that specifically binds to the analyte and can emit light of a certain wavelength, wherein the detection probe diffuses in the sample; (c) providing an optical enhancer dispersed in the sample, the optical enhancer binding to the cell and capable of emitting light of a certain wavelength, the light of the optical enhancer overlapping with the wavelength of the light emitted by the detection probe, or the wavelength thereof being within 150 nm; (d) sandwiching the sample, the detection probe and the optical enhancer between the sample contacting areas of the two plates so as to form a thin layer having a thickness of 200 μm or less; as well as (e) after step (d) and without any washing step, imaging the thin layer using an imager to detect cells having analyte bound to the detection probe; wherein the sample thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell does not overlap with other cells in the imaging; The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that in step (e), positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in the cell. 2 . The method of claim 1 , wherein the detection probe is deposited onto at least one of the sample contact areas.

3. The method of claim 1, wherein the detection probe and the optical enhancer are deposited onto at least one of the sample contact areas.

4. A device for enhancing homogeneous detection of an analyte in cells in a sample, comprising: (a) a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact area contacts a sample containing or suspected of containing cells of an analyte, (b) a detection probe that specifically binds to the analyte and is capable of emitting light of a certain wavelength, wherein the detection probe is dispersed in the sample; (c) an optical enhancer that binds to the cell and emits light at a wavelength overlapping, or within 150 nm of, the wavelength of light emitted by the detection probe; wherein the first plate and the second plate are configured to sandwich the sample, the detection probe and the optical enhancer between the sample contacting areas of the two plates so as to form a thin layer having a thickness of 200 micrometers or less; as well as wherein the sample thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell does not overlap with other cells in the imaging; The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured so that positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer during imaging, wherein the bound detection probes are detection probes that bind to an analyte in the cell.

5. The device of claim 4, further comprising an imager that images the thin layer to detect bound detection probes.

6. The device of claim 4, wherein the optical enhancing agent is coated on one or both of the sample contacting areas.

7. A system for enhancing homogeneous detection of an analyte of cells in a sample, comprising: (a) the device according to any one of claims 4 to 6; and (b) Communication device.

8. A device for rapid homogeneous detection of an analyte in a cell membrane in a sample, comprising: (a) a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact area contacts a sample containing cells containing or suspected of containing an analyte within a cell membrane; (b) a detection probe that specifically binds to the analyte and can emit light of a certain wavelength, wherein the detection probe is dispersed in the sample; (c) an optical enhancer that binds to the cell and is capable of emitting light of a wavelength that overlaps with the wavelength of light emitted by the detection probe or is within 150 nm of the wavelength of the light emitted by the detection probe; (d) a permeabilizing agent that makes the cell membrane permeable to the detection probe; wherein the first plate and the second plate are configured to sandwich the sample, the detection probe, the optical enhancer, and the permeabilizing agent between the sample contacting areas of the two plates to form a thin layer having a thickness of 200 microns or less; wherein the thickness of the thin layer is configured such that for a given concentration of cells in the sample, each single cell does not overlap with other cells during imaging, and wherein the imaging operation does not include a washing step; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured such that: in the imaging of the thin layer, locations in the thin layer with bound detection probes are distinguishable from locations without bound detection probes; and The washing step is a step of removing unbound detection probes, permeabilizing agents, or both from the sample contact area.

9. A method for rapid homogeneous detection of an analyte in a cell membrane in a sample, comprising: (a) providing a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact area contacts a sample containing cells containing or suspected of containing an analyte in the cell membrane, (b) providing a detection probe that specifically binds to the analyte and can emit light of a certain wavelength, wherein the detection probe is dispersed in the sample; (c) providing an optical enhancer that binds to the cell and is capable of emitting light of a wavelength that overlaps with the wavelength of light emitted by the detection probe, or is within 150 nm of the wavelength of the light emitted by the detection probe; (d) providing a permeabilizing agent that makes the cell membrane permeable to the detection probe; (e) sandwiching the sample, the detection probe, the optical enhancer and the permeabilizing agent between the sample contacting areas of the two plates so as to form a thin layer having a thickness of 200 μm or less; as well as (f) after step (e) and without any washing step, imaging the thin layer to detect cells having analyte bound to the detection probe; wherein the thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell does not overlap with other cells in the imaging; wherein the thickness of the thin layer, the concentration of the optical enhancer in the sample, and the concentration of the detection probe in the sample are configured such that: in the thin layer, locations with bound detection probes are distinguishable from locations without bound detection probes; and The washing step is a step of removing unbound detection probes, permeabilizing agents, or both from the sample contact area.

