Biomolecule detection method and system

Abstract

The invention provides a biomolecule detection method and system.The detection method comprises the steps that a micro-fluidic chip is prepared, the micro-fluidic chip comprises a resealable flowing unit and a biological tissue to be tested, and the biological tissue corresponds to the flowing unit; the biological tissue in the micro-fluidic chip is pretreated; carrying out marker marking and imaging treatment on the biological tissue in the micro-fluidic chip based on the compound with the marker capable of being sheared; and carrying out hematoxylin eosin tissue dyeing and color imaging on the biological tissue marked by the marker and subjected to imaging treatment. According to the embodiment of the invention, the staining fluid of the compound with the shearable marker is used for performing multi-marker marking on the biological tissue based on the micro-fluidic chip, so that the biological tissue and the staining fluid of the compound with the shearable marker are mixed more uniformly, and the multi-marker marking effect is better; and after multiple markers are marked, no great chemical influence is caused on biological tissues.

Description

Technical Field

[0001] The present application relates to the technical field of iterative detection of biomolecules, and in particular to a biomolecule detection method and system. Background Art

[0002] A microfluidic chip is a device that can manipulate or detect fluids at the micrometer scale. Microfluidic chips have the ability to miniaturize the basic functions of biological and chemical laboratories onto a chip of a few square centimeters, so they are also called chip laboratories. Usually, microfluidic chips include microchannels, and the microchannels can form a network, so that various functions of conventional chemical or biological laboratories can be achieved by controlling the flow of fluids in the network formed by microchannels. Therefore, microfluidic chips have the advantages of small size, reagent saving, portability, flexible combination of functions, and high integration.

[0003] Imaging markers is a common technique in chemistry, biology, medicine, and other fields, including, but not limited to, fluorescence imaging. Immunofluorescence microscopy has been widely used to quantify the identity, location, and abundance of proteins in single cells. Therefore, it may be well suited for analyzing cellular heterogeneity and the spatial complexity of proteins. Similarly, fluorescence-based detection schemes have been developed for DNA- and RNA-based targets. However, the capabilities of traditional immunofluorescence techniques may be limited due to the spectral overlap of commonly used fluorophores.

[0004] Various techniques have been developed to address the limitations of traditional marker imaging techniques and enable more comprehensive DNA, RNA, and protein analysis. One class of techniques includes multiplexed or iterative immunofluorescence with sequential cycles of fluorescent staining, imaging, and erasure of the fluorescent signal. Example methods for iterative immunofluorescence may rely on photobleaching or DNA displacement reactions to photobleach fluorophores, chemically inactivate fluorophores, remove fluorescent antibodies, or dissociate fluorescent DNA complexes.

[0005] While each of the above approaches may offer advantages over traditional marker imaging techniques, they still have a number of limitations. On the one hand, partial photobleaching during imaging and incomplete removal of fluorescent signals after bleaching may hinder consistent quantification of protein abundance. On the other hand, harsh chemical reagents often cause sample degradation and interfere with subsequent staining cycles. Therefore, marker imaging techniques that can overcome the above limitations are needed.

[0006] In view of this, there is an urgent need to provide a biomolecule detection method and system to solve at least one of the above problems. Summary of the invention

[0007] In view of this, an object of the present invention is to provide a method and system for detecting biological molecules, by using a staining solution of a complex with a shearable marker in a resealable microfluidic chip to mark biological tissue with multiple markers, so that the biological tissue and the staining solution of the complex with a shearable marker are mixed more evenly, the multiple marker labeling effect is better, and there is no significant chemical impact on the biological tissue itself after multiple marker labeling.

[0008] According to a first aspect of the present application, a biomolecule detection method is provided, the biomolecule detection method comprising:

[0009] preparing a microfluidic chip, the microfluidic chip comprising a resealable flow unit and a biological tissue to be tested, the biological tissue corresponding to the flow unit;

[0010] Pre-treat biological tissues in microfluidic chips;

[0011] Marking and imaging biological tissues in microfluidic chips based on complexes with shearable markers;

[0012] Perform hematoxylin and eosin tissue staining and color imaging on biological tissues that have been labeled with markers and processed for imaging.

[0013] In some specific embodiments, labeling and imaging of biological tissues in a microfluidic chip based on a complex having a cleavable marker includes:

[0014] Marking the cell nuclei of biological tissues with markers;

[0015] Perform background marker imaging;

[0016] Multiple rounds of labeling and imaging of biological tissues in microfluidic chips were performed using complexes with cleavable markers.

