Enhanced, fast, homogeneous cell staining and assays

Abstract

The present invention provides, inter alia, devices, kits, apparatuses and methods for rapid homogeneous cell staining and imaging. Specifically, in some embodiments, the present invention can immunochemically stain cells or tissues in less than 60 seconds without rinsing. In some embodiments, the present invention stains and observes an analyte (protein or nucleic acid) within a cell within 60 seconds without rinsing.

Description

[0001] Cross-referencing

[0002] This application is a divisional application of Chinese national phase application No. 201980069174.4, filed on August 23, 2019. Like the parent application, this application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 722,172, filed August 23, 2018, the contents of which are incorporated herein by reference in their entirety. The entire disclosure of any publications or patent documents mentioned herein is incorporated herein by reference in their entirety. Technical Field

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

[0004] There is a need for rapid cell staining and imaging, and / or the use of mobile phones to perform these. Summary of the Invention

[0005] This invention particularly provides apparatus, kits, devices, and methods for rapid homogeneous cell staining and imaging. Specifically, in some embodiments, the invention performs immunochemical staining of cells or tissues in less than 60 seconds without rinsing. In some embodiments, the invention stains and observes intracellular analytes (proteins or nucleic acids) within 60 seconds without rinsing.

[0006] One aspect of the invention is to use a QMAX card with a drying reagent to stain a sample (e.g., cells or tissue) on the sample contact area of ​​one or two plates.

[0007] Another aspect of the invention is the use of a QMAX card to configure the sample thickness as a thin layer to reduce background signal, making stained cells observable without any rinsing and / or without opening both plates of the QMAX card. Unbound labeled detection agents in the sample (i.e., detection agents that do not bind to target cells) are the primary source of background light signal, where the detection agents can be proteins or nucleic acids.

[0008] Another aspect of the invention is the use of a QMAX card to configure the thickness of the sample into a thin layer such that the stained cells do not overlap substantially in the direction perpendicular to the sample thickness. This allows the stained cells to be observed from the top of the QMAX card without being substantially obstructed by other stained cells.

[0009] Another aspect of the invention is to increase the light signal (i.e., optical signal) from stained cells by nonselectively staining cells in a sample with other dyes that emit light of the same wavelength as the dyes that selectively stain the cells. The addition (i.e., combination) of the optical signal makes the selectively stained cells observable (i.e., distinguishable from other parts of the sample), even without washing away unbound cells from the sample background.

[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 with a wavelength overlapping that 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 for staining target cells, wherein the multiple labeled antibodies, labeled probes, and dyes label light emitting wavelengths that overlap with the wavelength of a 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 present invention is to perform immunostaining.

[0015] In some embodiments, the present invention provides a method for homogeneous detection of an analyte in cells of 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 cells of the analyte; (b) having a detection probe that specifically binds to the analyte and is capable of emitting light of a specific wavelength, wherein the detection probe diffuses in the sample; and (c) having an optical enhancer that binds to the cells and is capable of emitting light at a wavelength that overlaps with or is within 30 nm of the wavelength of the light emitted by the detection probe.

[0016] (d) The sample, detection probe, and optical intensifier are sandwiched between two sample contact areas of two plates to form a thin layer with a thickness of 200 micrometers (μm) or less; and (e) after step (d) and without any rinsing step, the thin layer is imaged using an imager to detect cells with analytes bound to the detection probes; wherein the sample thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell substantially does not 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 intensifier in the sample is configured such that, in the imaging step (e), the locations of bound detection probes and the locations of unbound detection probes are distinguishable in the thin layer, wherein the bound detection probes are detection probes for analytes bound to the cells.

[0017] In some embodiments, the present invention provides a method for enhancing homogeneous detection of an analyte in cells of 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 the 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 specific wavelength, wherein the detection probe diffuses in the sample; (c) having an optical enhancer, the optical enhancer binding to the cells and emitting light at a wavelength overlapping with or within a wavelength range of 30 nm of the light emitted by the detection probe; and (d) sandwiching the sample, the detection probe, and the optical enhancer between two sample contact areas of the two plates to form a thin film. The thin layer has a thickness of 150 micrometers (μm) or less; and (e) imaging with an imager, after step (d) and without any rinsing step, using an imager to image the thin layer to detect a detection probe that has been specifically bound to the analyte; wherein the sample thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell substantially does not 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 intensifier in the sample are configured in the thin layer in imaging step (e) such that the locations of bound detection probes are distinguishable from the locations of unbound detection probes, wherein the bound detection probes are detection probes of the analyte bound to the cells.

[0018] In some embodiments, the present invention provides a method for homogeneous detection of an analyte in cells of 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 cells of the 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 specific 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 with a wavelength overlapping with or within a wavelength range of 30 nm of the light emitted by the detection probe, wherein the optical enhancer diffuses in the sample; and (d) placing the sample, the detection probe, and the optical enhancer together. An 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) after step (d) and without using any rinsing step, the thin layer is imaged using an imager to detect detection probes that have been specifically bound to the analyte; wherein the sample thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell substantially does not 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 such that, in imaging step (e), the locations of bound detection probes and the locations of unbound detection probes are distinguishable in the thin layer, wherein the bound detection probes are detection probes of the analyte bound to the cells.

[0019] In some embodiments, the present invention provides a kit for homogeneous detection of an analyte in cells of 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 or suspected of containing the analyte; (b) a detection probe that specifically binds to the analyte and is capable of emitting light of a specific wavelength, wherein the detection probe diffuses in the sample; and (c) an optical enhancer that binds to the cells and emits light at a wavelength overlapping with or within 30 nm of the light emitted by the detection probe; wherein the first plate and the second plate are configured to place the sample... A detection probe and an optical intensifier are sandwiched between the two sample contact areas to form a thin layer with a thickness of 200 micrometers (μm) or less; and wherein the sample thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell substantially does not 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 intensifier in the sample are configured such that, in the imaging step (e), the positions of the bound detection probes and the positions of the unbound detection probes are distinguishable in the thin layer, wherein the bound detection probes are detection probes of analytes bound to the cells.

[0020] In some embodiments, the present invention provides an apparatus for enhancing homogeneous detection of an analyte in cells of 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 or suspected of containing the analyte; (b) a detection probe that specifically binds to the analyte and emits light of a specific wavelength, wherein the detection probe is coated on at least one of the sample contact areas and diffuses in the sample; (c) an optical enhancer that binds to the cells and emits light with a wavelength overlapping with or within a wavelength range of 30 nm of the light emitted by the detection probe; and (d) the sample contact areas in the first and second plates facing each other and accommodating the sample, the detection probe, and... An optical intensifier 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 been specifically bound to the analyte; wherein the sample thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell substantially does not 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 intensifier in the sample is configured such that, in the imaging step (e), the locations of bound detection probes and the locations of unbound detection probes are distinguishable in the thin layer, wherein the bound detection probes are detection probes of the analyte bound to the cells.

[0021] In some embodiments, the present invention provides an apparatus for enhancing homogeneous detection of an analyte in cells of 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 or suspected of containing the analyte; (b) a detection probe that specifically binds to the analyte and emits light of a specific wavelength, wherein the detection probe is coated on one or both of the sample contact areas and diffuses in the sample; (c) an optical enhancer that binds to the cells and emits light with a wavelength overlapping with or within a wavelength range of 30 nm of the light emitted by the detection probe, wherein the optical enhancer is coated on one or both of the sample contact areas and diffuses in the sample; and (d) the sample contact areas in the first and second plates facing each other and the sample contact areas are used to... The sample, detection probe, and optical intensifier are 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) after step (d) and without using any rinsing step, the thin layer is imaged using an imager to detect the detection probe that has been specifically bound to the analyte; wherein the sample thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell substantially does not 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 intensifier in the sample is configured such that, in the imaging step (e), the locations of bound detection probes and the locations of unbound detection probes are distinguishable in the thin layer, wherein the bound detection probes are the detection probes of the analyte bound to the cells.

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

[0023] In some embodiments, the present invention provides a method for homogeneous detection of an analyte using enhanced cells in a sample, comprising: (a) having an apparatus or kit according to any of the foregoing embodiments; and (b) having a communication device.

[0024] In some embodiments, the present invention provides a method for rapid homogeneous detection of an analyte within 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 or suspected to contain cells within the cell membrane that contain the analyte; (b) having a detection probe that specifically binds to the analyte and is capable of emitting light of a specific wavelength, wherein the detection probe diffuses in the sample; (c) having a permeabilizing agent that allows the cell membrane to be permeable to the detection probe; and (d) sandwiching the sample, the detection probe, and the permeabilizing agent between two sample contact areas of the two plates to form a thickness of 200 μm. A thin layer of micrometers (μm) or smaller; and (e) after step (d) and without using any rinsing step, imaging the thin layer to detect cells having analytes bound to a detection probe; wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell substantially does not overlap with other cells in the imaging; wherein the thickness of the thin layer and the concentration of the detection probe in the sample are configured such that the location of the detection probe having analytes bound to the cell membrane is distinguishable from the location without cells in the thin layer; and wherein the rinsing step is a step of removing unbound detection probes, permeabilizing agents, or both from the sample contact area.

[0025] In some embodiments, the present invention provides a method for rapid homogeneous detection of an analyte within 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 or suspected to contain cells containing the 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 specific wavelength, wherein the detection probe diffuses within the sample; (c) having a permeabilizing agent that allows the cell membrane to be permeable to the detection probe; (d) sandwiching the sample, the detection probe, and an optical enhancer between 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) Following step (d) and without any rinsing step, a thin-layer image is used to detect detection probes that have specifically bound to the analyte; wherein the thickness of the thin-layer sample is configured such that for a given concentration of cells in the sample, each individual cell substantially does not overlap with other cells in the image; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample is configured such that, in imaging step (e), the locations of bound detection probes and the locations of unbound detection probes are distinguishable in the thin layer, wherein the bound detection probes are detection probes of the analyte bound to the cells; and wherein the rinsing step is a step of removing unbound detection probes, permeabilizing agents, or both from the sample contact area.

[0026] In some embodiments, the present invention provides a method for rapid homogeneous detection of an analyte within 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 the 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 specific wavelength, wherein the detection probe diffuses within 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 with a wavelength overlapping with or within a wavelength range of 30 nm of the light emitted by the detection probe, wherein the optical enhancer diffuses within the sample; and (d) sandwiching the sample, the detection probe, and the optical enhancer between two sample contact areas of the two plates. To form a thin layer having a thickness of 150 micrometers (μm) or less; and (e) after step (d) and without using any rinsing step, to image the thin layer using an imager to detect detection probes that have been specifically bound to the analyte; wherein the sample thickness of the thin layer is configured such that for a given concentration of cells in the sample, each individual cell substantially does not 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 intensifier in the sample is configured such that in the imaging step (e) the locations of bound detection probes and the locations of unbound detection probes are distinguishable in the thin layer, wherein the bound detection probes are detection probes of the analyte bound to the cells; and wherein the rinsing step is a step of removing unbound detection probes, permeabilizing agents, or both from the sample contact area.

[0027] In some embodiments, the present invention provides a method for homogeneous detection of Gram-positive 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 or Gram-negative cells; (b) depositing a Gram-positive staining agent, a Gram-negative staining agent, or both on at least one of the sample contact areas, wherein the Gram-positive and Gram-negative staining agents have two distinguishable colors; wherein the Gram-positive staining agent stains only Gram-positive cells, while the Gram-negative staining agent stains both Gram-positive and Gram-negative cells; wherein, in Gram-positive cells stained by both Gram-positive and Gram-negative staining agents, the Gram-positive staining agent may be designed by the Gram-negative staining agent; (c) clamping the sample between the two sample contact areas of the two plates to allow the sample to... A thin layer with a thickness of 150 micrometers (μm) or less is formed; and (d) after step (c) without any rinsing step, the thin layer is imaged using an imager to detect cells stained by (i) only Gram-negative staining, (ii) Gram-positive staining; and (iii); wherein the thickness of the thin layer sample is configured such that for a given concentration of cells in the sample, each individual cell substantially does not overlap with other cells in the imaging; wherein Gram-positive cells exhibit the color of Gram-positive staining, and Gram-negative cells exhibit the color of Gram-negative staining only; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample is configured in the thin layer in imaging step (e) such that the locations of bound detection probes are distinguishable from the locations of unbound detection probes, wherein the bound detection probes are detection probes of analytes bound to the cells.

[0028] In some embodiments, the thickness of the thin layer is configured in the imaging step (e) such that locations with bound detection probes are distinguishable from locations without bound detection probes, wherein the bound detection probes are detection probes that bind to analytes in the cells.

[0029] In some embodiments, the concentration of the detection probe in the sample is configured in the imaging step (e) such that the locations with bound detection probes are distinguishable from the locations without bound detection probes in the thin layer, wherein the bound detection probes are detection probes that bind to analytes in the cells.

[0030] In some embodiments, the concentration of the optical intensifier in the sample is configured in the imaging step (e) such that the locations with bound detection probes are distinguishable from the locations without bound detection probes in the thin layer, wherein the bound detection probes are detection probes of analytes bound to cells.

[0031] In some embodiments, the first plate and the second plate are movable relative to each other in different configurations, including open and closed configurations, wherein: in the open configuration, the first plate and the second plate are partially or completely separated, and a sample is deposited in a sample contact area on one or both of the plates, and a separation piece 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.

[0032] In some embodiments, the invention further includes a plurality of spacers, wherein: (i) the first plate and the second plate are movable relative to each other in different configurations, including an open configuration and a closed configuration, and (ii) one or both of these plates include spacers fixed together with the respective plates, wherein in the open configuration, the two plates are completely or partially separated, the spacing between the plates is not adjusted by the spacers, and the sample is deposited on one or both plates; and wherein in the closed configuration, which is configured after the sample deposition in the open configuration, at least a portion of the VC sample is between and in contact with the two plates, and has a very uniform thickness adjusted by the spacers and the two sample surfaces of the plates, and the thickness is equal to or less than 150 μm.

[0033] In some embodiments, imaging includes machine learning steps.

[0034] In some embodiments, imaging includes a machine learning step during the detection of the analyte.

[0035] In some embodiments, the present invention further includes a non-volatile storage medium having machine learning algorithms.

[0036] In some embodiments, the imager also includes a non-volatile storage medium with machine learning algorithms for detecting analytes.

[0037] In some embodiments, the analyte comprises molecules of different shapes (e.g., proteins, peptides, DNA, RNA, nucleic acids or other molecules), cells, tissues, viruses or nanoparticles.

[0038] In some embodiments, the analyte comprises molecules, proteins, peptides, DNA, RNA, and nucleic acids.

[0039] In some embodiments, the analytes within the cell membrane include molecules, proteins, peptides, DNA, RNA, and nucleic acids.

[0040] In some embodiments, the sample is a vapor from a biological sample, an environmental sample, a chemical sample, or a clinical sample.

[0041] In some embodiments, the detection of the labeled staining reagent is based on fluorescence (referring to other temporary substances), chemiluminescence (referring to other temporary substances), colorimetry (referring to other temporary substances), or plasma (referring to other temporary substances).

[0042] In some embodiments, the target cells are prokaryotes, such as bacteria and archaea, or eukaryotes, such as animal cells and plant cells.

[0043] In some embodiments, the number of target cells in the sample may be one or more.