10. The method according to any one of claims 1 to 3, further comprising a permeabilizing agent that makes the cell membrane permeable to the detection probe, The first plate and the second plate are configured to sandwich the sample, detection probe, optical enhancer and permeabilizing agent between the sample contact areas of the two plates to form a thin layer with a thickness of 200 microns or less.

11. The method of claim 9, wherein the detection probe, the permeabilizing agent, or the optical enhancing agent is deposited on one or both of the sample contacting areas.

12. A method for homogeneously detecting Gram-positive cells or Gram-negative cells in a sample, comprising: (a) providing a first plate and a second plate, each plate having a sample contact area on a surface thereof, wherein the sample contact area contacts a sample containing or suspected of containing Gram-positive cells or Gram-negative cells, (b) depositing a Gram-positive stain, a Gram-negative stain, or both on at least one of the sample contacting regions, wherein the Gram-positive and Gram-negative stains have two distinguishable colors; wherein the Gram-positive stain stains only Gram-positive cells, and the Gram-negative stain stains both Gram-positive and Gram-negative cells; in, In a Gram-positive cell stained by both Gram-positive and Gram-negative stains, the Gram-positive stain is designed by modifying the Gram-negative stain; (c) sandwiching the sample between the sample contact areas of the two plates so that the sample forms a thin layer having a thickness of 150 μm or less; as well as (d) after step (c) and without any washing steps, imaging the thin layer using an imager to detect cells stained by (i) only a Gram-negative stain, (ii) a Gram-positive stain; and (iii) both; wherein the thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell does not overlap with other cells in the imaging; Gram-positive cells show the color of Gram-positive stain, while Gram-negative cells only show the color of Gram-negative stain; wherein the thickness of the thin layer is configured such that, in the imaging step (d), positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in the cell, the analyte is a nucleic acid and the detection probes are fluorescence in situ hybridization (FISH) probes 13. A method according to any one of claims 1 to 3, 9, wherein the thickness of the thin layer is configured so that in the imaging step, positions with bound detection probes are distinguishable from positions without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to an analyte in the cell, the analyte is a nucleic acid and the detection probe is a fluorescent in situ hybridization (FISH) probe.

14. The method according to any one of claims 1 to 3, 9, and 12, wherein the analyte is detected by an immunoprobe.

15. The method according to any one of claims 1 to 3, 9, and 12, wherein the analyte is CD4.

16. The method according to any one of claims 1 to 3, 9, and 12, 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, wherein: In the open configuration, the first plate and the second plate are partially or completely separated and the sample is deposited in a sample contact area on one or both plates; as well as In the closed configuration, at least a portion of the deposited sample is compressed into a thin layer by the two plates.

17. The method according to any one of claims 1 to 3, 9, and 12, further comprising a plurality of spacers, wherein: (i) 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, and (ii) one or both of the plates include a spacer fixed to the respective plate, wherein in the open configuration, the two plates are fully or partially separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; as well as Wherein in the closed structure configured after the sample is deposited in the open structure, at least a portion of the sample is between and in contact with the two plates, and has a very uniform thickness adjusted by the spacer and the two sample contact areas of the plates, and the thickness is equal to or less than 150 μm.

18. The method according to any one of claims 1 to 3, 9 and 12, wherein: The imaging also includes a machine learning step.

19. The method according to any one of claims 1 to 3, 9 and 12, wherein: The optical enhancers are cell permeable dyes that can penetrate the cell wall and stain its constituents without the presence of an additional permeabilizing agent.

20. The system according to claim 7, wherein: Further comprising a non-volatile storage medium having a machine learning algorithm.

21. The method according to claim 17, wherein: The spacer has a height of 30 um or less.

22. The method according to claim 17, wherein: The spacer has a height of 10 um or less.

23. The method according to any one of claims 1 to 3, 9, and 12, wherein the analyte comprises a molecule, a cell, a tissue, or a nanoparticle having a different shape, wherein the molecule comprises a protein, a peptide, and a nucleic acid.