[0017] In some specific embodiments, multiple rounds of labeling and imaging of biological tissues in a microfluidic chip using a complex having a cleavable label include:

[0018] Labeling biological tissues with markers based on a staining solution containing a complex of an active enzyme marker and a cleavable marker;

[0019] Performing marker imaging on biological tissues labeled with markers;

[0020] Removal of active enzyme tags;

[0021] Cutting markers to mark biological tissues;

[0022] Repeat the above steps for the biological tissue to be tested until the processing results meet the requirements.

[0023] In some specific embodiments, the staining solution based on the active enzyme label and the complex with the cleavable label is used to label the biological tissue, comprising:

[0024] Use specific primary antibodies to recognize the corresponding antigen on the tissue;

[0025] Use enzyme-linked secondary antibodies to recognize primary antibodies;

[0026] The region of the antigen labeled with the enzyme is labeled with the marker using a staining solution containing a complex with a cleavable marker.

[0027] In some embodiments, a specific primary antibody is used to recognize the corresponding antigen on the tissue, including:

[0028] Injecting a primary antibody into the microfluidic chip for incubation so that the primary antibody binds to the antigen;

[0029] Rinse the microfluidic chip.

[0030] In some embodiments, a secondary antibody with an enzyme is used to recognize a primary antibody, including:

[0031] Injecting a secondary antibody with an enzyme into the microfluidic chip for incubation so that the secondary antibody binds to the primary antibody;

[0032] Rinse the microfluidic chip.

[0033] In some specific embodiments, using a staining solution having a complex with a cleavable marker to label an enzyme-labeled antigen region comprises:

[0034] Injecting a staining solution containing a complex of a shearable marker into the microfluidic chip for incubation to label the marker antigen with the marker;

[0035] Rinse the microfluidic chip.

[0036] In some specific embodiments, the active enzyme label includes a primary antibody that recognizes the corresponding antigen on the biological tissue, and a secondary antibody with an enzyme label that recognizes the primary antibody;

[0037] Removal of active enzyme labels, including: simultaneous removal of primary and secondary antibodies, removal of secondary antibodies only, or inactivation of enzyme labels.

[0038] In some specific embodiments, the marker for cutting out the biological tissue includes:

[0039] Injecting a shearing fluid into the microfluidic chip, and incubating the biological tissue in the microfluidic chip under heating conditions to shear the markers of the biological tissue;

[0040] Rinse the microfluidic chip.

[0041] In some embodiments, the complex having a cleavable marker comprises:

[0042] Molecular groups, markers, and molecular links connecting the molecular groups and markers that can bind to protein tissues in the biological tissues under the catalysis of enzymes;

[0043] The molecular link can be effectively cut in a cellular environment without damaging biological tissues, and does not affect the effective binding of the complex with protein tissues in the biological tissues.

[0044] In some embodiments, the molecular linkage comprises an azido-type linkage or an allylic-type linkage or a structure that is cleaved by an enzyme, a nucleophile, an electrophile, a reducing agent, an oxidizing agent, light irradiation, or metal catalysis.

[0045] In some specific embodiments, the chemical formula of the complex with a cleavable marker linked using an azide group is as follows:

[0046]

[0047] Among them, T is a molecular group that can bind to protein tissue in biological tissue under the catalysis of enzyme, and R is a marker.

[0048] In some specific embodiments, the molecular group that can bind to protein tissue in biological tissue under the catalysis of an enzyme includes tyramine.

[0049] In some specific embodiments, the chemical structure of the complex with a cleavable marker using an azide-type linker that can bind to protein tissues in biological tissues under the catalysis of an enzyme is as follows:

[0050]

[0051] Among them, R is a marker.

[0052] In some specific embodiments, the label includes a fluorescent group, a tyramide that can bind to protein tissue in biological tissue under the catalysis of an enzyme, and a cleavable Cy5 fluorescent group. The chemical structure of the complex with the cleavable label is as follows:

[0053]

[0054] In some specific embodiments, the molecular group that can bind to protein tissue in biological tissue under the catalysis of an enzyme includes phenylacetamide.

[0055] In some specific embodiments, the chemical structure of the complex with a cleavable marker is as follows:

[0056]

[0057] Wherein, Linker is the cleavable molecular link, R is a marker; X is hydrogen, and each group R1, R2, R3, R4, R5, R6 and R7 can independently be hydrogen, alkyl, alkenyl, alkynyl, halogen, carboxyl, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxyl, phosphoryl, sulfonyl, carbonyl, boroyl, aryl or heteroaryl.