[0044] 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 (1a1), 11b, 11c, 11d, CTAC, Tween 20, Tween 40, and Tween 60, Tween80, Sodium lauryl sulfate (SLS), Ammonium lauryl sulfate, CTAB, Sodium lauryl ether sulfate (SLES), Sodium myristyl ether sulfate, Docusate, Perfluorooctyl sulfonate, Alkyl-aryl ether phosphate, Alkyl ether phosphate, CTAB, Hexadecylpyridine chloride (CPC), Benzalkonium chloride (BAC), Benzyl chloride (BZT), Dimethyl dioctadecyl ammonium chloride, Dioctadecyl dimethyl ammonium bromide (DODAB), Cocamidopropyl hydroxysulfonate betaine, Cocamidopropyl betaine, Narrow-range ethoxylates, Octadecyl glycol monododecyl ether, Pentaethylene glycol monododecyl ether, Nonxynols, Triton X-100, polyethoxylated tallow amine, cocamidoyl monoethanolamine, cocamidoyl diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, dehydrosorbitan laurate, sorbitan monostearate, dehydrosorbitan tristearate, alkyl polyglucoside, lauryl glucoside, octyl glycoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0045] In some embodiments, the reagent coated on the device includes a reagent that makes the cell membrane or cell nucleus permeable, either by dilution of the osmotic pressure difference between cell membranes during dilution or by adjusting the salt concentration.

[0046] In some embodiments, the reagents coated on the device include agents that crosslink proteins, including but not limited to formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, and potassium permanganate.

[0047] In some embodiments, the dye used for WBC staining is applied to a first plate or a second plate or both.

[0048] In some embodiments, the dyes used to stain WBC and PLT are applied to a first plate or a second plate or both.

[0049] In some embodiments, the dye used to dye the PLT is applied to a first plate or a second plate or both.

[0050] In some embodiments, reagents are coated into an array by droplet printing.

[0051] In some embodiments, the open flow-guiding properties of the structure are used to coat the reagent onto the plate.

[0052] In some embodiments, the reagent is applied by spraying.

[0053] In some embodiments, the reagent is applied by contact printing.

[0054] In some embodiments, the reagent is coated by transfer.

[0055] In some embodiments, the dye used to stain RBCs is applied to a first plate or a second plate or both.

[0056] In some embodiments, a surfactant for separating and rounding RBCs is applied to a first plate or a second plate or both.

[0057] In some embodiments, chemicals that cause RBC pyrolysis are applied to a first plate or a second plate or both.

[0058] In some embodiments, acridine orange is coated onto a first plate or a second plate or both.

[0059] In some embodiments, Zwittergent is coated onto a first plate or a second plate or both.

[0060] In some embodiments, methylene blue and Zwittergent are coated onto a first plate or a second plate or both.

[0061] In some embodiments, acridine orange and Zwittergent are coated onto a first plate or a second plate or both.

[0062] In some embodiments, YOYO dye and Zwittergent are coated onto a first plate or a second plate or both.

[0063] In some embodiments, the device further comprises a plurality of reagent layers on one or two plates, the plurality of reagent layers including anti-adhesion, cell lysis, cell staining, release time control material layers, and combinations thereof;

[0064] In some embodiments, each layer coated on the plate has a thickness of 10 nm, 100 nm, 200 nm, 500 nm, 1 μm, or within a range of any two of these values.

[0065] In some embodiments, the anti-adhesion agent comprises ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetic acid, K2EDTA, or K3EDTA.

[0066] In some embodiments, the cell staining agents include Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue, and azure B), May-grünwald stain, Leishman's solution ("multicolored" methylene blue (i.e., demethylated to azure) and eosin), erythrosine B stain (erythrosine B), and other fluorescent staining agents, including but not limited to acridine orange dye, 3,3-dihexyloxycarbonyl cyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dye, ethidium bromide, leucine sulfaflavin and diaminostilbene disulfonic acid derivatives, erythrosine B or trypan blue, Hoechst 33342, trihydrochloride, trihydrate, DAPI (4',6-bisamidine yellow-2-phenylindole, dihydrochloride), and YOYO.

[0067] In some embodiments, cell staining agents include Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue, and azure B), May-grünwald stain, Leishman's solution ("multicolored" methylene blue (i.e., demethylated azure) and eosin), erythrosine B stain (erythrosine B), and other fluorescent stains, including but not limited to acridine orange, 3,3-dihexyloxycarbonyl cyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dye, ethidium bromide, leucine sulfaflavin and diaminostilbene disulfonic acid derivatives, erythrosine B or trypan blue, Hoechst 33342, trihydrochloride, trihydrate, DAPI (4',6-bisamidine yellow-2-phenylindole, dihydrochloride), YOYO, acid fuchsin, hematoxylin, and Hoechst stain, which includes Hoechst 33258 and Hoechst 33342, Methyl Green, Methylene Blue, Nile Blue, Nile Red, Osmium Tetraoxide, Rhodamine, Safranin, Meosic-AAPV-AMC, CFSE, BCECF / AM, Silver Nitrate, Neutral Red, Pyronine Y, Calcein-AM, Ethidium Dihydro, Xylyl Cyanocyanate FF, Rhodamine 123, 4-Methyl Umbelliferone Palmitate, Fast Blue B Salt, Fluorescein, CH Dipotassium Salt, DAPI Dilactate, Propidium Iodide.

[0068] In some embodiments, the cell lysis agent comprises ammonium chloride, sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), acetic acid, citric acid, or other acids and bases.

[0069] In some embodiments, the release time control material comprises albumin, carbomer, carboxymethyl cellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinylpyrrolidone, or polyvinyl alcohol.

[0070] In some embodiments, a chemical of a certain concentration is coated onto the plate and dissolved into the blood to achieve a uniform distribution of red blood cells in the device.

[0071] In some embodiments, a certain concentration is coated onto a plate and dissolved into blood to lyse red blood cells in the device, wherein the coating may be on a first plate or a second plate, or both.

[0072] In some embodiments, the chemicals coated on the device include, but are not limited to, surfactants, Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tris(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (1a1), 11b, 11c, 11d, CTAC, Tween 20, Tween 40, Tween 60, and Tween 80, Sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium lauryl ether sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctyl sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, CTAB, hexadecylpyridine chloride (CPC), benzalkonium chloride (BAC), benzyl chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysulfonyl betaine, cocamidopropyl betaine, narrow-range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamidoyl monoethanolamine, cocamidoyl diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, dehydrosorbitan laurate, sorbitan monostearate, dehydrosorbitan tristearate, alkyl polyglucoside, lauryl glucoside, octyl glycoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0073] In some embodiments, the agents coated on the device that induce erythrocyte lysis include, but are not limited to, 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, saponins, 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, phenapyridine (marofen), rho immunoglobulin, ribavirin, sulfonamides, and sulfones.

[0074] 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 (comydine), acenaphthylcoumarol, phenylpropargyl coumarin, atromanin, phenylindobenzoate, heparin, fondaparinux sodium, and edaparin, dabigatran etexilate, rivaroxaban, apixaban, edoxaban, betrixaban, NOAC, hirudin, recombinant hirudin, bivalirudin, agratroban, dabigatran etexilate, batroxobin, hirudin, vitamin E, sodium citrate, glucose citrate, oxalates such as fluoride oxalate, deltaparin, disiludin, and enoxaparin.

[0075] In some embodiments, in order to achieve a uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 3 ng / mm², 5 ng / mm², 8 ng / mm², 12 ng / mm², 15 ng / mm², 25 ng / mm², 35 ng / mm², 50 ng / mm², 80 ng / mm², 100 ng / mm², or within any two of the stated values.

[0076] In some embodiments, for red blood cells in a lysis apparatus, Zwittergent is coated on a plate at a preferred area concentration of 100 ng / mm², 120 ng / mm², 150 ng / mm², 180 ng / mm², 200 ng / mm², 300 ng / mm², 400 ng / mm², 500 ng / mm², 800 ng / mm², 1000 ng / mm², or within the range of any two of these values.

[0077] In some embodiments, in order to achieve a uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration 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 within the range of any two of these values ​​in blood.

[0078] In some embodiments, to lyse red blood cells in the device, Zwittergent is coated on a plate at a preferred final concentration in 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 within the range of any two of these values.

[0079] In some embodiments, in order to achieve a uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 3 ng / mm², 5 ng / mm², 8 ng / mm², 12 ng / mm², 15 ng / mm², 25 ng / mm², 35 ng / mm², 50 ng / mm², 80 ng / mm², 100 ng / mm², or within any two of the stated values.

[0080] In some embodiments, for red blood cells in a lysis apparatus, Zwittergent is coated on a plate at a preferred area concentration of 100 ng / mm², 120 ng / mm², 150 ng / mm², 180 ng / mm², 200 ng / mm², 300 ng / mm², 400 ng / mm², 500 ng / mm², 800 ng / mm², 1000 ng / mm², or within the range of any two of the stated values.

[0081] In some embodiments, in order to achieve a uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration 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 within the range of any two of these values ​​in blood.

[0082] In some embodiments, to lyse red blood cells in the device, Zwittergent is coated on a plate at a preferred final concentration in 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 within the range of any two of these values.

[0083] In some embodiments, acridine orange is coated on a plate at an area concentration of 0.5 ng / mm², 1 ng / mm², 2 ng / mm², 3 ng / mm², 5 ng / mm², 8 ng / mm², 10 ng / mm², 15 ng / mm², 20 ng / mm², 30 ng / mm², or within the range of any two of the stated values.

[0084] In some embodiments, acridine orange is coated onto the plate at an area concentration of 3-10 ng / mm², and Zwittergent is coated onto the plate at an area concentration of 3-10 ng / mm².

[0085] Acridine orange was coated onto the plate at an area concentration of 5-20 ng / mm², and Zwittergent was coated onto the plate at an area concentration of 10-30 ng / mm².

[0086] In some embodiments, the label is an immunoassay antibody marker, an RNA marker, or a staining dye, which is coated on the plate at a preferred final concentration 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 within the range of any two of these values.

[0087] In some embodiments, the label is an immunoassay antibody label, an RNA label, or a staining dye, which is coated on the plate at a preferred final concentration of 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL, or within the range of any two of these values.

[0088] In some embodiments, the label is an immunoassay antibody label, an RNA label, or 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 within the range of any two of said values.

[0089] In some embodiments, the label is an immunoassay antibody label, an RNA label, or a staining dye, which is applied to the plate at a final concentration 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 within any two of these values.

[0090] In some embodiments, the label is an immunoassay antibody label, an RNA label, or a staining dye, which is applied to the plate at a final concentration of 0.05 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1.0 mg / mL, or within any two of these values.

[0091] In some embodiments, the label is an immunoassay antibody label, an RNA label, or a staining dye, which is applied to the plate at a final concentration 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 within any two of the stated values.

[0092] In some embodiments, the label is an immunoassay antibody label, an RNA label, or a staining dye, which is coated on the plate at a final concentration of 100 mg / mL, 200 mg / mL, 300 mg / mL, or preferably within a range of any two of the stated values.

[0093] In some embodiments, the thickness of the sample is 1µm, 2µm, 3µm, 5µm, 10µm, 20µm, 30µm, 50µm, 100µm, 150µm, or within the range of any two of the stated values.

[0094] In some embodiments, the preferred thickness of the sample is 2 μm, 3 μm, 5 μm, 10 μm, 30 μm, or within the range of any two of these values.

[0095] 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 within the range of any two of the stated values.

[0096] In some embodiments, the preferred concentration of the probe in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, or within the range of any two of these values.

[0097] In some embodiments, the concentration of the enhancer in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 500 μM, or within the range of any two of the stated values.

[0098] 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 within the range of any two of these values.

[0099] In some embodiments, the optical wavelength of the optical enhancer and the optical wavelength of the probe are in the range of 1 nm, 5 nm, 10 nm, 20 nm, 50 nm, 100 nm, 150 nm, or between any two of these values.

[0100] In some embodiments, the optical wavelengths of the optical enhancer and the probe are preferably within 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, or within the range of any two of these values.

[0101] In some embodiments, the spacer is fixed to the plate by direct impression plate or injection molding plate.

[0102] In some embodiments, the materials for the plates and spacers are selected from polystyrene, PMMA, PC, COC, COP, and other plastics.

[0103] In some embodiments, the spacing between the spacers is in the range of 1 μm to 200 μm.

[0104] In some embodiments, the spacing between the spacers is in the range of 200 μm to 1000 μm.

[0105] In some embodiments, the spacers of the uniform thickness layer have a fill factor of at least 1%, wherein the fill factor is the ratio of the area of ​​the spacers in contact with the uniform thickness layer to the total plate area in contact with the uniform thickness layer.

[0106] In some embodiments, for spacers in a uniform thickness layer, the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 10 MPa, wherein the fill 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.

[0107] In some embodiments, for a flexible plate, the thickness of the flexible plate multiplied by the Young's modulus of the flexible plate is in the range of 60 to 750 GPa-μm.

[0108] In some embodiments, for a flexible plate, the fourth power of the spacer spacing (ISD) divided by the thickness (h) of the flexible plate and the Young's modulus (E) of the flexible plate, ISD4 / (hE), is equal to or less than 106 μm3 / GPa.

[0109] In some embodiments, one or both plates include location markers located on or inside the surface of the plates, the location markers providing information about the position of the plates.

[0110] In some embodiments, one or both plates include scale markings located on or inside the surface of the plates, the scale markings providing information on the lateral dimensions of the sample and / or plate structure.

[0111] In some embodiments, one or two plates include imaging markers located on or inside the surface of the plates, which assist in imaging the sample.

[0112] In some embodiments, the spacer serves as a position marker, scale marker, imaging marker, or any combination thereof.

[0113] In some embodiments, the average thickness of the uniform thickness layer is approximately equal to the minimum size of the analyte in the sample.

[0114] In some embodiments, the spacing between the spacers is in the range of 1 μm to 50 μm.

[0115] In some embodiments, the spacing between the spacers is in the range of 50 μm to 120 μm.

[0116] In some embodiments, the spacing between the spacers is in the range of 120 μm to 200 μm.

[0117] In some embodiments, the spacing between the spacers is substantially fixed.

[0118] In some embodiments, the spacer is a column having a cross-sectional shape selected from a circle, polygon, perfect circle, square, rectangle, oval, ellipse, and any combination thereof.

[0119] In some embodiments, the spacer has a columnar shape and a substantially flat top surface, wherein for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1.

[0120] In some embodiments, for each spacer, the ratio of the spacer's lateral dimension to its height is at least 1.

[0121] In some embodiments, the minimum lateral dimension of the spacer is less than or substantially equal to the minimum dimension of the analyte in the sample.

[0122] In some embodiments, the minimum lateral dimension of the spacer is in the range of 0.5 μm to 100 μm.

[0123] In some embodiments, the minimum lateral dimension of the spacer is in the range of 0.5 μm to 10 μm.

[0124] In some embodiments, the spacers have a density of at least 100 / mm².

[0125] In some embodiments, the spacers have a density of at least 1000 / mm².

[0126] In some embodiments, at least one of the panels is transparent.

[0127] In some embodiments, at least one of the plates is made of a flexible polymer.

[0128] In some embodiments, for pressure on the compression plates, the spacers are incompressible and / or, independently, only one of the plates is flexible.