24. The method of claim 17, wherein: The spacer has a height of 2 um or less.

25. The method according to any one of claims 1 to 3, 9, and 12, wherein: The thickness of the sample is 2 um or less.

26. The method according to any one of claims 1 to 3, 9, and 12, wherein: The sample is derived from a biological sample, an environmental sample, a chemical, or a clinical sample.

27. The method according to any one of claims 1 to 3, 9, and 12, wherein: The cell is a prokaryotic organism or a eukaryotic organism; wherein prokaryotes include bacteria and archaea, and the eukaryotic organism includes animal cells and plant cells.

28. The method according to any one of claims 1 to 3, 9, and 12, wherein: The number of said cells in said sample may be one or more than one.

29. The method according to claim 9, wherein: The permeabilizing agent makes the cell membrane or the cell nucleus permeable, and includes: Triton X-100, Zwittergent, ASB-14, ASB-16, CHAPS, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium lauryl ether sulfate (SLES), sodium myristyl alcohol polyether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysulfobetaine, cocamidopropyl Betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamers, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

30. The method according to any one of claims 1 to 3, 9, and 12, wherein the permeabilizing agent, as a diluent, makes the cell membrane or the cell nucleus permeable by causing an osmotic pressure difference between cell membranes or adjusting salt concentration.

31. The method according to any one of claims 1 to 3, 9, and 12, wherein: Also included are reagents coated on the plate to cross-link proteins, including formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, and potassium permanganate.

32. The method according to any one of claims 1 to 3, 9, and 12, wherein: The optical enhancer emits light at a wavelength that overlaps with or is within 30 nm of the wavelength of light emitted by the detection probe.

33. The method according to any one of claims 1 to 3, 9, and 12, wherein: Acridine orange is applied to the first plate or the second plate or both.

34. The method according to any one of claims 1 to 3, 9, and 12, wherein: Zwittergent is applied to the first plate or the second plate or both.

35. The method according to any one of claims 1 to 3, 9, and 12, wherein: Methylene blue and Zwittergent were applied to the first plate or the second plate or both.

36. The method according to any one of claims 1 to 3, 9, and 12, wherein: Acridine Orange and Zwittergent were applied to the first plate or the second plate or both.

37. The method according to any one of claims 1 to 3, 9, and 12, wherein: YOYO dye and Zwittergent are applied to the first plate or the second plate or both.

38. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both plates further comprise a plurality of reagent layers, the plurality of reagent layers comprising anti-adhesion, cell lysis, cell staining, release time control material layers, and combinations thereof.

39. The method according to any one of claims 1 to 3, 9, and 12, wherein: Also included are anti-adhesive agents comprising ethylenediaminetetraacetic acid (EDTA), disodium EDTA, K2EDTA, or K3EDTA.

40. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both sample contact areas are coated with a cell stain selected from: Wright's stain, Giemsa stain, May-grünwald stain, Leishman stain, Erythrosin B stain, Acridine Orange dye, 3,3-dihexyloxycarbocyanine DiOC6, Propidium iodide PI, Fluorescein isothiocyanate FITC and Basic Orange 21 dyes, Ethidium bromide, Brilliant Sulfaflavine and diaminostilbene disulfonic acid derivatives, Trypan Blue, Hoechst 33342, Trihydrochloride, Trihydrate, 4,6-Diamidinylflavone-2-phenylindole, Dihydrochloride, and YOYO.

41. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both sample contact areas are coated with cell stains including: Wright's stain, Giemsa stain, May-grünwald stain, Leishman stain, Erythrosin B stain, Acridine orange dye, 3,3-dihexyloxycarbocyanine DiOC6, Propidium iodide PI, fluorescein isothiocyanate FITC and basic orange 21 dyes, Ethidium bromide, Leucoflavin and diaminostilbene disulfonic acid derivatives, Trypan blue, trihydrochloride, Trihydrate, 4,6-diamidinoflavone-2-phenylindole, dihydrochloride, YOYO, acid fuchsin, hematoxylin, Hoechst 33258 and Hoechst Hoechst stain of 33342, methyl green, methylene blue, Nile blue, Nile red, osmium tetroxide, rhodamine, safranin, MeOSuc-AAPV-AMC, CFSE, BCECF / AM, silver nitrate, neutral red, pyronine Y, calcein-AM, dihydroethidium, ditolyl cyanide FF, 4-methylumbelliferyl palmitate, fast blue B salt, fluorescein yellow, CH dipotassium salt, DAPI double lactate, propidium iodide.