[0058] In some specific embodiments, the chemical structure of the complex with a cleavable marker is as follows:

[0059]

[0060] Wherein, Linker is the cleavable molecular link, R is a marker; X is hydrogen, and each group R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 can independently be hydrogen, alkyl, alkenyl, alkynyl, halogen, carboxyl, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxyl, phosphoryl, sulfonyl, carbonyl, boroyl, aryl or heteroaryl.

[0061] In some specific embodiments, the chemical structure of the complex with a cleavable marker is as follows:

[0062]

[0063] Wherein, Linker is a cleavable molecular link, R is a marker; X is hydrogen, and each group R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 can independently be hydrogen, alkyl, alkenyl, alkynyl, halogen, carboxyl, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxyl, phosphoryl, sulfonyl, carbonyl, boroyl, aryl or heteroaryl.

[0064] In some specific embodiments, pre-treating the biological tissue in the microfluidic chip includes:

[0065] injecting antigen repair solution, incubating the biological tissue for antigen repair under heating conditions, and cooling;

[0066] Inject peroxidase and incubate the biological tissue at room temperature to block endogenous peroxidase activity;

[0067] Inject antibody blocking buffer and incubate the tissue at room temperature to block nonspecific binding.

[0068] In some specific embodiments, the microfluidic chip comprises:

[0069] A resealable fluidic device having a resealable flow cell;

[0070] a glass slide provided with biological tissue, the glass slide being fixedly mounted to a resealable fluidic device;

[0071] A snap fit is used to securely mount the slide to the resealable fluidic device.

[0072] In some specific embodiments, the resealable fluidic device comprises:

[0073] frame;

[0074] a cover glass, fixed to the frame as the top of the flow cell, which is provided with fluid inlet and outlet;

[0075] A layer of optical sealing material is combined with the cover glass to form a resealable flow cell.

[0076] In some specific embodiments, preparing a microfluidic chip includes:

[0077] Baking slides with biological tissue;

[0078] Dewaxing biological tissues on glass slides and assembling microfluidic chips;

[0079] Wherein, assembling the microfluidic chip comprises:

[0080] Mount the coverslip to the frame;

[0081] mounting an optical sealing material to a coverslip to form a resealable flow chamber;

[0082] Remove the film from the bottom of the coverslip;

[0083] Place the slide in the frame;

[0084] Install the clips to complete the assembly of the microfluidic chip.

[0085] In some specific embodiments, preparing a microfluidic chip includes:

[0086] Obtain slides;

[0087] Assembling microfluidic chips;

[0088] Baking the microfluidic chip;

[0089] Dewaxing the biological tissue in the microfluidic chip;

[0090] Wherein, assembling the microfluidic chip comprises:

[0091] Mount the coverslip to the frame;

[0092] mounting an optical sealing material to a coverslip to form a resealable flow chamber;

[0093] Remove the film from the bottom of the coverslip;

[0094] Place the slide in the frame;

[0095] Install the clips to complete the assembly of the microfluidic chip.

[0096] According to a second aspect of the present application, a biomolecule detection system is provided, the biomolecule detection system comprising:

[0097] A microfluidic chip comprising a resealable flow cell and a biological tissue, the biological tissue corresponding to the flow cell;

[0098] a microfluidic chip processing unit configured to pre-process biological tissue in the microfluidic chip;

[0099] A staining processing unit, which is configured to: perform marker marking and imaging processing on biological tissues in the microfluidic chip based on a complex having a shearable marker;

[0100] The hematoxylin and eosin tissue staining unit is configured to perform hematoxylin and eosin tissue staining and color imaging on biological tissues that have been marked with markers.

[0101] In some specific embodiments, the preprocessing comprises:

[0102] injecting antigen repair solution, incubating the biological tissue for antigen repair under heating conditions, and cooling;

[0103] Inject peroxidase and incubate the biological tissue at room temperature to block endogenous peroxidase activity;

[0104] Inject antibody blocking buffer and incubate the tissue at room temperature to block nonspecific binding.

[0105] In some specific embodiments, labeling and imaging of biological tissues in a microfluidic chip based on a complex having a cleavable marker includes:

[0106] Marking the cell nuclei of biological tissues with markers;

[0107] Perform background marker imaging;

[0108] Multiple rounds of labeling and imaging of biological tissues in microfluidic chips were performed using complexes with cleavable markers.