[0129] In some embodiments, the flexible plate has a thickness in the range of 10 μm to 200 μm.

[0130] In some embodiments, the change is less than 30%.

[0131] In some embodiments, the change is less than 10%.

[0132] In some embodiments, the change is less than 5%.

[0133] In some embodiments, the collection plate and the cover plate are connected and configured to change from an open configuration to a closed configuration by means of a folding plate.

[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.

[0135] In some embodiments, the collection plate and the cover plate are connected by a hinge and configured to change from an open configuration to a closed configuration by folding the plate along the hinge, the hinge being a material separate from the plate.

[0136] In some embodiments, the collecting plate and the cover plate are made of a single sheet of material and are configured to change from an open structure to a closed structure by folding the plate.

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

[0138] In some embodiments, in a closed configuration, the final sample thickness device is configured to analyze the sample in 60 seconds or less.

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

[0141] In some embodiments, the collection plate or cover plate has at least two adjacent analyte measurement sites on its surface, the at least two adjacent analyte measurement sites being not separated by a distance substantially greater than the thickness of the uniform layer when the plate is in the closed position, wherein at least a portion of the uniform layer is above the measurement sites, and wherein the sample has one or more analytes capable of diffusing in the sample.

[0142] In some embodiments, the present invention provides a system for rapidly analyzing steam condensate samples using a mobile phone, comprising: (a) the apparatus of claim 1; and (b) a mobile communication device comprising: i. one or more cameras for detecting signals and / or imaging steam condensate samples; and ii. electronic devices, a signal processor, and hardware and software for receiving and / or processing the detected signals and / or images of the steam condensate samples and for remote communication.

[0143] In some embodiments, the system may further include a light source from a mobile communication device or an external source.

[0144] In some embodiments, one of the plates has a binding site for the analyte, wherein at least a portion of the uniformly thick sample layer is above the binding site and is substantially smaller than the average lateral linear dimension of the binding site.

[0145] In some embodiments, the invention further includes: (d) a housing configured to hold a sample and be mounted to a mobile communication device.

[0146] In some embodiments, the housing includes optics for facilitating imaging and / or signal processing of the sample by the mobile communication device, and a base configured to hold the optics on the mobile communication device.

[0147] In some embodiments, the optical components within the housing are movable relative to the housing.

[0148] In some embodiments, the mobile communication device is configured to transmit test results to medical personnel, medical institutions, or insurance companies.

[0149] In some embodiments, the mobile communication device is also configured to communicate information about the test and the subject with medical personnel, medical facilities, or insurance companies.

[0150] In some embodiments, the mobile communication device is further configured to transmit test information to a cloud network, which is configured to process the information to improve the test results.

[0151] In some embodiments, the mobile communication device is also configured to transmit test and subject information to a cloud network, which is configured to process the information to improve test results, and the improved test results are sent back to the subject.

[0152] In some embodiments, the mobile communication device is configured to receive prescriptions, diagnoses, or advice from medical personnel.

[0153] In some embodiments, the spacers have a predetermined substantially uniform height and a predetermined constant spacer spacing, and at least one of the spacers is located within the sample contact area.

[0154] In some embodiments, 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.

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

[0156] In some embodiments, a detection agent and an optical enhancer are coated on the inner surface of the plates before the sample is deposited between the two plates.

[0157] In some embodiments, the layer thickness of at least a portion of the sample is configured such that the time between the detection agent and the optical enhancer is 30 seconds or less.

[0158] In some embodiments, the layer thickness of at least a portion of the sample is configured such that the time between the detector and the optical enhancer is 60 seconds or less.

[0159] In some embodiments, the layer thickness of at least a portion of the sample is configured such that the time between the detection agent and the optical enhancer is 120 seconds or less.

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

[0161] In some embodiments, the adhesion of the optical enhancer to the target cells is nonspecific.

[0162] In some embodiments, the attachment of the optical enhancer to the target cell is nonspecific, wherein nonspecific attachment is the binding of the target cell's nucleic acid.

[0163] In some embodiments, the thickness of the thin sample layer is configured such that for a given concentration of cells in the sample, each individual cell substantially does not overlap with other cells in the imaging.

[0164] In some embodiments, the device further includes an optical enhancer that is bound to the cell and capable of emitting light at a wavelength that overlaps with or is within 30 nm of the light emitted by the detection probe. Attached Figure Description

[0165] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the invention in any way. Some of the drawings are not to scale. In the drawings that provide experimental data points, the lines connecting the data points are used only to guide the observation of the data and have no other purpose.

[0166] Figure 1 An example of QMAX (Q: Quantization); M: Scale-up; A: Addition of reagent; X: Acceleration; also known as a Compressed 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 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 with the sample located between the two plates.

[0167] Figure 2 An embodiment of the QMAX device is shown, comprising a first plate and a second plate. Figure (a) shows a cross-sectional view of the plate in an open configuration when the plates are separated, with two reagents (1 and 2) coated on the two plates; Figure (b) shows a cross-sectional view of a sample deposited on one of the plates in the open configuration; Figure (c) shows a perspective view of the QMAX device in a closed configuration, with the sample located between the two plates.

[0168] Figure 3 Exemplary effects of using a combination of labeled antibodies and cell enhancers (e.g., nonspecific cell dyes or other specific antibodies) for signal detection of cellular or non-cellular analytes from within a sample are illustrated. Figure (a) shows cells without antibody-labeled enhancers exhibiting signals below the detection threshold; Figure (b) shows cells with antibody-labeled enhancers exhibiting signals above the detection threshold.

[0169] Figure 4Exemplary effects of varying spacer height on signal detection of cells or non-cell analytes within a sample are illustrated. Figure (a) shows antibody-labeled cells at a large spacer height with signal overlaid by a high background from non-cell analytes in the sample; Figure (b) shows antibody-labeled cells at a small spacer height with signal overlaid by a background from non-cell analytes in the sample.

[0170] Figure 5 The effects of different Zwittergent concentrations on leukocyte permeation during rapid FISH using a GAPDH probe in QMAX are shown. Figure (a) shows the GAPDH probe observed using smartphone-based microscopy; 0.625 mg / mL Zwittergent in blood does not produce a FISH signal. Figure (b) shows Zwittergent providing a strong fluorescent signal from GAPDH-labeled leukocytes observed using smartphone-based microscopy. Figure (c) shows fluorescence microscopy (DAPIWBC nuclear repeat staining) used in (c).

[0171] Figure 6 Exemplary photographs of chlamydia staining using the QMAX apparatus without an optional washing step are shown. Figure (a) shows chlamydia-positive cells stained with strong fluorescence; Figure (b) shows chlamydia-negative cells without fluorescence staining. In the experiment, slides of human tissue cells fixed with chlamydia-infected tissue were incubated for 2 min with 1 μL of 40 μg / mL DyLight633-labeled anti-chlamydia antibody. X-ray plates with a column height of 30 μm were used for QMAX immunostaining. Images were taken under a microscope without washing.

[0172] Figure 7 Exemplary photographs are shown illustrating the use of different DNA dyes to distinguish between Gram-positive and Gram-negative bacteria. These three images were taken from different bacterial mixtures and analyzed using the same rapid staining assay. The bacterial mixtures analyzed are: A) a mixture of bacterial species, B) *Escherichia coli* only, and C) *Staphylococcus epidermidis* only.

[0173] Figure 8 An exemplary photograph of CD4T cell counting using a QMAX device, taken with an iPhone-based microscope, is shown. Cells were stained with a detection antibody along with SYTO62 nonspecific nuclear dye. Detailed Implementation

[0174] The following detailed description illustrates some embodiments of the invention by way of example and not limitation. Where applicable, chapter headings and any subtitles used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. The content under chapter headings and / or subheadings is not limited to the chapter headings and / or subheadings, but applies to the entire description of the invention.

[0175] References to any publication refer to its disclosure prior to the filing date and should not be construed as an admission that this claim is exempt from prior rights to such publication due to a prior invention. Furthermore, the provided disclosure date may differ from the actual disclosure date, which may need to be independently determined.

[0176] It should be noted that the accompanying drawings are not intended to show these elements at strict scale. For clarity, some elements are shown enlarged in the drawings. The dimensions of the elements should be described by the description provided herein and incorporated by reference.

[0177] The terms "optical intensifier" and "intensifier" are used interchangeably.

[0178] The terms "cell" and "target cell" are used interchangeably.

[0179] Typically, staining analytes on cells or within cell membranes for imaging is a multi-step process (approximately 10 steps) that usually takes several hours. One aspect of the present invention is the ability to perform immunochemical staining of cells or tissues in less than 60 seconds without rinsing. In some embodiments, the present invention stains and observes intracellular analytes (proteins or nucleic acids) within 60 seconds without rinsing.

[0180] Example of using optical enhancers to enhance the detection of specifically stained cells.

[0181] Figures 1 to 4 Several embodiments of the invention are shown, which rapidly stain cells or tissues without rinsing.

[0182] In some embodiments, a method for enhancing homogeneous detection of analytes in cells of a sample includes:

[0183] (a) A sample 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 cells of the analyte.

[0184] (b) A detection probe that specifically binds to an analyte and is capable of emitting light of a specific wavelength, wherein the detection probe diffuses in the sample;

[0185] (c) It has an optical enhancer that binds to the cell and is capable of emitting light with a wavelength that overlaps with or is within 30 nm of the light emitted by the detection probe;

[0186] (d) The sample, detection probe, and optical enhancer are sandwiched between two sample contact areas of two plates to form a thin layer with a thickness of 200 micrometers (μm) or less; and

[0187] (e) After step (d) and without using any rinsing step, use an imager to image a thin layer in order to detect cells with analytes that bind to the detection probe;

[0188] The thickness of the thin sample layer 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.

[0189] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample are configured in the thin layer in imaging step (e) such that the positions of the detection probes with binding and the positions of the detection probes without binding are distinguishable, wherein the bound detection probes are detection probes of analytes bound to cells.

[0190] In some embodiments, a method for enhancing homogeneous detection of analytes in cells of a sample includes:

[0191] (a) A sample 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 cells of the analyte.

[0192] (b) A detection probe is deposited on at least one sample contact area, the detection probe specifically binds to the analyte and emits light of a certain wavelength, wherein the detection probe diffuses in the sample;

[0193] (c) It has an optical enhancer that binds to the cell and emits light with a wavelength that overlaps with or is within a wavelength range of 30 nm of the light emitted by the detection probe;

[0194] (d) The sample, detection probe, and optical intensifier are sandwiched between two sample contact areas of two plates, wherein the thickness of the thin layer is 150 μm or less; and

[0195] (e) After step (d) and without using any rinsing step, use an imager to perform thin-layer imaging to detect the detection probe that has been specifically bound to the analyte;

[0196] The thickness of the thin sample layer 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.

[0197] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample are configured in the thin layer in imaging step (e) such that the positions of the detection probes with binding and the positions of the detection probes without binding are distinguishable, wherein the bound detection probes are detection probes of analytes bound to cells.

[0198] In some embodiments, a method for enhancing homogeneous detection of analytes in cells of a sample includes:

[0199] (a) A sample 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 cells of the analyte.

[0200] (b) A detection probe is deposited on at least one sample contact area, the detection probe specifically binds to the analyte and emits light of a certain wavelength, wherein the detection probe diffuses in the sample;

[0201] (c) An optical enhancer is deposited on at least one of the sample contact areas, the optical enhancer binds to the cell and emits light that overlaps with or is within a wavelength range of 30 nm of the light emitted by the detection probe, wherein the optical enhancer diffuses in the sample;

[0202] (d) The sample, detection probe, and optical intensifier are sandwiched between two sample contact areas of two plates, wherein the thickness of the thin layer is 150 μm or less; and

[0203] (e) After step (d) and without using any rinsing step, use an imager to perform thin-layer imaging to detect the detection probe that has been specifically bound to the analyte;

[0204] The thickness of the thin sample layer 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.

[0205] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample are configured in the thin layer in imaging step (e) such that the positions of the detection probes with binding and the positions of the detection probes without binding are distinguishable, wherein the bound detection probes are detection probes of analytes bound to cells.

[0206] In some embodiments, a kit for enhancing homogeneous detection of analytes in cells of a sample comprises:

[0207] (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 or suspected of containing the analyte.

[0208] (b) A detection probe that specifically binds to the analyte and is capable of emitting light of a specific wavelength, wherein the detection probe diffuses in the sample;

[0209] (c) An optical enhancer that binds to cells and emits light at a wavelength that overlaps with or is within 30 nm of the light emitted by the detection probe;

[0210] The first and second plates are configured to sandwich the sample, detection probe, and optical intensifier between two sample contact areas to form a thin layer with a thickness of 200 micrometers (μm) or less; and

[0211] The thickness of the thin sample layer 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.

[0212] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample are configured in the thin layer in imaging step (e) such that the positions of the detection probes with binding and the positions of the detection probes without binding are distinguishable, wherein the bound detection probes are detection probes of analytes bound to cells.

[0213] In some embodiments, an apparatus for enhancing homogeneous detection of analytes in cells of a sample includes:

[0214] (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 or suspected of containing the analyte.

[0215] (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 diffuses in the sample;

[0216] (c) An optical enhancer that binds to cells and emits light with a wavelength that overlaps with or is within a wavelength range of 30 nm of the light emitted by the detection probe;

[0217] (d) The sample contact areas in the first and second plates face each other, and the sample, detection probe, and optical intensifier 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

[0218] (e) An imager that enables thin-layer imaging to detect a detection probe that has been specifically bound to the analyte;

[0219] The thickness of the thin sample layer 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.

[0220] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample are configured in the thin layer in imaging step (e) such that the positions of the detection probes with binding and the positions of the detection probes without binding are distinguishable, wherein the bound detection probes are detection probes of analytes bound to cells.

[0221] In some embodiments, an apparatus for enhancing homogeneous detection of analytes in cells of a sample includes:

[0222] (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 or suspected of containing the analyte.

[0223] (b) A detection probe that specifically binds to the analyte and emits light of a specific wavelength, wherein the detection probe is coated on one or two of the sample contact areas and diffuses in the sample;

[0224] (c) An optical enhancer that binds to cells and emits light that overlaps with or is within a wavelength range of 30 nm of the light emitted by the detection probe, wherein the optical enhancer is coated on one or both of the sample contact areas and diffuses in the sample;

[0225] (d) The sample contact areas in the first and second plates face each other, and the sample, detection probe, and optical intensifier 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

[0226] (e) After step (d) and without using any rinsing step, use an imager to perform thin-layer imaging to detect the detection probe that has been specifically bound to the analyte;

[0227] The thickness of the thin sample layer 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.

[0228] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample are configured in the thin layer in imaging step (e) such that the positions of the detection probes with binding and the positions of the detection probes without binding are distinguishable, wherein the bound detection probes are detection probes of analytes bound to cells.

[0229] In some embodiments, a system for enhancing homogeneous detection of analytes in cells of a sample includes:

[0230] (a) the apparatus or kit according to any of the above embodiments; and

[0231] (b) Communication device.

[0232] In some embodiments, a method for rapid homogeneous detection of analytes within cell membranes in a sample includes:

[0233] (a) A sample 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 to contain the analyte within the cell membrane.