42. The method according to any one of claims 1 to 3, 9, and 12, further comprising a release time-controlling material, wherein the release time-controlling material comprises albumin, carbomer, carboxymethyl cellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinyl pyrrolidone, or polyvinyl alcohol.

43. The method according to any one of claims 1 to 3, 9, and 12, wherein one or both sample contact areas are coated with chemicals, the chemicals comprising: Zwittergent, ASB-14, ASB-16, CHAPS, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium laureth sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide,.

44. The method according to any one of claims 1 to 3, 9, and 12, wherein one or both sample contact areas are coated with a reagent, the reagent comprising Pluronic F-127, polyoxyethylene castor oil, Pluronic F-68, Myrj 52, Brij 35, sodium oleate, sodium lauryl sulfate, Tween 20, Tween 40, Tween 60, Tween 80, SLS, CTAB, CTAC, tamoxifen, saponin, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, ABS-14, ABS-16, antimalarial drugs arsenic, dapsone, metals, nitrites, nitrofurantoin, penicillin, phenazopyridine, rho immunoglobulin, ribavirin, sulfonamides, and sulfones; The metals include: Chromium, copper, lead.

45. The method according to any one of claims 1 to 3, 9, and 12, wherein one or both sample contact areas are coated with Zwittergent at the following area concentration: 3 ng / mm 2 , 5 ng / mm 2 , 8 ng / mm 2 , 12 ng / mm 2 , 15 ng / mm 2 , 25 ng / mm 2 , 35 ng / mm 2 , 50 ng / mm 2 , 80ng / mm 2 , 100 ng / mm 2 Or within a range between any two values.

46. ​​The method according to any one of claims 1 to 3, 9, and 12, wherein one or both sample contact areas are coated with a concentration of 3 to 10 ng / mm 2 Acridine orange, and Zwittergent at 3~30ng / mm 2 The area concentration is coated on the plate.

47. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both sample contact areas are coated with a marker, which is an immunoassay antibody marker, an RNA marker, a staining dye, which is coated on the plate at a final concentration in the sample of 0.1 nM / mL, 0.5 nM / mL, 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL, 50 nM / mL or in a range between any two values.

48. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both sample contact areas are coated with a marker, which is an immunoassay antibody marker, an RNA marker, a staining dye, which is coated on the plate at a final concentration of 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL in the sample, or in a range between any two values.

49. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both sample contact areas are coated with a marker, which is an immunoassay antibody marker, an RNA marker, a staining dye, which is coated on the plate at a final concentration in the sample of 0.05 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1.0 mg / mL, or in a range between any two values.

50. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both sample contact areas are coated with a marker, which is an immunoassay antibody marker, an RNA marker, a staining dye, which is coated on the plate at a final concentration in the sample of 0.5 nM / mL, 1.0 nM / mL, 1.5 nM / mL, 2.0 nM / mL, 5 nM / mL, 10 nM / mL, 50 nM / mL, or in a range between any two values.

51. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both sample contact areas are coated with a marker, which is an immunoassay antibody marker, an RNA marker, a staining dye, which is coated on the plate at a final concentration in the sample of 100 mg / mL, 200 mg / mL, 300 mg / mL or in a range between any two values.

52. The method according to any one of claims 1 to 3, 9, and 12, wherein: The thickness of the sample was 30 μm or less.

53. The method according to any one of claims 1 to 3, 9, and 12, wherein: The thickness of the sample was 10 μm or less.

54. The method according to any one of claims 1 to 3, 9, and 12, wherein: The concentration of the probe in the sample is 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM or a range between any two of the values.

55. The method according to any one of claims 1 to 3, 9, and 12, wherein: The concentration of the probe in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, or within a range between any two of the values.

56. The method according to any one of claims 1 to 3, 9, and 12, wherein: The concentration of the optical enhancer in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 uM, 2 uM, 5 uM, 10 uM, 20 uM, 50 uM, 100 uM, 500 uM, or a range between any two of the values.

57. The method according to any one of claims 1 to 3, 9, and 12, wherein: The concentration of the optical enhancer in the sample is 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, or a range between any two values.