[0234] (b) A detection probe that specifically binds to an analyte and is capable of emitting light of a specific wavelength, wherein the detection probe diffuses in the sample;

[0235] (c) It has a permeabilizing agent that allows the cell membrane to pass through the detection probe;

[0236] (d) The sample, detection probe, and permeabilizer are sandwiched between two sample contact areas of two plates to form a thin layer with a thickness of 200 micrometers (μm) or less; and

[0237] (e) After step (d) and without using any rinsing steps, perform thin-layer imaging to detect cells with analytes that bind to the detection probe;

[0238] The thickness of the thin sample layer 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.

[0239] The thickness of the thin layer and the concentration of the detection probe in the sample are configured such that the locations of detection probes containing analytes bound to the cell membrane are distinguishable from locations without cells; and

[0240] The rinsing step is a step of removing unbound detection probes, permeation reagents, or both from the sample contact area.

[0241] In some embodiments, a method for rapid homogeneous detection of analytes within cell membranes in a sample includes:

[0242] (a) A sample 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 cells of the analyte.

[0243] (b) A detection probe is deposited on at least one sample contact area, the detection probe specifically binds to the analyte and emits light of a certain wavelength, wherein the detection probe diffuses in the sample;

[0244] (c) It has a permeabilizing agent that allows the cell membrane to pass through the detection probe;

[0245] (d) The sample, detection probe, and optical intensifier are sandwiched between two sample contact areas of two plates, wherein the thickness of the thin layer is 150 μm or less; and

[0246] (e) After step (d) and without using any rinsing step, use an imager to perform thin-layer imaging to detect the detection probe that has been specifically bound to the analyte;

[0247] The thickness of the thin sample layer 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.

[0248] The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample are configured such that, in imaging step (e), the locations of bound detection probes and non-bound detection probes are distinguishable within the thin layer, wherein the bound detection probes are detection probes for analytes bound to cells; and

[0249] The rinsing step is a step of removing unbound detection probes, permeation reagents, or both from the sample contact area.

[0250] According to the method and apparatus of any of the above embodiments, the optical signal from the optical enhancer is equal to or less than the optical signal from the region of the sample layer that does not contain target cells.

[0251] According to the method and apparatus of any of the above embodiments, a detection agent and an optical enhancer are coated on the inner surface of the plates before the sample is deposited between the two plates.

[0252] According to the method and apparatus of any of the above embodiments, a detection agent and an optical enhancer are coated on the inner surface of the plates before the sample is deposited between the two plates.

[0253] According to the method and apparatus of any of the above embodiments, the layer thickness of at least a portion of the sample is configured such that the time between the detector and the optical enhancer is 30 seconds or less.

[0254] According to the method and apparatus of any of the above embodiments, the layer thickness of at least a portion of the sample is configured such that the time between the detector and the optical enhancer is 60 seconds or less.

[0255] According to the method and apparatus of any of the above embodiments, the layer thickness of at least a portion of the sample is configured such that the time between the detector and the optical enhancer is 120 seconds or less.

[0256] According to the method and apparatus of any of the above embodiments, the attachment of the optical enhancer to the target cells is specific.

[0257] According to the method and apparatus of any of the above embodiments, the attachment of the optical enhancer to the target cells is non-specific.

[0258] In some embodiments, the reagent is coated with a sustained-release layer that releases the reagent (i.e., the staining agent) for at least 3 seconds after the sample comes into contact with the reagent, and in some embodiments for at least 10 seconds or at least 60 seconds.

[0259] Immunostaining

[0260] In some embodiments, immunostaining can be used to detect cellular or non-cellular analytes in a sample. In some embodiments, immunostaining can be used to detect cellular or non-cellular analytes by amplifying the signal from the cellular or non-cellular analyte relative to a background signal. Immunostaining generally refers to any method that uses one or more antibodies to detect cellular 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 may consist of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region may consist of three domains, CH1, CH2, and CH3. Each light chain may consist of a light chain variable region (VL) and a light chain constant region. The light chain constant region may consist of one domain, CL. The VH and VL regions may be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), and interspersed with more conserved regions, called framework regions (FRs). Each VH and VL may consist of three CDRs and four FRs arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The constant region of the antibody mediates 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 (C1q) of the classical complement system. Antibodies may be any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), subclass, or modified form thereof.

[0261] Antibodies can comprise complete immunoglobulins or fragments thereof. An antibody fragment can refer to one or more segments of an antibody that maintain the ability to bind specifically to cellular or non-cellular analytes (e.g., antigens). Furthermore, aggregates, polymers, and conjugates of immunoglobulins or fragments thereof may be used where appropriate, provided that binding affinity to a specific molecule is maintained. Examples of antibody fragments include Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, divalent fragments comprising two Fab fragments linked by disulfide bonds in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of the antibody; single-domain antibody (dAb) fragments consisting of the VH domain; and isolated CDR and single-chain fragments (scFv) where the VL and VH regions pair to form a monovalent molecule (collectively referred to as single-chain Fv (scFv)). Therefore, antibody fragments include Fab, F(ab)2, and Fd2 fragments. b) 2, scFv, Fv, dAb, etc. Although the two VL and VH domains are encoded by separate genes, they can be linked using recombinant methods by artificial peptide linkers that enable them to be made into single protein chains. Such single-chain antibodies comprise one or more antigen-binding moieties. 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 for intact antibodies. Antibodies can be human antibodies, humanized antibodies, chimeric antibodies, isolated antibodies, canine antibodies, cat antibodies, donkey antibodies, sheep antibodies, any plant antibodies, animal antibodies, or mammalian antibodies.

[0262] In any embodiment of this disclosure, any number and / or type of antibody may 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 may be used. For example, as Figure 3 As shown, labeled antibodies can be used to detect cellular analytes within a sample. However, as... Figure 3As shown, two different labeled antibodies can be used to detect cellular or non-cellular analytes (e.g., antigens) in a sample, thereby enhancing the signal detected from the cellular or non-cellular analytes relative to the background signal. In some embodiments, two or more antibodies can be used to detect cellular or non-cellular analytes, and the two or more antibodies can bind to the same molecule. In some embodiments, two or more antibodies can be used to detect cellular or non-cellular analytes, and the two or more antibodies can bind to the same epitope. In some embodiments, two or more antibodies can be used to detect cellular or non-cellular analytes, and the two or more antibodies can bind to different epitopes on the same antigen (e.g., polyclonal antibodies, etc.). In some embodiments, two or more antibodies can be used to detect cellular or non-cellular analytes, and the two or more antibodies can bind to the same epitope on different molecules. In some embodiments, two or more antibodies can be used to detect cellular or non-cellular analytes, and the two or more antibodies can bind to different molecules.

[0263] Any antibodies used in the embodiments of this disclosure may be labeled or unlabeled. A label may refer to a molecule that, when linked to another molecule (directly or indirectly, e.g., via antibody linkage), provides or enhances the means of detecting that other molecule. The signal emitted from the label may allow the detection of the molecule or complex to which it is attached, and / or the label itself. A label may be a class of molecules that elicit a physical or chemical response that can be observed or detected by the naked eye or by instruments such as, but not limited to, cameras (e.g., mobile phone cameras), scintillation counters, colorimeters, UV spectrophotometers, 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 label or tag emits detectable light at a specific wavelength. Radioactive labels or radiolabeled particles are detectable by instruments such as, but not limited to, scintillation counters. Other signal generation and detection methods include chemiluminescence detection, electrochemiluminescence detection, Raman energy detection, colorimetric detection, hybridization protection assays, and mass spectrometry. Non-limiting examples of labels 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 xanthan dyes, 4,7-dichlorofluorescein, 4,7-dichlororhodamine, anthocyanin dyes, enzymes, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, luciferase, chloramphenicol acetyltransferase (CAT), avidin, streptoavidin, biotin, biotinylated protein, or any combination, fragment, or derivative thereof. In any embodiment of this disclosure, one or more labels may be used to detect cellular or non-cellular analytes. In any embodiment of this disclosure, two or more markers may be used to detect cellular or non-cellular analytes. In some embodiments, when two or more markers are used to detect cellular or non-cellular analytes, the excitation and / or emission spectra of the two or more markers may be identical. For example, a sample may be contacted with two antibodies (e.g., each antibody binds to the same epitope on the same antigen), each antibody having a different marker that excites at the same wavelength. In another example, a sample may be contacted with two antibodies (e.g., each antibody binds to a different epitope on the same antigen), each antibody having the same marker that excites at a predetermined wavelength. In some embodiments, when two or more markers are used to detect cellular or non-cellular analytes, the excitation and / or emission spectra of the two or more markers may be different.In some embodiments, when three or more markers are used to detect cellular 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 cellular or non-cellular analytes, the excitation and / or emission spectra of at least two or more markers may be different.

[0264] If the antibody is labeled, detection methods can include fluorescence, luminescence, radioactivity, etc. If the antibody is unlabeled, binding detection can be based on changes in some physical properties of the target analyte. These physical properties can include, for example, refractive index or electrical impedance. Detection of binding to unlabeled antibodies can include, for example, mass spectrometry. In competitive methods, binding site occupancy can be determined indirectly. In this method, target cells or non-cell analytes can be exposed to a solution containing both a labeled antibody and an unlabeled antibody. The labeled homoantibody 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 homoantibody is determined by the reduction in the amount of labeled antibody bound.

[0265] dyeing

[0266] In some embodiments, dye staining can be used to detect cellular or non-cellular analytes within a sample. In some embodiments, dye staining can be used to detect cellular or non-cellular analytes by enhancing the signal from the cellular or non-cellular analyte relative to a background signal. Dye staining can refer to a technique used to enhance contrast in an image and, specifically, to enhance the detectable signal from a target cell or non-cellular analyte in a sample. Staining agents and dyes can be used to highlight structures within biological tissues or cells for observation. Staining agents can be used to identify and examine cell populations (e.g., classifying different blood cells or bacteria, such as Gram-positive and Gram-negative bacteria) or organelles within a single cell. Dye staining can involve contacting a class-specific (e.g., DNA, protein, lipid, or carbohydrate) dye with a sample to qualitatively or quantitatively determine the presence of a specific cellular or non-cellular analyte within the sample.

[0267] In any embodiment of this disclosure, any number and / or type of dye may be used. Specifically, about one dye, about two dyes, about three dyes, about four dyes, about five dyes, about ten dyes, about 25 dyes, or more than about 25 dyes may be used. In some embodiments, two or more dyes may be used to detect cellular or non-cellular analytes, and the two or more dyes may bind to the same molecule. In some embodiments, two or more dyes may be used to detect cellular or non-cellular analytes, and the two or more dyes may stain the same region (e.g., the same organelle) of the same target cell or non-cellular analyte. In some embodiments, two or more dyes may be used to detect cellular or non-cellular analytes, and the two or more dyes may stain different regions (e.g., cytoplasm and nucleus) of the same target cell or non-cellular analyte.

[0268] The signal emitted by the dye allows for the detection of the molecules or complexes to which it is attached. The dye can be a type of molecule that elicits a physical or chemical response that can be observed or detected by the naked eye or by instruments such as, but not limited to, cameras (e.g., mobile phone cameras), scintillation counters, colorimeters, UV spectrophotometers, etc. Dyes may include, but are not limited to, 5-ethynyl-2'-deoxyuridine, 7-aminoactinomycin D, acid fuchsin, acridine orange, acridine yellow, Alcin Blue staining agent, aniline blue WS, aniline yellow, auramine O, Bismarck brown Y, brilliant green (dye), bromodeoxyuridine, Karl Cochlear fluorescent whitening agent, carmine fuchsin, succinimide acetate, carmine, Congo red, Coomassie brilliant blue, crystal violet, DAPI, DiI, DiOC6, eosin, ethidium bromide, ethyl green, fast green FCF, Fulgen staining agent, fluorescein, fluorescein isothiocyanate, fuchsin, gel green, gel red, Gimza staining agent, edible green S, H&E staining agent, hematoxylin, heme, Hoechst staining agent, Janus green B, Jaswant Singh–Bhattacharji (JSB) staining agent, light green SF, Lugol's iodine solution, malachite green, Mallory's trichrome staining agent, methylene blue, methyl violet, methylene blue, neutral red, neomethylene 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, staining agent, Sudan Black B, Sudan III, Sudan IV, Sudan Red B, SYBR Green I, SYBR Safe, SYTOX, template: staining agent, Texas red, toluidine blue staining agent, trypan blue, User: Kyle MoJo / sandbox, Victoria Blue BO, Aqua Blue, Wayson stain and Ziehl-Neelsen stain.

[0269] In some embodiments, the selected dye is a metachromatic dye. The term "metachromatic dye" can refer to a fluorescent dye that contains two or more peaks in its emission spectrum when bound to a cell or cellular component. Metachromatic dyes can fluoresce at different wavelengths when bound to different types of cells or molecules, such as RNA, DNA, or other cellular components. For example, when bound to double-stranded DNA, single-stranded DNA, or single-stranded RNA, the metachromatic dyes used in any embodiment of this disclosure fluoresce at different wavelengths.

[0270] Various heterochromatic dyes are known in the art, including but not limited to xanthan dyes, carbonyl cyan dyes, polymethazine dyes (including Astra Violet FR), thiofalvine T, pseudocyanine, oxacarbocyanine dyes, acridine dyes, azine dyes, diphenylmethane dyes, methylene dyes, azine dyes, cyan dyes, styryl dyes, nonylacridine orange dye (3,6-di(dimethylamino)-10-nonylacridine bromide), molecular probes, Eugene, Oreg., rhodamine, acridine red dyes, toluidine blue dye (2-amino-7-dimethylamino-3-methylphenthiazine chloride), hydrosystilbamidine, and anthocyanin dyes, including SYTO dyes, TOTO dyes, YOYO dyes, BOBO dyes, and combinations or derivatives thereof.

[0271] In some embodiments, the dye may be a non-heterochromic dye. The term "non-heterochromic dye" can refer to a fluorescent dye that provides single-wavelength excitation when irradiated at a predetermined wavelength. Such dyes can be used in methods for distinguishing multiple cell types, or in situations where a second fluorescent dye or antibody with a heterochromatic wavelength that may interfere with sample analysis may be present. Such dyes can be used to stain cellular components of cells, including eosinophilic granules, basophilic granules, and cell membranes. Non-limiting examples of non-heterogeneous 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'-dimethyloxacarbonyl cyanine), pyronin Y dye, methylene blue dye (3-bis(dimethylamino)-phenothiazine-5-enhalpyr), auramine O dye (4,4'-bis(N,N-dimethylaniline) monohydrochloride), LDS 751 dye (quinolineon, 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-phenanthidium iodide, hexidium Iodide (hexanediol iodide), ethidium dihydrogenate, ethidium monoazide, thiazole orange dye, and combinations thereof and their derivatives.

[0272] 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 (1a1), 11b, 11c, 11d, CTAC, Tween 20, Tween 40, and Tween 60, Tween80, Sodium lauryl sulfate (SLS), Ammonium lauryl sulfate, CTAB, Sodium lauryl ether sulfate (SLES), Sodium myristyl ether sulfate, Docusate, Perfluorooctyl sulfonate, Alkyl-aryl ether phosphate, Alkyl ether phosphate, CTAB, Hexadecylpyridine chloride (CPC), Benzalkonium chloride (BAC), Benzyl chloride (BZT), Dimethyl dioctadecyl ammonium chloride, Dioctadecyl dimethyl ammonium bromide (DODAB), Cocamidopropyl hydroxysulfonate betaine, Cocamidopropyl betaine, Narrow-range ethoxylates, Octadecyl glycol monododecyl ether, Pentaethylene glycol monododecyl ether, Nonxynols, Triton X-100, polyethoxylated tallow amine, cocamidoyl monoethanolamine, cocamidoyl diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, dehydrosorbitan laurate, sorbitan monostearate, dehydrosorbitan tristearate, alkyl polyglucoside, lauryl glucoside, octyl glycoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0273] In some embodiments, the reagent coated on the device includes a reagent that makes the cell membrane or cell nucleus permeable, either by dilution of the osmotic pressure difference between cell membranes during dilution or by adjusting the salt concentration.

[0274] In some embodiments, the reagents coated on the device include agents that crosslink proteins, including but not limited to formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, and potassium permanganate.

[0275] In any embodiment of this disclosure, Gram-positive and Gram-negative staining agents, 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 lectin can specifically stain Gram-positive bacteria. Hexanediol iodide can specifically stain Gram-positive bacteria. Crystal violet and iodine stain both Gram-positive and Gram-negative bacteria. Destaining is performed using ethanol and acetone. Safranin / carbohydrate fuchsin counterstaining is also used.

[0276] Any dye used in the embodiments of this disclosure may be a cell-permeable dye or a cell-impermeable dye. The term "cell-permeable agent" can refer to a dye that readily penetrates the cell wall and stains its components without requiring the presence of an additional permeabilizing agent. Typically, cell-permeable dyes are used to stain live cells or unlystomped cell components. In some embodiments, cell-permeable dyes are used to analyze samples containing whole blood. In some embodiments of this disclosure, if a cell-impermeable dye is used, a cell permeabilizing agent may be used to improve cell permeability to the dye.

[0277] In some embodiments, the dye may be used in combination with labeled antibodies. For example, such as Figure 3 As shown, labeled antibodies can be used to detect cellular analytes within a sample. However, as... Figure 3 As shown, a combination of labeled antibodies and cellular dyes can be used to detect cellular or non-cellular analytes (e.g., antigens) in a sample, thereby enhancing the signal detected from the cellular or non-cellular analyte relative to a background signal. Simple staining can refer to staining with only one staining agent / dye. Various multiple staining methods exist, many of which are examples of counterstaining, differential staining, or both, including double staining and triple staining. In any embodiment of this disclosure, one or more dyes can be used to detect cellular or non-cellular analytes. In any embodiment of this disclosure, two or more dyes can be used to detect cellular or non-cellular analytes. In some embodiments, when two or more dyes are used to detect cellular or non-cellular analytes, the excitation and / or emission spectra of the two or more dyes may be identical. For example, a sample may be contacted with two antibodies (e.g., each antibody binds to the same epitope on the same antigen), each antibody having a different dye that excites at the same wavelength. In another example, a sample may be contacted with two antibodies (e.g., each antibody binds to a different epitope on the same antigen), each antibody having the same dye that excites at a predetermined wavelength. In some embodiments, when two or more dyes are used to detect cellular 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 cellular 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 cellular or non-cellular analytes, the excitation and / or emission spectra of at least two or more dyes may be different.

[0278] In any of the embodiments disclosed herein, labeled antibodies, staining agents, and / or dyes may be coated onto one or more surfaces of a first plate and / or a second plate. Coating labeled antibodies, staining agents, and / or dyes onto one or more surfaces of a first or second plate is intended to reduce the number of steps a user must perform to analyze a sample. For example, if the first plate is coated with a cell staining reagent, the user will simply need to deposit the sample on or within the QMAX card (e.g., in the sample contact area). Upon contact with the cell staining reagent, cells within the sample can be automatically (e.g., without user intervention). This reduction in the number of steps performed by the user can reduce errors (e.g., human error), increase the accuracy of data analysis, and reduce the amount of time required to analyze a sample. In some embodiments, the staining agent or dye may be coated onto one or more surfaces of the first plate, and the labeled antibody may be coated onto one or more surfaces of the second plate. In another embodiment, the labeled antibody and staining agent or dye may be coated onto one or more surfaces of both the first and second plates.

[0279] It is also anticipated that blocking agents competing with staining agents or dyes can be used to enhance signals from target cells or non-cell analytes. For example, a blocking agent can be used to bind to unbound antibodies, thereby quenching reporter molecules on the antibody and reducing background signals that might otherwise be generated by unbound antibodies. Blocking agents can block non-specific interactions between dyes and cells or cellular components. Blocking agents can compete with other dyes present at binding sites on the cells or non-cell analytes to be analyzed. Blocking agents can compete with low-affinity dyes for specific binding sites on cells or non-cell analytes. The blocking agent itself can be a dye; in this case, the dye is selected such that it can be detected differently from dyes or staining agents that bind to target cells or non-cell analytes. Alternatively, a blocking agent can be any compound that blocks non-specific interactions of dyes or staining agents without affecting the specific binding of the dye or staining agent to its target.

[0280] The blocking agent is non-fluorescent, or selected from dyes that fluoresce at a wavelength different from the wavelength of the dye used to detect target cells or non-cellular analytes. The blocking agent may be non-fluorescent at the wavelength that activates the fluorescence of the dye or staining agent. Many suitable blocking agents are known in the art and can be readily used in the embodiments of this disclosure, including, but not limited to, bisbenzamide (N,N'-(dithiodi-2,1-phenyl)-bisbenzamide), Hoechst™ 33258 dye (bisbenzamide, 2'-p-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5-bis-1H-benzimidazole trihydrochloride pentahydrate, Hoechst 34580 dye, Hoechst... 33342 dyes (bisbenzoimide, 2'-(ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5-bi-1H-benzimidazole trihydrochloride trihydrate), 4',6-bisamidine-2-phenylindole dihydrochloride (DAPI), 4',6-bis-[2-imidazoline-4H,5H]-2-phenyl-indole (DIPI), Eosin Y dye, Lichen Red dye, Fluorescent Pink B dye (2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein disodium salt), Hexanone theobromine dye, Quinacrine dye (6-chloro-9-(4-diethyl-1)-methylbutylamino)-2-methoxyacridine dihydrochloride), combinations thereof, and their derivatives.

[0281] RNA fluorescence in situ hybridization

[0282] RNA fluorescence in situ hybridization (RNA-FISH) is a molecular cytogenetics technique used to detect and locate specific RNA-FigA targets (mRNA, lncRNA, and miRNA) in single cells using fluorescence microscopy. Traditional RNA-FISH methods typically involve multiple steps, such as fixation, permeabilization, hybridization, and imaging. Despite the wide range of medical applications of FISH, the complexity of the technique limits its potential in rapid diagnostics. Therefore, there is a need to develop rapid, accurate, portable, and / or inexpensive RNA-FISH assays that can be performed by non-specialists.

[0283] 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 amplifying the signal from the cellular or non-cellular analyte relative to a background signal. FISH can generally refer to any method that uses one or more probes to detect cellular or non-cellular analytes within a sample, including DNA probes that detect and locate the presence or absence of a specific DNA sequence on a chromosome and RNA probes that detect or locate specific RNA targets (mRNA, lncRNA, and miRNA). Therefore, FISH is commonly used to discover specific features in DNA for use in 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 chain (typically 10-25 nucleotides) of DNA or RNA complementary to a given target sequence typically used for target localization. In some embodiments, it can be used... RNA FISH probes are RNA visualization methods that allow for the simultaneous detection, localization, and quantification of individual mRNA molecules.

[0284] In one embodiment, the surfactant used to make the probe accessible includes, but is not limited to, surfactants such as Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tris(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (1a1a), 11b, 11c, 11d, CTAC, Tween 20, Tween 40, and Tween 60, Tween80, Sodium lauryl sulfate (SLS), Ammonium lauryl sulfate, CTAB, Sodium lauryl ether sulfate (SLES), Sodium myristyl ether sulfate, Docusate, Perfluorooctyl sulfonate, Alkyl-aryl ether phosphate, Alkyl ether phosphate, CTAB, Hexadecylpyridine chloride (CPC), Benzalkonium chloride (BAC), Benzyl chloride (BZT), Dimethyl dioctadecyl ammonium chloride, Dioctadecyl dimethyl ammonium bromide (DODAB), Cocamidopropyl hydroxysulfonate betaine, Cocamidopropyl betaine, Narrow-range ethoxylates, Octadecyl glycol monododecyl ether, Pentaethylene glycol monododecyl ether, Nonxynols, Triton X-100, polyethoxylated tallow amine, cocamidoyl monoethanolamine, cocamidoyl diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, dehydrosorbitan laurate, sorbitan monostearate, dehydrosorbitan tristearate, alkyl polyglucoside, lauryl glucoside, octyl glycoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0285] In some embodiments, the agents coated on the device that induce erythrocyte lysis include, but are not limited to, 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, saponins, 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, phenapyridine (marofen), rho immunoglobulin, ribavirin, sulfonamides, and sulfones.

[0286] Any RNA / DNA probe used in the embodiments of this 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 of detecting that other 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 class of molecules that elicit a physical or chemical response that can be observed or detected by the naked eye or by instruments such as, but not limited to, cameras (e.g., mobile phone cameras), scintillation counters, colorimeters, UV spectrophotometers, 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 label or tag emits detectable light at a specific wavelength. Radioactive labels or radiolabeled particles are detectable by instruments such as, but not limited to, scintillation counters. Other signal generation and detection methods include chemiluminescence detection, electrochemiluminescence detection, Raman energy detection, colorimetric detection, hybridization protection assays, and mass spectrometry. Non-limiting examples of labels 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 xanthan dyes, 4,7-dichlorofluorescein, 4,7-dichlororhodamine, anthocyanin dyes, enzymes, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, luciferase, chloramphenicol acetyltransferase (CAT), avidin, streptoavidin, biotin, biotinylated protein, or any combination, fragment, or derivative thereof. In any embodiment of this disclosure, one or more labels may be used to detect cellular or non-cellular analytes. In any embodiment of this disclosure, two or more markers may be used to detect cellular or non-cellular analytes. In some embodiments, when two or more markers are used to detect cellular or non-cellular analytes, the excitation and / or emission spectra of the two or more markers may be identical. For example, a sample may be contacted with two antibodies (e.g., each antibody binds to the same epitope on the same antigen), each antibody having a different marker that excites at the same wavelength. In another example, a sample may be contacted with two antibodies (e.g., each antibody binds to a different epitope on the same antigen), each antibody having the same marker that excites at a predetermined wavelength. In some embodiments, when two or more markers are used to detect cellular or non-cellular analytes, the excitation and / or emission spectra of the two or more markers may be different.In some embodiments, when three or more markers are used to detect cellular 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 cellular or non-cellular analytes, the excitation and / or emission spectra of at least two or more markers may be different.

[0287] protocol

[0288] 1. Design, synthesis, and purification of target RNA fluorescent probes.

[0289] 2. Use Biodot to print probes (1 nM) and surfactants (e.g., Zwittergent) onto the X-plate.

[0290] 3. Apply 1 drop of blood to the substrate, close the wafer, press, and incubate at room temperature for 1 minute.

[0291] 4. Insert the chip into the device, take a picture, and analyze the data.

[0292] Spacer height & sample thickness

[0293] In some embodiments of this disclosure, the height of the spacer can be varied to adjust the thickness of the sample to be analyzed, thereby enhancing the detection of signals from target cells or non-cellular analytes. For example, as Figure 4 As shown, relative to the background signal, the signal detected from cellular or non-cellular analytes can be obtained from a larger thickness ( Figure 4 (a)) to smaller thicknesses ( Figure 4(b) Enhancement by varying the sample thickness. In some embodiments, the spacer height and / or thickness of the sample can be up to about 500 micrometers, up to about 250 micrometers, up to about 100 micrometers, up to about 90 micrometers, up to about 80 micrometers, up to about 70 micrometers, up to about 60 micrometers, up to about 50 micrometers, up to about 40 micrometers, up to about 30 micrometers, up to about 25 micrometers, up to about 20 micrometers, up to about 15 micrometers, up to about 10 micrometers, up to about 5 micrometers, up to about 4 micrometers, up to about 3 micrometers, up to about 2 micrometers, up to about 1 micrometer, up to about 0.5 micrometers, up to about 0.1 micrometers, or up to about 0.05 micrometers. For example, the spacer height can be up to about 30 micrometers. In some embodiments, the spacer height and / or sample thickness can be from about 25 micrometers to about 35 micrometers. In some embodiments, the spacer height and / or sample thickness can be from about 20 micrometers to about 40 micrometers. In some embodiments, the spacer height and / or sample thickness may be from about 10 micrometers to about 30 micrometers. In some embodiments, the spacer height and / or sample thickness may be from about 30 micrometers to about 50 micrometers. In some embodiments, the spacer height and / or sample thickness may be from about 50 micrometers to about 75 micrometers. In some embodiments, the spacer height and / or sample thickness may be from about 1 micrometer to about 25 micrometers. For example, the spacer height and / or sample thickness may be from about 25 micrometers to about 35 micrometers. In some embodiments, the spacer height and / or thickness of the sample may be determined based on the size or shape of the target cells or non-cellular analytes. For example, lymphocytes in suspension may have an average diameter of about 15 micrometers. Therefore, in some embodiments, the spacer height and / or thickness of the sample may be about 15 micrometers, about 20 micrometers, about 25 micrometers, about 30 micrometers, or greater than about 30 micrometers.

[0294] Examples of the present invention

[0295] A1. A method for analyzing a sample, the method comprising:

[0296] (a) Obtaining an apparatus according to an embodiment disclosed herein, the apparatus 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 micrometers;

[0297] (b) Contact 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;

[0298] (c) When the plates are configured in an open configuration, the product is deposited on one or both of the plates, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers;

[0299] (d) After (c), the two plates are forced 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 contact surface of the plates and adjusted by the plates and spacers.

[0300] A2. A method for analyzing a sample, the method comprising:

[0301] (a) Obtaining an apparatus according to an embodiment disclosed herein, the apparatus 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 micrometers, 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.

[0302] (b) When the plates are configured in an open configuration, a sample is deposited on one or both of the plates, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers; 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 target cells within the sample.

[0303] (d) Following (c), the two plates are forced 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 contact surface of the plates and adjusted by the plates and spacers.

[0304] A3. The method according to any of the above embodiments, wherein the spacer has a predetermined substantially uniform height equal to or less than about 100 micrometers, equal to or less than about 75 micrometers, equal to or less than about 50 micrometers, equal to or less than about 30 micrometers, equal to or less than about 25 micrometers, equal to or less than about 10 micrometers, equal to or less than about 5 micrometers, or equal to or less than about 1 micrometer.

[0305] A4. The method according to any of the above embodiments, wherein a predetermined substantially uniform height of the spacer is determined based on the size or shape of the target cell.

[0306] A5. The method according to any of the above embodiments, wherein two or more labeled antibodies bind to different epitopes on target cell molecules.

[0307] A6. The method according to any of the above embodiments, wherein two or more labeled antibodies bind to the same epitope.

[0308] A7. The method according to any of the above embodiments, wherein two or more labeled antibodies bind to different molecules on the target cells.

[0309] A7.1 The method according to any of the above embodiments, wherein the label conjugated with two or more labeled antibodies can be excited at the same wavelength.