58. The method according to any one of claims 1 to 3, 9, and 12, wherein: The light wavelength of the optical enhancer and the light wavelength of the probe are within 1 nm, 5 nm, 10 nm, 20 nm, 50 nm, 100 nm, 150 nm, or within a range between any two of the values.

59. The method according to any one of claims 1 to 3, 9, and 12, wherein: The optical wavelength of the optical enhancer and the optical wavelength of the probe are within 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, or within a range between any two of the values.

60. The method of claim 17, wherein: The pitch of the spacers is in the range of 1 μm to 200 μm.

61. The method of claim 17, wherein: The spacer pitch is in the range of 200 μm to 1000 μm.

62. A method according to claim 17, wherein one or both plates are flexible, and for a spacer that adjusts a uniform thickness layer, the Young's modulus of the spacer multiplied by the filling factor of the spacer is equal to or greater than 10 MPa, wherein the filling factor is the ratio of the area of ​​the spacer in contact with the uniform thickness layer to the total plate area in contact with the uniform thickness layer.

63. The method of claim 62, wherein: 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.

64. The method of claim 62, wherein: The fourth power of the spacer spacing ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE), equal to or less than 10 6 um 3 / GPa.

65. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both plate sample contact areas contain ruler markings, position marks or imaging marks.

66. The method according to any one of claims 1 to 3, 9, and 12, wherein: One or both plates contain imaging markers located on the surface or within the plate that aid in imaging of the sample.

67. The method of claim 17, wherein: The spacers serve as position markers, scale markers, imaging markers, or any combination thereof.

68. The method of claim 62, wherein: The uniform thickness layer has an average thickness equal to the smallest dimension of an analyte in the sample.

69. The method of claim 17, wherein: The spacer pitch is in the range of 1 μm to 50 μm.

70. The method of claim 17, wherein: The spacer pitch is in the range of 50 μm to 120 μm.

71. The method of claim 17, wherein: The spacer pitch is in the range of 120 μm to 200 μm.

72. The method of claim 17, wherein: The spacers are spaced at fixed intervals.

73. The method of claim 17, wherein: The spacers are posts having a cross-sectional shape selected from the group consisting of circular, annular, rectangular, oval, elliptical, or any combination thereof.

74. The method of claim 17, wherein: The spacers have a columnar shape and have a flat top surface, wherein for each spacer, a ratio of a lateral dimension of the spacer to its height is at least 1.

75. The method of claim 17, wherein: The lateral dimension of the spacer is in the range of 0.5 μm to 100 μm.

76. The method according to any one of claims 1 to 3, 9, and 12, wherein the first plate and the second plate are connected by a hinge.

77. The method of claim 17, wherein: In the closed configuration, the final sample thickness device is configured to analyze the sample in 60 seconds or less.

78. The system of claim 7, wherein: The communication device is also configured to transmit the test and subject information to a cloud network, which processes the information and is configured to improve the results of the test, and the improved test results are sent back to the subject.

79. The system of claim 7, wherein: The communication device is configured to receive a prescription, diagnosis or advice from a medical professional.

80. The method according to any one of claims 1 to 3, 9, and 12, wherein: The layer thickness of at least a portion of the sample is configured such that a saturation incubation time of the sample with the detection probe is 60 seconds or less.

81. The method according to any one of claims 1 to 3, 9, and 12, wherein: The attachment of the optical enhancer to the cells is specific.

82. The method according to any one of claims 1 to 3, 9, and 12, wherein: The attachment of the optical enhancer to cells is non-specific.

83. The method according to any one of claims 1 to 3, 9, and 12, wherein: The attachment of the optical enhancer to the cell is non-specific, wherein the non-specific attachment is binding to the nucleic acid of the cell.

84. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, further comprising a permeabilizing agent, wherein the first plate and the second plate are configured to sandwich the sample, the detection probe and the permeabilizing agent between the sample contact areas of the two plates to form a thin layer having a thickness of 200 microns or less; wherein the detection probe is dispersed in the sample, binds to the cell, and is capable of emitting light of a wavelength overlapping with or within 150 nm of the wavelength of light emitted by the detection probe; wherein the thickness of the thin layer is configured such that for a given concentration of cells in the sample, each single cell does not overlap with other cells in the imaging, wherein the imaging operation does not include a washing step; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured such that upon imaging, locations with bound detection probe are distinguishable from locations without bound detection probe; and wherein the bound detection probe is bound to an analyte in a cell; and; The washing step is a step of removing unbound detection probes, permeabilizing agents, or both from the sample contact area.

85. The device of claim 8, wherein the detection probe, the permeabilizing agent, or the optical enhancing agent is deposited on one or both of the sample contacting areas.

86. An apparatus according to any one of claims 4 to 6, 8, or a system according to claim 7, wherein the thickness of the thin layer is configured such that, during imaging, positions in the thin layer with bound detection probes are distinguishable from positions without bound detection probes, wherein the bound detection probes are detection probes that bind to an analyte in the cell, the analyte is a nucleic acid and the detection probe is a fluorescent in situ hybridization probe.

87. The device of any one of claims 4 to 6, 8, or the system of claim 7, wherein the analyte is detected by an immunoprobe.

88. The device of any one of claims 4 to 6, 8, or the system of claim 7, wherein the analyte is CD4.

89. The device of any one of claims 4 to 6, 8, or the system of claim 7, 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, wherein: In the open configuration, the first plate and the second plate are partially or completely separated and the sample is deposited in a sample contact area on one or both plates and In the closed configuration, at least a portion of the deposited sample is compressed into a thin layer by the two plates.

90. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, further comprising a plurality of spacers, wherein: (i) 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, and (ii) one or both of the plates include a spacer fixed to the respective plate, wherein in the open configuration, the two plates are fully or partially separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; as well as Wherein in the closed configuration configured after the sample is deposited in the open configuration, at least a portion of the sample is between and in contact with the two plates, and has a very uniform thickness adjusted by the spacer and the two sample surfaces of the plates, and the thickness is equal to or less than 150 μm.

91. The device according to any one of claims 4 to 6 and 8 further comprises a non-volatile storage medium having a machine learning algorithm.

92. The device or system of claim 90, wherein: The spacer has a height of 30 um or less.

93. The device or system of claim 90, wherein: The spacer has a height of 10 um or less.

94. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein the analytes include molecules, cells, tissues, or nanoparticles having different shapes, wherein the molecules include proteins, peptides, and nucleic acids.

95. The device or system of claim 90, wherein: The spacer has a height of 2 um or less.

96. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The thickness of the sample is 2 um or less.

97. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The sample is derived from a biological sample, an environmental sample, a chemical, or a clinical sample.

98. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The cell is a prokaryotic organism or a eukaryotic organism; the prokaryotic organisms include bacteria and archaea, and the eukaryotic organisms include animal cells and plant cells.

99. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The number of cells in the sample may be one or more than one.

100. The device or system of claim 84, wherein: The permeabilizing agent makes the cell membrane or the cell nucleus permeable, and includes: Triton X-100, Zwittergent, ASB-14, ASB-16, CHAPS, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium lauryl ether sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysulfobetaine, cocamidopropyl Betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamers, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

101. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein the permeabilizing agent acts as a diluent to make the cell membrane or the cell nucleus permeable by causing an osmotic pressure difference between the cell membranes or adjusting the salt concentration.

102. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: Also included are reagents coated on the device for cross-linking proteins, including formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, and potassium permanganate.

103. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The optical enhancer emits light at a wavelength that overlaps with or is within 30 nm of the wavelength of light emitted by the detection probe.

104. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: Acridine orange is applied to the first plate or the second plate or both.

105. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: Zwittergent is applied to the first plate or the second plate or both.

106. According to the device of any one of claims 4 to 6, 8, or the system of claim 7, methylene blue and Zwittergent are coated on the first plate or the second plate or both.

107. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: Acridine Orange and Zwittergent were applied to the first plate or the second plate or both.

108. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: YOYO dye and Zwittergent are applied to the first plate or the second plate or both.

109. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The device further comprises a plurality of reagent layers on one or both plates, the plurality of reagent layers comprising anti-adhesion, cell lysis, cell staining, release time control material layers, and combinations thereof.

110. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: Also included are anti-adhesive agents comprising ethylenediaminetetraacetic acid (EDTA), disodium EDTA, K2EDTA, or K3EDTA.