[0310] A8. A method for analyzing a sample, the method comprising:

[0311] (a) Obtaining an apparatus according to an embodiment disclosed herein, the apparatus 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 micrometers, wherein the first plate comprises a surface coated with a first antibody and the second plate comprises a surface coated with a second antibody.

[0312] (b) When the plate is configured in an open configuration, a sample is deposited on one or both of the plates, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers, wherein when the sample is deposited on one or both plates, the sample is in contact 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 target cells within the sample.

[0313] (d) Following (c), the two plates are forced 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 contact surface of the plates and adjusted by the plates and spacers.

[0314] A9. A method for analyzing a sample, the method comprising:

[0315] (a) Obtaining an apparatus according to an embodiment disclosed herein, the apparatus 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 micrometers;

[0316] (b) Contacting the sample with two or more dyes to obtain a mixture, wherein the two or more labeled dyes are able to bind to target cells within the sample;

[0317] (c) When the plates are configured in an open configuration, the product is deposited on one or both of the plates, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers;

[0318] (d) After (c), the two plates are forced 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 contact surface of the plates and adjusted by the plates and spacers.

[0319] A10. A method for analyzing a sample, the method comprising:

[0320] (a) Obtaining an apparatus according to an embodiment disclosed herein, the apparatus 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 micrometers, 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.

[0321] (b) When the plates are configured in an open configuration, a sample is deposited on one or both of the plates, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers; wherein when the sample is deposited on one or both plates, the sample is in contact with two or more dyes, wherein the two or more dyes are capable of binding to target cells within the sample;

[0322] (d) Following (c), the two plates are forced 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 contact surface of the plates and adjusted by the plates and spacers.

[0323] A11. The method according to any of the above embodiments, wherein the spacer has a predetermined substantially uniform height equal to or less than about 100 micrometers, equal to or less than about 75 micrometers, equal to or less than about 50 micrometers, equal to or less than about 30 micrometers, equal to or less than about 25 micrometers, equal to or less than about 10 micrometers, equal to or less than about 5 micrometers, or equal to or less than about 1 micrometer.

[0324] A12. The method according to any of the above embodiments, wherein a predetermined substantially uniform height of the spacer is determined based on the size or shape of the target cell.

[0325] A13. The method according to any of the above embodiments, wherein two or more dyes nonspecifically bind to target cells.

[0326] A14. The method according to any of the above embodiments, wherein two or more dyes are capable of being excited at the same wavelength.

[0327] A15. A method for analyzing a sample, the method comprising:

[0328] (d) Obtaining an apparatus according to an embodiment disclosed herein, the apparatus 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 micrometers, wherein the first plate comprises a surface coated with an antibody and the second plate comprises a surface coated with a dye.

[0329] (e) When the plate is configured in an open configuration, the sample is deposited on one or both of the plates, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers; wherein when the sample is deposited on one or both plates, the sample is in contact with antibodies and dyes, wherein the antibodies and dyes are capable of binding to target cells within the sample.

[0330] (f) Following (b), the two plates are forced 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 contact surface of the plates and adjusted by the plates and spacers.

[0331] A16. A method for analyzing a sample, the method comprising:

[0332] (e) Obtaining an apparatus according to an embodiment disclosed herein, the apparatus 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 micrometers;

[0333] (f) Contact the sample with labeled antibodies and dyes to obtain a mixture, wherein the labeled antibodies and dyes are capable of binding to target cells within the sample;

[0334] (c) When the plates are in an open configuration, the mixture is deposited on one or both of the plates, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers;

[0335] (h) After (c), the two plates are forced 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 contact surface of the plates and adjusted by the plates and spacers.

[0336] A17. A method for analyzing a sample, the method comprising:

[0337] (d) Obtaining an apparatus according to an embodiment disclosed herein, the apparatus 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 micrometers, and wherein at least one of the first plate, the second plate and the spacer comprises at least one surface coated with labeled antibodies and dyes.

[0338] (e) When the plates are configured in an open configuration, a sample is deposited on one or both of the plates, wherein in the open configuration, the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers; wherein when the sample is deposited on one or both plates, the sample is in contact with labeled antibodies and dyes, wherein the labeled antibodies and dyes are capable of binding to target cells within the sample;

[0339] (f) Following (b), the two plates are forced 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 contact surface of the plates and adjusted by the plates and spacers.

[0340] A18. The method according to any of the above embodiments, wherein the spacer has a predetermined substantially uniform height equal to or less than about 100 micrometers, equal to or less than about 75 micrometers, equal to or less than about 50 micrometers, equal to or less than about 30 micrometers, equal to or less than about 25 micrometers, equal to or less than about 10 micrometers, equal to or less than about 5 micrometers, or equal to or less than about 1 micrometer.

[0341] A19. The method according to any of the above embodiments, wherein a predetermined substantially uniform height of the spacer is determined based on the size or shape of the target cell.

[0342] A20. The method according to any of the above embodiments, wherein the dye nonspecifically binds to the target cells.

[0343] A21. The method according to any of the above embodiments, wherein the labeled antibody and the label on the dye are excited at the same wavelength.

[0344] In one embodiment, the reagent and additive are coated on the same plate (first plate or second plate) or on separate plates (first plate and second plate).

[0345] In one embodiment, the additive is a chemical that has a physical, chemical, or physiological effect on target cells. Examples are surfactants (as opposed to other temporary substances) and ions (as opposed to other temporary substances).

[0346] In one embodiment, the staining reagent is a labeled antibody, peptide, oligonucleotide, aptamer, small molecule, or any other substance having a binding affinity for one or more specific components inside or outside the target cell.

[0347] In one embodiment, the detection of the labeled staining reagent is based on fluorescence (referring to other temporary substances), chemiluminescence (referring to other temporary substances), colorimetry (referring to other temporary substances), or plasma (referring to other temporary substances).

[0348] In one embodiment, the target cell is a prokaryote, such as bacteria and archaea, or a eukaryote, such as animal and plant cells. Common examples include mammalian cells, yeast, and algae.

[0349] In one embodiment, the number of target cells in the sample may be one or more.

[0350] 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 (1a1), 11b, 11c, 11d, CTAC, Tween 20, Tween 40, and Tween 60, Tween80, Sodium lauryl sulfate (SLS), Ammonium lauryl sulfate, CTAB, Sodium lauryl ether sulfate (SLES), Sodium myristyl ether sulfate, Docusate, Perfluorooctyl sulfonate, Alkyl-aryl ether phosphate, Alkyl ether phosphate, CTAB, Hexadecylpyridine chloride (CPC), Benzalkonium chloride (BAC), Benzyl chloride (BZT), Dimethyl dioctadecyl ammonium chloride, Dioctadecyl dimethyl ammonium bromide (DODAB), Cocamidopropyl hydroxysulfonate betaine, Cocamidopropyl betaine, Narrow-range ethoxylates, Octadecyl glycol monododecyl ether, Pentaethylene glycol monododecyl ether, Nonxynols, Triton X-100, polyethoxylated tallow amine, cocamidoyl monoethanolamine, cocamidoyl diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, dehydrosorbitan laurate, sorbitan monostearate, dehydrosorbitan tristearate, alkyl polyglucoside, lauryl glucoside, octyl glycoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0351] In some embodiments, the reagent coated on the device includes a reagent that makes the cell membrane or cell nucleus permeable, either by dilution of the osmotic pressure difference between cell membranes during dilution or by adjusting the salt concentration.

[0352] In some embodiments, the reagents coated on the device include agents that crosslink proteins, including but not limited to formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, and potassium permanganate.

[0353] According to any of the above embodiments, the apparatus, kit, system, or method wherein the dye for WBC staining is applied to a first plate, a second plate, or both.

[0354] According to any of the above embodiments, the apparatus, kit, system, or method wherein a dye for staining WBC and PLT is applied to a first plate or a second plate or both.

[0355] According to any of the above embodiments, the apparatus, kit, system, or method involves coating a PLT-stained dye onto a first plate, a second plate, or both.

[0356] According to any of the above embodiments, the apparatus, kit, system, or method wherein reagents are coated into an array by droplet printing.

[0357] The method, apparatus, system, and kit according to any of the above embodiments, wherein the reagent is coated on the plate by utilizing the open guided flow properties of the structure.

[0358] The apparatus, kit, system, or method according to any of the above embodiments, wherein the reagent is coated by spraying.

[0359] The apparatus, kit, system, or method according to any of the above embodiments, wherein the reagent is coated by contact printing.

[0360] In the apparatus, reagent kit, system, or method described in any of the above embodiments, the reagent is coated by transfer.

[0361] According to any of the above embodiments, the apparatus, kit, system, or method wherein the dye for RBC staining is applied to a first plate or a second plate or both.

[0362] According to any of the above embodiments, the apparatus, kit, system, or method wherein a surfactant used for separating and circulating RBCs is coated onto a first plate or a second plate or both.

[0363] According to any of the above embodiments, the apparatus, kit, system, or method involves coating a first plate, a second plate, or both with chemicals that cause RBC lysis.

[0364] According to any of the above embodiments, the apparatus, kit, system, or method involves coating acridine orange onto a first plate, a second plate, or both.

[0365] According to any of the above embodiments, the apparatus, kit, system, or method wherein Zwittergent is coated onto a first plate, a second plate, or both.

[0366] According to any of the above embodiments, the apparatus, kit, system, or method wherein methylene blue and Zwittergent are coated onto a first plate, a second plate, or both.

[0367] According to any of the above embodiments, the apparatus, kit, system, or method involves coating acridine orange and Zwittergent onto a first plate, a second plate, or both.

[0368] According to the apparatus, kit, system, or method described in any of the above embodiments, YOYO dye and Zwittergent are coated onto a first plate or a second plate or both.

[0369] The method and apparatus according to any of the above embodiments, wherein the apparatus further comprises a plurality of reagent layers on one or two plates, the plurality of reagent layers including anti-adhesion, cell lysis, cell staining, release time control material layers, and combinations thereof;

[0370] The thickness of each layer coated on the plate is 10 nm, 100 nm, 200 nm, 500 nm, 1 μm, or within any two of the stated values.

[0371] The anti-adhesion agents include ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetic acid, K2EDTA, K3EDTA, etc.

[0372] The cell staining agents include Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue, and azure B), May-grünwald stain, Leishman's solution ("multicolored" methylene blue (i.e., demethylated to azure) and eosin), erythrosine B stain (erythrosine B), and other fluorescent staining agents, including but not limited to acridine orange dye, 3,3-dihexyloxycarbonyl cyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dye, ethidium bromide, leucine sulfaflavin and diaminostilbene disulfonic acid derivatives, erythrosine B or trypan blue, Hoechst 33342, trihydrochloride, trihydrate, DAPI (4,6-bisamidine yellow-2-phenylindole, dihydrochloride), and YOYO;

[0373] The cell staining agents include Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue, and azure B), May-grünwald stain, Leishman's solution ("multicolored" methylene blue (i.e., demethylated azure) and eosin), erythrosine B stain (erythrosine B), and other fluorescent stains, including but not limited to acridine orange, 3,3-dihexyloxycarbonyl cyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dye, ethidium bromide, leucine sulfaflavin and diaminostilbene disulfonic acid derivatives, erythrosine B or trypan blue, Hoechst 33342, trihydrochloride, trihydrate, DAPI (4,6-bisamidine yellow-2-phenylindole, dihydrochloride), YOYO, acid fuchsin, hematoxylin, and Hoechst stain, which includes Hoechst... 33258 and Hoechst 33342, Methyl Green, Methylene Blue, Nile Blue, Nile Red, Osmium Tetraoxide, Rhodamine, Safranin, Meosic-AAPV-AMC, CFSE, BCECF / AM, Silver Nitrate, Neutral Red, Pyronine Y, Calcein-AM, Ethidium Dihydro, Xylyl Cyanocyanate FF, Rhodamine 123, 4-Methyl Umbelliferone Palmitate, Fast Blue B Salt, Fluorescein, CH Dipotassium Salt, DAPI Dilactate, Propyridine Iodide;

[0374] Cell lysis agents include ammonium chloride, sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), acetic acid, citric acid, and other acids and bases;

[0375] The release time control materials include albumin, carbomer, carboxymethyl cellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinylpyrrolidone, or polyvinyl alcohol, etc.

[0376] In some embodiments, a chemical of a certain concentration is coated onto the plate and dissolved into the blood to achieve a uniform distribution of red blood cells in the device.

[0377] In some embodiments, a chemical of a certain concentration is coated onto a plate and dissolved in blood to lyse red blood cells in the device, wherein the coating may be on a first plate or a second plate, or both.

[0378] In one embodiment, the chemicals coated on the device include, but are not limited to, surfactants, Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tris(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (1a1a), 11b, 11c, 11d, CTAC, Tween 20, Tween 40, Tween 60, and Tween 80, Sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium lauryl ether sulfate (SLES), sodium myristyl ether sulfate, docusate, perfluorooctyl sulfonate, alkyl-aryl ether phosphate, alkyl ether phosphate, CTAB, hexadecylpyridine chloride (CPC), benzalkonium chloride (BAC), benzyl chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysulfonyl betaine, cocamidopropyl betaine, narrow-range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamidoyl monoethanolamine, cocamidoyl diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, dehydrosorbitan laurate, sorbitan monostearate, dehydrosorbitan tristearate, alkyl polyglucoside, lauryl glucoside, octyl glycoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0379] In some embodiments, the agents coated on the device that induce erythrocyte lysis include, but are not limited to, 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, saponins, 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, phenapyridine (marofen), rho immunoglobulin, ribavirin, sulfonamides, and sulfones.

[0380] 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 (comydine), acenaphthylcoumarol, phenylpropargyl coumarin, atromanin, phenylindobenzoate, heparin, fondaparinux sodium, and edaparin, dabigatran etexilate, rivaroxaban, apixaban, edoxaban, betrixaban, NOAC, hirudin, recombinant hirudin, bivalirudin, agratroban, dabigatran etexilate, batroxobin, hirudin, vitamin E, sodium citrate, glucose citrate, oxalates such as fluoride oxalate, deltaparin, disiludin, and enoxaparin.

[0381] In some embodiments, in order to achieve a uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 3 ng / mm², 5 ng / mm², 8 ng / mm², 12 ng / mm², 15 ng / mm², 25 ng / mm², 35 ng / mm², 50 ng / mm², 80 ng / mm², or within any two of the stated values.

[0382] In some embodiments, for red blood cells in a lysis apparatus, Zwittergent is coated on a plate at a preferred area concentration of 100 ng / mm², 120 ng / mm², 150 ng / mm², 180 ng / mm², 200 ng / mm², 300 ng / mm², 400 ng / mm², 500 ng / mm², 800 ng / mm², 1000 ng / mm², or within the range of any two of these values.

[0383] In some embodiments, in order to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on a 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 within a range of any two of these values.

[0384] In one embodiment, to lyse red blood cells in the device, Zwittergent is coated on a 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 within the range of any two of the stated values.

[0385] In some embodiments, in order to achieve a uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 3 ng / mm², 5 ng / mm², 8 ng / mm², 12 ng / mm², 15 ng / mm², 25 ng / mm², 35 ng / mm², 50 ng / mm², 80 ng / mm², 100 ng / mm², or within any two of the stated values.

[0386] In some embodiments, for red blood cells in a lysis apparatus, Zwittergent is coated on a plate at a preferred area concentration of 100 ng / mm², 120 ng / mm², 150 ng / mm², 180 ng / mm², 200 ng / mm², 300 ng / mm², 400 ng / mm², 500 ng / mm², 800 ng / mm², 1000 ng / mm², or within the range of any two of the stated values.

[0387] In some embodiments, in order to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on a 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 within a range of any two of these values.

[0388] In one embodiment, to lyse red blood cells in the device, Zwittergent is coated on a 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 within the range of any two of the stated values.

[0389] According to the apparatus, kit, system, or method described in any of the above embodiments, acridine orange is coated on the plate at an area concentration of 0.5 ng / mm², 1 ng / mm², 2 ng / mm², 3 ng / mm², 5 ng / mm², 8 ng / mm², 10 ng / mm², 15 ng / mm², 20 ng / mm², 30 ng / mm², or within the range of any two of the above values.

[0390] According to any of the above embodiments, the apparatus, kit, system, or method wherein acridine orange is coated on the plate at an area concentration of 3 to 10 ng / mm², and Zwittergent is coated on the plate at an area concentration of 3 to 10 ng / mm².

[0391] According to the apparatus, kit, system, or method described in any of the above embodiments, acridine orange is coated on the plate at an area concentration of 5 to 20 ng / mm², and Zwittergent is coated on the plate at an area concentration of 10 to 30 ng / mm².

[0392] In one embodiment, a label (e.g., as an immunoassay antibody label, RNA label, staining dye) is applied to the plate at a preferred final concentration 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 within the range of any two of these values.

[0393] In some embodiments, a label (e.g., as an immunoassay antibody label, RNA label, staining dye) is applied to the plate at a preferred final concentration of 1 nM / mL, 5 nM / mL, 10 nM / mL, 15 nM / mL, 20 nM / mL, or within the range of any two of these values.

[0394] In some embodiments, a label (e.g., an immunoassay antibody label, an RNA label, or a staining dye) is applied to the plate at a preferred final concentration 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 within any two of these values.

[0395] In some embodiments, a label (e.g., an immunoassay antibody label, an RNA label, or a staining dye) is applied to the plate at a final concentration 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 preferably within any two of these values.

[0396] In some embodiments, a label (e.g., an immunoassay antibody label, an RNA label, a staining dye) is applied to the plate at a final concentration of 0.05 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1.0 mg / mL, or preferably within any two of these values.

[0397] In some embodiments, a label (e.g., an immunoassay antibody label, an RNA label, a staining dye) is applied to the plate at a final concentration 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 preferably within any two of these values.

[0398] In some embodiments, a label (e.g., an immunoassay antibody label, an RNA label, a staining dye) is applied to the plate at a final concentration of 100 mg / mL, 200 mg / mL, 300 mg / mL, or preferably within any two of the stated values ​​in the sample.

[0399] Example 1:

[0400] Rapid detection of Gram-negative and Gram-positive bacteria

[0401] Material

[0402] 1. Hexanediol iodide: This nucleic acid dye is permeable to mammalian cells and selectively stains most Gram-positive bacteria orange (Thermofisher, H7593).

[0403] 2. Example 9: Staining Gram-positive and Gram-negative bacteria green (Thermofisher, S34854).

[0404] 3.500nm wavelength excitation filter.

[0405] 4. Staphylococcus epidermidis (Gram-positive bacteria), Escherichia coli (Gram-negative bacteria).

[0406] method:

[0407] Bacterial culture preparation:

[0408] 1. Staphylococcus epidermidis and Escherichia coli were used as Gram-positive and Gram-negative bacteria, respectively. These different bacterial species were cultured overnight at 37°C in nutrient liquid medium (R061582, Remel) and then stained.

[0409] Q-card preparation:

[0410] 1. A mixture containing 12 μg / mL hexanediol iodide and 8 μM SYTO 9 suspended in PBS was printed at 3 μL / cm² onto a batch of X plates (containing columns 2 μm high) and air-dried.

[0411] 2.2 μL of PBS or blood containing Staphylococcus epidermidis and Escherichia coli was added to the substrate. The printed X-plate and the substrate containing the bacterial-blood sample were then firmly pressed together. Figure 7 ).

[0412] Imaging:

[0413] After a 60-second incubation period, fluorescence and bright-field images were captured using an iPhone 6s. Only Gram-positive bacteria were shown in orange, while both Gram-positive and Gram-negative bacteria were shown in green. Figure 7 ).

[0414] In this experiment, the QMAX card has two plates: a flat PMMA substrate with a thickness of 1 mm and a PMMA film with a thickness of 175 μm, on which there is a column array with column size of 30 × 40 μm, 80 μm spacing (column) and 2 μm column height.

[0415] Example 2:

[0416] Rapid CD4 Immunostaining

[0417] Material

[0418] 1. CD4 antibodies: Ab34276 (Abcam) and 10R-CD4KHUP (Fitzgerald Industrial International)

[0419] 2. Antibody labeling kit:

[0420] 2. Alexa Fluor 647NHS ester reagent kit (A37573, Thermofisher)

[0421] 2.2 Zenon mouse IgG1 antibody labeling kit (Z25008, Thermofisher)

[0422] 3. 5 mM of SYTO62 red fluorescent nucleic acid dye (S11344, Thermofisher) in DMSO.

[0423] 4. Bioworld Antibody Biostabilizer 10X (22050005-2, Fisher Scientific)

[0424] 5.Triton X-100 (X-100, 100ml, Sigma)

[0425] method:

[0426] 1. Antibody double labeling method:

[0427] 1.1. Primary Fluorescent Labeling: Following the manufacturer's protocol, 100 μg of each CD4 detection antibody (Ab34276 and 10R-CD4KHUP) was labeled using the Alexa Fluor 647NHS ester labeling kit (Thermofisher). After antibody labeling, residual dye was removed using a Sephadex-G25 column (17-0853-01, GE Health, Switzerland). The purified antibody was resuspended to a final concentration of 1 μg / μL in a mixture of 30% glycerol and 1% BSA. The antibody was stored at -20°C before further use.

[0428] 1.2. Secondary Fluorescent Labeling: Following the manufacturer's instructions, the CD4 antibodies labeled with Alexa Fluor 647 NHS ester were second-labeled using the Zenon Alexa Fluor 647 Labeling Kit (Fc domain labeling). Simply put, 1 μg of each CD4 antibody was mixed with 8 μL of mouse IgG1 Alexa Fluor 647 labeling reagent in 15 μL of PBS. The reaction mixture was incubated at room temperature for 5 minutes and then maintained at 4°C before further use. This double-labeling method significantly enhanced the fluorescence signal of the CD4-specific antibodies.

[0429] 2. Detection of antibody mixtures:

[0430] 2D assay: 6 μg / mL of each double-labeled assay antibody was mixed with 5 μM SYTO 62 (a red fluorescent DNA dye from Thermofisher), 0.5X antibody biostabilizer (Bioworld), and 0.1% Triton X-100 (Sigma) in PBS. This mixture thus yielded a final antibody concentration of 1 μg / mL, which was then mixed with the dye used to stain the DNA.

[0431] 2A Assay: Double-labeled 6 μg / mL of each assay antibody was mixed with 0.5X antibody biostabilizer (Bioworld) and 0.1% Triton X-100 (Sigma) in PBS. This mixture also yielded a final antibody concentration of 12 μg / mL, but DNA dye was omitted.

[0432] 1A Assay: A double-labeled 6 μg / mL assay antibody was mixed with 0.5X antibody biostabilizer (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.

[0433] 1D assay: A double-labeled 6 μg / ml detection antibody was mixed with 0.5X antibody biostabilizer (Bioworld) and 0.1% Triton X-100 (Sigma) in PBS. This mixture contained only one antibody with DNA dye.

[0434] 3. Surface treatment of Q cards:

[0435] 3.1. Treat both the X plate (containing a 10 μm column) and the substrate with 1% NaOH at 50 °C for 1 hour. Then rinse both plates once with distilled water, PBS, and distilled water, each rinse lasting 5 minutes.

[0436] 3.2. After rinsing the plates, seal them with 4% BSA at room temperature for 2 hours. Rinse the plates twice with distilled water to remove residual BSA for 5 minutes each time, then air dry.

[0437] 4. Print the antibody mixture onto the Q-card:

[0438] 4.1 The prepared antibody mixture was printed onto two plates (X plate and substrate) at approximately 3 μL / cm². Once the antibody was printed, the plates were air-dried and protected from light before further use.

[0439] 5. Use iPhone 6s to detect CD4:

[0440] 5.1. Add fresh whole blood to the prepared substrate. Then firmly press the prepared X-plate onto the blood sample. Take a photo after incubating for 1 minute using an iPhone 6s.

[0441] 6. CD4 count per micrometer:

[0442] 6.1.X Slab Column Height: 10µm

[0443] 6.2 fov: Field of view (total area of ​​the Q-card in a photograph taken using an iPhone or fluorescence microscope)

[0444] 6.3. FOV sample quantity:

[0445] 6.3.1. Length (um) x Width (um) x Height (10um) = Volume of fov (um³)

[0446] 6.4.1 μl = 109 μm3

[0447] 6.5. CD4 count per micrometer:

[0448] 6.5.1: CD4 count in FOV x 10⁹ μm³ / FOV volume (μm³)

[0449] Total CD4 count

[0450] FOV volume um3

[0451] 6.6. Example: In such Figure 8 The iPhone image shown contains 31 positive CD4 signals. Figure 8 An exemplary photograph of CD4T cell counting using a QMAX device, taken with an iPhone-based microscope, is shown. Cells were stained with a detection antibody along with SYTO62 nonspecific nuclear dye.

[0452] 6.6.1. CD4 count per micrometer:

[0453] 31x 109um3 / (1680um x 2200um x 10um)=31x 109um3 / 3.7x 107um3=838 / ul

[0454] Example 3:

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

[0456] RNA fluorescence in situ hybridization (RNA-FISH) is a molecular cytogenetics technique used for fluorescence microscopy to detect and localize specific RNA targets (mRNA, lncRNA, and miRNA) within single cells. Traditional RNA-FISH methods typically involve multiple steps, such as fixation, permeabilization, hybridization, and imaging. Despite the wide range of medical applications of FISH, the complexity of the technique limits its potential in rapid diagnostics. Therefore, there is a need to develop rapid, accurate, portable, and / or inexpensive RNA-FISH assays that can be performed by non-specialist personnel. This invention addresses these needs.

[0457] 1. Materials

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

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

[0460] 2. Procedure:

[0461] Coat the board. Apply 0.25 μM. 670-labeled FISH probes (with varying concentrations of Zwittergent (Sigma-Aldrich) as 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. Drop 3 μL of fresh whole blood onto the center of the substrate and cover it with an X-plate.

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

[0464] The stained card was imaged using an iPhone 6S under external laser illumination.

[0465] 3. Results:

[0466] An embodiment of an X-FISH device includes a first plate (X-plate) and a second plate (substrate). Specific probes and a surfactant (e.g., Zwittergent) are printed onto the X-plate. After a blood sample is dropped onto the substrate, the X-plate is covered and pressed onto the substrate. After 1 minute, the device is inserted into a smartphone for imaging and analysis. (b) is a diagram of the detection of specific RNA expressed in leukocytes via X-FISH. Columns on the X-plate form a gap between the two plates, where the assay is performed. The printed probes and surfactant (e.g., Zwittergent) dissolve in the blood; the surfactant (e.g., Zwittergent) lyses red blood cells and also permeates leukocytes to facilitate probe entry into the cells to bind target RNA. Notably, the probes are designed to bind target RNA tandemly to amplify the signal.

[0467] A flowchart describing X-FISH is provided. Preparation involves printing X-ray plates with fluorophore-labeled detection probes and surfactants. Measurements include applying the sample to a substrate, covering it with the X-ray plate, incubating at room temperature for 1 minute, and inserting a smartphone device for reading.

[0468] Figure 5The effects of different Zwittergent concentrations on leukocyte permeation during rapid FISH using a GAPDH probe in QMAX are shown. Figure (a) shows the GAPDH probe observed using smartphone-based microscopy; 0.625 mg / mL of Zwittergent in blood does not produce a FISH signal. Figure (b) shows strong fluorescence signals from GAPDH-labeled leukocytes observed by smartphone-based microscopy at concentrations of 2.5 mg / mL and above (5 mg / mL). Figure (c) shows fluorescence microscopy (DAPIWBC nuclear repeat staining) used in (c).

[0469] Example 4:

[0470] Chlamydia staining

[0471] In the experiment, 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 for fixing samples.

[0472] In one experiment, slides of human tissue cells fixed with Chlamydia trachomatis were incubated for 30 seconds with 1 μL DyLight633-labeled anti-Chlamydia antibody at a concentration of 40 μg / mL. X-ray plates with a column height of 30 μm were used for QMAX immunostaining. The slides were then washed three times with PBST and images were captured using a fluorescence microscope. BF: Bright field. DL633: DyLight633 fluorescence.

[0473] Figure 6 An exemplary photograph is shown of chlamydia staining using a QMAX device without an optional washing step. In the experiment, a slide of human tissue cells fixed with chlamydia-infected tissue was incubated for 1 min with 1 μL of 40 μg / mL LdyLight633-labeled anti-chlamydia antibody. An X-ray plate with a column height of 30 μm was used for QMAX immunostaining. Images were taken using a fluorescence microscope without washing.

[0474] In routine staining experiments, slides of human tissue cells fixed with Chlamydia trachomatis were incubated for 30 min with 20 μL LdyLight633-labeled anti-Chlamydia antibody at a concentration of 40 μg / mL. The slides were then washed three times with PBST and images were captured using a fluorescence microscope.

[0475] Examples of the present invention

[0476] One-step staining assay for Chlamydia detection

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

[0478] (a) Obtaining a first plate, the first plate having a sample contact area configured to contact a sample on its inner surface;

[0479] (b) Depositing the sample in the sample contact area, wherein the sample is suspected to contain cells infected with Chlamydia; and

[0480] (c) Depositing a chlamydia-stained medium onto a sample, wherein the staining medium contains an antibody that binds to chlamydia, and the staining medium forms a mixture with the sample;

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

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

[0483] (f) A predetermined incubation period of approximately 60 seconds or less; and

[0484] (g) Detect chlamydia-related signals from the mixture.

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

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

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

[0488] AA5. The method according to any of the above embodiments, wherein the thin layer has a uniform thickness of less than 100 μm.

[0489] AA6. The method according to any of the above embodiments, wherein the thin layer has a uniform thickness of less than 50 μm.

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

[0491] AA8. The method according to any of the above embodiments, wherein chlamydia-related signals are detected by imaging the sample.

[0492] One-step sandwich assay for Chlamydia detection

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

[0494] (a) Obtain a first plate, the first plate having a sample contact area with binding sites on its inner surface, wherein the binding sites contain immobilized capture antibodies that bind to chlamydia in a sample suspected of containing chlamydia.

[0495] (b) Obtain a second plate, the second plate having a sample contact area with storage sites on its inner surface, wherein the storage sites contain detection antibodies that are able to diffuse 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) Deposit the sample onto one or two sample contact areas of the plate;

[0497] (d) After (c), the two plates are made into a closed structure, wherein, in the closed structure, at least a portion of the sample deposited in (c) is confined between the 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) Detect the chlamydia-related signals captured by the capture antibody.