111. According to the device of any one of claims 4 to 6, 8, or the system of claim 7, one or both sample contact areas are coated with a cell stain, and the cell stain comprises: Wright's stain, Giemsa stain, May-grünwald stain, Leishman stain, Erythrosin B stain, Acridine Orange dye, 3,3-dihexyloxycarbonylcyanine, Propidium iodide, Fluorescein isothiocyanate and Basic Orange 21 dye, Ethidium bromide, Brilliant Sulfaflavine and diaminostilbene disulfonic acid derivatives, Trypan Blue, Hoechst 33342, Trihydrochloride, Trihydrate, 4,6-Diamidinyl yellow-2-phenylindole, Dihydrochloride, and YOYO.

112. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: One or both sample contact areas are coated with cell stains including: Wright's stain, Giemsa stain, May-grünwald stain, Leishman stain, Erythrosin B stain, Acridine orange dye, 3,3-dihexyloxycarbocyanine, Propidium iodide, Fluorescein isothiocyanate and Basic Orange 21 dyes, Ethidium bromide, Leucoflavin and diaminostilbene disulfonic acid derivatives, Trypan blue, Trihydrochloride, Trihydrate, 4,6-diamidinoflavone-2-phenylindole, Dihydrochloride, YOYO, Acid fuchsin, Hematoxylin, Hoechst 33258 and Hoechst Hoechst stain 33342, methyl green, methylene blue, Nile blue, Nile red, osmium tetroxide, rhodamine, safranin, MeOSuc-AAPV-AMC, CFSE, BCECF / AM, silver nitrate, neutral red, pyronine Y, calcein-AM, dihydroethidium, dicrenilide FF, rhodamine 123, 4-methylumbelliferyl palmitate, fast blue B salt, fluorescein yellow, CH dipotassium salt, DAPI double lactate, propidium iodide.

113. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, further comprising a release time-controlled material, wherein the release time-controlled material comprises albumin, carbomer, carboxymethyl cellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinyl pyrrolidone, or polyvinyl alcohol.

114. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein one or both sample contacting areas are coated with chemicals, the chemicals comprising: Zwittergent, ASB-14, ASB-16, CHAPS, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium laureth sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctane sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucosides, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

115. The device of any one of claims 4 to 6, 8, or the system of claim 7, wherein one or both sample contact areas are coated with a reagent comprising Pluronic F-127, polyoxyethylene castor oil, Pluronic F-68, Myrj 52, Brij 35, sodium oleate, sodium lauryl sulfate, Tween 20, Tween 40, Tween 60, Tween 80, SLS, CTAB, CTAC, tamoxifen, saponin, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, ABS-14, ABS-16, antimalarial drugs, arsenic, dapsone, chromium, copper, lead, nitrite, nitrofurantoin, penicillin, phenazopyridine, rho immunoglobulin, ribavirin, sulfonamides, and sulfones.

116. The device of any one of claims 4 to 6, 8, or the system of claim 7, wherein one or both sample contact areas are coated with Zwittergent at the following area concentration: 3 ng / mm 2 , 5 ng / mm 2 , 8 ng / mm 2 , 12 ng / mm 2 , 15 ng / mm 2 , 25 ng / mm 2 , 35 ng / mm 2 , 50 ng / mm 2 , 80 ng / mm 2 , 100 ng / mm 2 Or within a range between any two values.

117. The device of any one of claims 4 to 6, 8, or the system of claim 7, wherein one or both sample contact areas are coated with a concentration of 3 to 10 ng / mm 2 Acridine orange and Zwittergent at 3 to 30 ng / mm 2 The area concentration of is coated on the plate.

118. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: One or both sample contact areas are coated with a marker, which is an immunoassay antibody marker, an RNA marker, a staining dye, which is coated on the plate at a final concentration in the sample of 0.1 nM / mL, 0.5 nM / mL, 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL, 50 nM / mL or in a range between any two values.

119. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: One or both sample contact areas are coated with a marker, which is an immunoassay antibody marker, an RNA marker, a staining dye, which is coated on the plate at a final concentration of 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL in the sample, or in a range between any two values.

120. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: A marker is coated on one or both sample contact areas, the marker being an immunoassay antibody marker, an RNA marker, a staining dye, coated on the plate at a final concentration in the sample of 0.05 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 1.0 mg / mL, or in a range between any two of the values.

121. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: One or both sample contact areas are coated with a marker, which is an immunoassay antibody marker, an RNA marker, a staining dye, which is coated on the plate at a final concentration in the sample of 0.5 nM / mL, 1.0 nM / mL, 1.5 nM / mL, 2.0 nM / mL, 5 nM / mL, 10 nM / mL, 50 nM / mL, or in a range between any two values.

122. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: A marker is coated on one or both sample contact areas, the marker being an immunoassay antibody marker, an RNA marker, a staining dye, coated on the plate at a final concentration in the sample of 100 mg / mL, 200 mg / mL, 300 mg / mL or in a range between any two values.

123. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The thickness of the sample was 30 μm or less.

124. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The thickness of the sample was 10 μm or less.

125. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The concentration of the probe in the sample is 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, or in a range between any two of the values.

126. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The concentration of the probe in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, or within a range between any two of the values.

127. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The concentration of the optical enhancer in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 uM, 2 uM, 5 uM, 10 uM, 20 uM, 50 uM, 100 uM, 500 uM, or a range between any two of the values.

128. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The concentration of the optical enhancer in the sample is 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, or a range between any two values.

129. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The light wavelength of the optical enhancer and the light wavelength of the probe are within 1 nm, 5 nm, 10 nm, 20 nm, 50 nm, 100 nm, 150 nm, or within a range between any two of the values.

130. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The optical wavelength of the optical enhancer and the optical wavelength of the probe are within 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, or within a range between any two of the values.

131. The device or system of claim 90, wherein: The spacer pitch is in the range of 1 μm to 200 μm.

132. The device or system of claim 90, wherein: The spacer pitch is in the range of 200 μm to 1000 μm.

133. An apparatus or system according to claim 90, wherein one or both plates are flexible, and for a spacer that adjusts a uniform thickness layer, the Young's modulus of the spacer multiplied by the filling factor of the spacer is equal to or greater than 10 MPa, wherein the filling factor is the ratio of the area of ​​the spacer in contact with the uniform thickness layer to the total area of ​​the plate in contact with the uniform thickness layer.

134. The device or system of claim 133, wherein: 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.

135. The device or system of claim 90, wherein: The fourth power of the spacer spacing ISD divided by the thickness h of the flexible board and the Young's modulus E of the flexible board, ISD 4 / (hE), equal to or less than 10 6 um 3 / GPa.

136. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The sample contact area of ​​one or both plates may contain scale markings, position markings or imaging markings.

137. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: One or both plates contain imaging markers located on the surface or within the plate that aid in imaging of the sample.

138. The device or system of claim 90, wherein: The spacers serve as position markers, scale markers, imaging markers, or any combination thereof.

139. The device or system of claim 90, wherein: The average thickness of the uniform thickness layer is equal to the smallest dimension of the analytes in the sample.

140. The device or system of claim 90, wherein: The spacer pitch is in the range of 1 μm to 50 μm.

141. The device or system of claim 90, wherein: The spacer pitch is in the range of 50 μm to 120 μm.

142. The device or system of claim 90, wherein: The spacer pitch is in the range of 120 μm to 200 μm.

143. The device or system of claim 90, wherein: The spacers are spaced at fixed intervals.

144. The device or system of claim 90, wherein: The spacers are posts having a cross-sectional shape selected from the group consisting of circular, annular, rectangular, oval, elliptical, or any combination thereof.

145. The device or system of claim 90, wherein: The spacers have a columnar shape and have a flat top surface, wherein for each spacer, a ratio of a lateral dimension of the spacer to its height is at least 1.

146. The device or system of claim 90, wherein: The lateral dimension of the spacer is in the range of 0.5 μm to 100 μm.

147. The device of any one of claims 4 to 6, 8, or the system of claim 7, wherein the first plate and the second plate are connected by a hinge.

148. The device or system of claim 90, wherein: In the closed configuration, the final sample thickness device is configured to analyze the sample in 60 seconds or less.

149. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The layer thickness of at least a portion of the sample is configured such that a saturation incubation time of the sample with the detection probe is 60 seconds or less.

150. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The attachment of the optical enhancer to the cells is specific.

151. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The attachment of the optical enhancer to cells is non-specific.

152. The device according to any one of claims 4 to 6, 8, or the system according to claim 7, wherein: The optical enhancer attaches to the cells in a non-specific manner, wherein the non-specific attachment is binding to nucleic acids of the cells.

153. The method of any one of claims 1 to 3, wherein the optical enhancer is coated on one or both sample contact areas.

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