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

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

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

[0502] AB5. The 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. 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 1 to 35 μm.

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

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

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

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

[0509] Additional features:

[0510] AC1. The method according to any of the above embodiments, wherein one or both of the sample contact areas include spacers, wherein when the plates are in a closed configuration, the spacers adjust the spacing between the sample contact areas of the plates.

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

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

[0513] AC4. The method according to any of the above embodiments, wherein the capture antibody at the binding site is on an amplification surface, which in any prior embodiment amplifies the optical signal of the analyte or the captured detector.

[0514] AC5. The method according to any of the above embodiments, wherein the trapping agent at the binding site is on the amplification surface, the amplification surface amplifies the optical signal of the analyte or the trapped detector in any prior embodiment, wherein the amplification is proximity-dependent because the amplification decreases significantly with increasing distance between the trapping agent and the analyte or detector.

[0515] AC6. The 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] Quantitative analysis of CD4-expressing cells

[0517] In a method for staining CD4-expressing cells according to some embodiments of the 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 some embodiments, the first plate and the second plate are not connected. In some 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 together. On the inner surface, each plate includes a sample contact area for contacting a liquid sample.

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

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

[0520] In step 3, the plates are pressed together to form a closed structure. In some embodiments, imprecise force is applied manually. In the closed structure, the plates are pressed together with a gap between them, 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. As the plates are pressed into the closed structure, the spacer adjusts the thickness of the sample layer. In some embodiments, the spacer has a columnar 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 had two plates. The first plate was an X-plate with a column height of 2 μm or 10 μm, a column size of 30 × 40 μm, and a spacing of 80 μm, and was made of 175 μm thick PMMA. The second plate was 1 mm thick glass or acrylic resin. AlexaFluor 647-labeled anti-CD4 antibody was positioned on the second plate in liquid or dry form. In its liquid form, the anti-CD4 antibody concentration was 5 to 50 μg / mL, and the volume was 0.5 to 1 μL. The anti-CD4 antibody was printed into an array with a fixed spacing of 300 μm and dried, resulting in a surface concentration of 1–100 ng / cm² after drying.

[0523] In the experiment, for step 2, the sample was approximately 1 μL of fresh whole blood.

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

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

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

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

[0528] The following lists the number of CD4-expressing T cells counted using the QMAX and iPhone-laser devices. The CD4 T cell counted using the QMAX system was 900 μL, and the known T cell counts for samples ranged from 500 to 1600 μL.

[0529] In some embodiments, dyeing includes the following steps:

[0530] (a) Obtaining a first plate and a second plate, wherein each plate includes a sample contact area configured to contact the liquid sample on its respective inner surface.

[0531] The detection reagent is located in the sample contact area of ​​one or two plates, and

[0532] The detection reagent is configured to specifically bind to biomarkers.

[0533] (b) Depositing the sample in the sample contact area, wherein the sample contains cells expressing biomarkers;

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

[0535] (d) A predetermined incubation period of approximately 60 seconds or less; and

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

[0537] An exemplary flowchart demonstrating the process of staining for CD4-expressing cells includes the following steps:

[0538] (a) Obtaining a first plate and a second plate, wherein each plate includes a sample contact area configured to contact the liquid sample on its respective inner surface.

[0539] The detection reagent is located in the sample contact area of ​​one or two plates, and

[0540] The detection reagent is configured to specifically bind to biomarkers.

[0541] (b) Depositing the sample in the sample contact area, wherein the sample contains cells expressing biomarkers;

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

[0543] (d) A predetermined incubation period of approximately 60 seconds or less; and

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

[0545] Examples of the present invention

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

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

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

[0549] (b) Deposit the sample in the sample contact area, wherein the sample contains cells expressing biomarkers; the sample is in contact with the detection reagent in the sample holder;

[0550] (c) Adjust the sample holder to compress the sample into a thin layer.

[0551] (d) The scheduled incubation period; and

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

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

[0554] (a) Obtaining a first plate and a second plate, wherein each plate includes a sample contact area configured to contact the liquid sample on its respective inner surface.

[0555] The detection reagent is located in the sample contact area of ​​one or two plates, and

[0556] The detection reagent is configured to specifically bind to biomarkers.

[0557] (b) Depositing the sample in the sample contact area, wherein the sample contains cells expressing biomarkers;

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

[0559] (d) A predetermined incubation period of approximately 60 seconds or less; and

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

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

[0562] (a) Obtaining a first plate and a second plate, wherein each plate includes a sample contact area configured to contact a blood sample on its respective inner surface.

[0563] The detection reagent is located in the sample contact area of ​​one or two plates, and

[0564] The detection reagent is configured to specifically bind to CD4.

[0565] (b) Depositing a blood sample in the sample contact area, wherein the blood sample contains cells expressing CD4;

[0566] (c) Press the first and second plates 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) A predetermined incubation period of approximately 60 seconds or less; and

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

[0569] BA2.1 A device for quantifying cells expressing biomarkers in a sample, comprising:

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

[0571] An adapter, configured to accommodate 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 within the imager's field of view (FOV) when the adapter is attached to the moving device, and

[0574] iii. The imager is configured to capture an image of the sample, thereby detecting / measuring the signal generated by the binding of the biomarker and the detector after the sample has been incubated with the detector for a predetermined period of time of about 60 seconds or less.

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

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

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

[0578] BB1.4 The method or apparatus according to any of the above embodiments, wherein a fluorophore-labeled antibody is used.

[0579] BB1.5 The method or apparatus according to any of the above embodiments, wherein the detector is used as a signal molecule label that emits a signal upon excitation.

[0580] BB1.6 The method or apparatus according to any of the above embodiments, wherein the thin layer has a uniform thickness of approximately equal to or less than 10 μm.

[0581] BB1.7 The method or apparatus according to any of the above embodiments, wherein the thin layer has a uniform thickness of approximately equal to or less than 2 μm.

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

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

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

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

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

[0587] In this invention, a machine learning model for detecting objects in image-based measurements is built based on training data. In some embodiments, machine learning-based detection is performed on images used for measurement, and in some embodiments, machine learning-based detection is performed on transformed images used for measurement that have higher contrast and lower signal-to-noise ratio. It begins with the collection of a training image set DBO for measurement taken by an imager. These images are collected by taking multiple images of the sample in a sample holding device such as a QMAX card. Each training image is then acquired from DB0, and objects of interest are labeled in the images for training. The labeled images are stored in a separate training set DB1 for building the machine learning model.

[0588] In machine learning model training, a labeled training database DBI is used. A machine learning model architecture is selected in the form of a deep neural network, and the model is trained on the training database DBI. In some embodiments, a RetinaNet machine learning model is used, and in some other embodiments, a Fast-RCNN machine learning model is selected. The machine learning model is trained using Tensorflow and PyTourch, and detection and segmentation are performed using the training database DB1. The machine learning model training process terminates after a certain number of iterations on the training data in DBI when the loss function on the training set DBI or a separate evaluation image dataset meets a preset stopping criterion. The machine learning model obtained from the machine learning training process is saved for evaluation applications.

[0589] During this measurement process, images of the samples serve as input to a machine learning-based inference module. This module performs machine learning-based detection and segmentation based on a model acquired during previous training on the DB1 training set. Detected objects and their locations are stored in a database, which can be searched and retrieved for image-based measurement 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 measurements, depending on computational complexity, on-device resources, network connectivity, measurement latency requirements, etc., machine learning-based detection and segmentation can be performed either on-device or using distributed computing resources, such as a hybrid cloud using both local and cloud resources.

[0591] Other embodiments

[0592] This disclosure includes various embodiments that can be combined in multiple ways as long as the various components do not contradict each other. These embodiments include not only the disclosure in this document, but also documents referenced, incorporated, or claiming priority herein.

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

[0594] The apparatus, 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 spacers that facilitate making at least a portion of the sample a very uniform layer.

[0595] The apparatus, 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 spacers that facilitate making at least a portion of the sample a highly uniform layer. The aforementioned PCT applications (designated as U.S.) PCT / US2016 / 046437 and PCT / US2016 / 051775, and the corresponding U.S. provisional applications, disclose the structure, materials, functions, variations, and dimensions of spacers, as well as the uniformity of the spacers with respect to the sample layer.

[0596] Other notes

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

[0598] As used herein, the terms “adapter” and “configuration” mean that a component, assembly, or other subject is designed and / or intended to perform a given function. Therefore, the use of the terms “adapter” and “configuration” should not be construed as meaning that a given component, assembly, or other subject is simply “capable” of performing a given function. Similarly, a subject stated as being configured to perform a particular function may additionally or optionally be described as being operable to perform that function.

[0599] As used herein, when reference is made to one or more components, features, details, structures, embodiments, and / or methods according to this disclosure, the phrases “for example,” “as an example,” and / or simply the terms “example” and “exemplary” are intended to convey illustrative, non-exclusive examples of the described components, features, details, structures, embodiments, and / or methods according to this disclosure. Therefore, the described components, features, details, structures, embodiments, and / or methods are not intended to be limiting, essential, 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 this disclosure.

[0600] As used herein, the phrases “at least one” and “one or more” in relation to a list of more than one entity refer to any one or more entities in the entity list, and are not limited to each and every one of the entities specifically listed in the entity list. 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”) could refer to A alone, B alone, or a combination of A and B.

[0601] As used herein, the term “and / or” placed between the first entity and the second entity refers to (1) the first entity, (2) the second entity, and (3) one of the first entity and the second entity. Multiple entities listed using “and / or” should be interpreted in the same way, i.e., “one or more” of the entities thus combined. Other entities may optionally exist in addition to those specifically identified by the “and / or” clause, whether related to or unrelated to those specifically identified.

[0602] When referring to numerical ranges herein, the invention includes embodiments that include endpoints, embodiments that exclude two endpoints, and embodiments that include one endpoint while excluding the other. It should be assumed that two endpoints are included unless otherwise stated. Furthermore, unless otherwise stated or obvious from the context and understanding by one of ordinary skill in the art.

Claims

1. A method for homogeneous detection of analytes in cells of a sample, comprising: (a) A first plate and a second plate are provided, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing or suspected of containing the analyte. (b) A detection probe is provided 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) Provide an optical enhancer dispersed in the sample, the optical enhancer binding to the cells and capable of emitting light of a wavelength that overlaps with or is within 150 nm of the wavelength of light emitted by the detection probe; (d) The sample, the detection probe and the optical enhancer are sandwiched between two sample contact areas of the two plates to form a thin layer with a thickness of 200 micrometers (μm) or less; as well as (e) After step (d) and without using any rinsing step, the thin layer is imaged using an imager to detect cells having analytes that bind to the detection probe; The thickness of the thin sample layer is configured such that, for a given concentration of cells in the sample, each individual cell substantially 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 intensifier in the sample are configured such that, in the imaging step (e), the locations of the bound detection probes in the thin layer are distinguishable from the locations of the unbound detection probes, wherein the bound detection probes are detection probes of analytes bound to the cells.

2. An apparatus for enhancing homogeneous detection of analytes in cells of 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 or suspected of containing the analyte. (b) A detection probe that specifically binds to the analyte and is capable of emitting light of a specific wavelength, wherein the detection probe is dispersed in the sample; (c) An optical enhancer that binds to the cell and emits light with a wavelength that overlaps with or is within 150 nm of the light emitted by the detection probe; The first plate and the second plate are configured to sandwich the sample, the detection probe and the optical enhancer between the two sample contact areas to form a thin layer with a thickness of 200 micrometers (μm) or less; as well as The thickness of the thin sample layer is configured such that, for a given concentration of cells in the sample, each individual cell substantially 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 intensifier in the sample are configured such that, in the imaging step (e), the locations of the bound detection probes in the thin layer are distinguishable from the locations of the unbound detection probes, wherein the bound detection probes are detection probes of analytes bound to the cells.

3. A system for enhancing homogeneous detection of analytes in cells of a sample, comprising: (a) the apparatus or kit according to any of the preceding claims; and (b) Communication device.

4. An apparatus for rapid homogeneous detection of analytes within cell membranes 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, the cells containing or suspected of containing the analyte within the cell membrane; (b) A detection probe that specifically binds to the analyte and is capable of emitting light of a specific wavelength, wherein the detection probe is dispersed in the sample; (c) A permeabilizer that makes the cell membrane permeable to the detection probe; The first and second plates are configured to hold the sample between the two sample contact areas of the two plates, the detection probe and the permeabilizer to form a thin layer with a thickness of 200 micrometers (µm) or less. The thickness of the thin layer is configured such that, for a given concentration of cells in the sample, each individual cell substantially does not overlap with other cells in the imaging process, and the imaging operation does not include a washing step. The thickness of the thin layer and the concentration of the detection probe in the sample are configured such that, in the imaging of the thin layer, the locations of the detection probes bound to the analyte within the cell membrane are distinguishable from locations without the cells; and The rinsing step is a step of removing unbound detection probes, permeation reagents, or both from the sample contact area.

5. A method for rapid homogeneous detection of analytes within cell membranes in a sample, comprising: (a) A first plate and a second plate are provided, each plate having a sample contact area on its surface, wherein the sample contact surface contacts a sample containing cells, the cells containing or suspected of containing the analyte within the cell membrane. (b) A detection probe is provided 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) Providing a permeabilizer that allows the cell membrane to be permeable to the detection probe; (d) The sample, the detection probe and the permeating agent are sandwiched between two sample contact areas of the two plates to form a thin layer with a thickness of 200 micrometers (μm) or less; as well as (e) After step (d) and without using any rinsing step, the thin film is imaged to detect cells having analytes that bind to the detection probe; The thickness of the thin sample layer is configured such that, for a given concentration of cells in the sample, each individual cell substantially does not overlap with other cells in the imaging. The thickness of the thin layer and the concentration of the detection probe in the sample are configured such that the locations of the detection probes containing analytes bound to the cell membrane are distinguishable from locations without the cells; and The rinsing step is a step of removing unbound detection probes, permeation reagents, or both from the sample contact area.

6. A method for homogeneous detection of Gram-positive or Gram-negative cells in a sample, comprising: (a) A first plate and a second plate are provided, 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 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 stains only Gram-positive cells, while the Gram-negative stain stains both Gram-positive and Gram-negative cells; wherein, In Gram-positive cells stained with both Gram-positive and Gram-negative staining agents, the Gram-positive staining agent can be designed from the Gram-negative staining agent; (c) The sample is sandwiched between two sample contact areas of the two plates to form a thin layer of the sample with a thickness of 150 micrometers (μm) or less; as well as (d) After step (c) and without using any rinsing step, the thin slice is imaged using an imager to detect cells stained by (i) only Gram-negative staining, (ii) Gram-positive staining, and (iii) both; The thickness of the thin sample layer is configured such that, for a given concentration of cells in the sample, each individual cell substantially does not overlap with other cells in the imaging. Gram-positive cells show the color of Gram-positive staining agent, while Gram-negative cells only show the color of Gram-negative staining agent; The thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical intensifier in the sample are configured such that, in the imaging step (e), the locations of the bound detection probes in the thin layer are distinguishable from the locations of the unbound detection probes, wherein the bound detection probes are detection probes of analytes bound to the cells.

Images