[0109] In some specific embodiments, multiple rounds of labeling and imaging of biological tissues in a microfluidic chip using a complex having a cleavable label include:

[0110] Labeling biological tissues with markers based on a staining solution containing a complex of an active enzyme marker and a cleavable marker;

[0111] Performing marker imaging on biological tissues labeled with markers;

[0112] Removal of active enzyme tags;

[0113] Cutting markers to mark biological tissues;

[0114] Repeat the above steps for the biological tissue to be tested until the processing results meet the requirements.

[0115] The beneficial effects of the embodiments of the present application include at least:

[0116] In this embodiment, biological tissues are labeled with multiple markers using a staining solution having a complex of shearable markers in a resealable microfluidic chip, so that the biological tissues and the staining solution having a complex of shearable markers are mixed more evenly, the multiple marker labeling effect is better, and there is no significant chemical impact on the biological tissues themselves after the multiple marker labeling; because the microfluidic chip is resealable, that is, the microfluidic chip can be reused, the glass slide containing the biological tissue that has been tested can be disassembled for subsequent storage and can be used for other biological tests (such as multiple RNA tests, hematoxylin and eosin tests, etc.), so as to obtain maximized biological detection diagnostic information on the same piece of biological tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0118] Figure 1 A flow chart of a biomolecule detection method provided in the first embodiment of the present application;

[0119] Figure 2 A schematic diagram of the structure of a microfluidic chip provided in one embodiment of the present application;

[0120] Figure 3This is an effect diagram of staining 12 different proteins of formalin-fixed paraffin-embedded (FFPE) human tonsil tissue and DAPI staining of cell nuclei in an embodiment of the present application;

[0121] Figure 4 This is a diagram showing the effect of hematoxylin and eosin (H&E) staining of formalin-fixed paraffin-embedded (FFPE) human tonsil tissue after staining for 12 proteins according to an embodiment of the present application;

[0122] Figure 5 This is a rendering of a traditional dyeing method according to an embodiment of the present application;

[0123] Figure 6 This is a diagram showing the effect of multiple staining based on antibodies of cleavable markers in a microfluidic chip according to an embodiment of the present application. DETAILED DESCRIPTION

[0124] The specific embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the description of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0125] In the description of the present invention, unless otherwise clearly specified and limited, the terms "set", "install", "connection" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0126] The directions or positional relationships indicated by terms such as “upper”, “lower”, “left”, “right”, “front”, “back”, “top”, “bottom”, “inside” and “outside” are based on the directions or positional relationships shown in the accompanying drawings, or are the directions or positional relationships in which the inventive product is usually placed when used. They are only for the convenience of description and simplified description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as a limitation on the present invention.

[0127] The terms "first", "second", "third" and the like are merely used to distinguish elements of similar nature, and do not indicate or imply relative importance or a particular order.

[0128] The terms "comprises," "comprising," or any other variations thereof, are intended to cover a non-exclusive inclusion of the elements listed and may also include additional elements not expressly listed.

[0129] Some embodiments of the present application provide a method for detecting biomolecules, see Figure 1 , biomolecular detection methods include:

[0130] Step S1, preparing a microfluidic chip, wherein the microfluidic chip comprises a resealable flow unit and a biological tissue to be tested, wherein the biological tissue to be tested corresponds to the flow unit.

[0131] In some specific embodiments, the biological tissue to be tested may be FFPE tissue (such as formalin-fixed paraffin-embedded (FFPE) human tonsil tissue), fixed cells, frozen fresh tissue, frozen fixed tissue, etc., but is not limited thereto.

[0132] In some specific embodiments, see Figure 2 , the microfluidic chip includes:

[0133] A resealable fluidic device having a resealable flow cell;

[0134] a glass slide provided with biological tissue, the glass slide being fixedly mounted to a resealable fluidic device;

[0135] A snap fit is used to securely mount the slide to the resealable fluidic device.

[0136] In some embodiments, the resealable fluidic device comprises:

[0137] frame;

[0138] a cover glass, fixed to the frame as the top of the flow cell, which is provided with fluid inlet and outlet;

[0139] An optical sealing material is combined with the cover glass to form a resealable flow cell.

[0140] In some specific embodiments, the optical sealing material includes but is not limited to an optical silicone layer.

[0141] In some specific embodiments, preparing a microfluidic chip includes:

[0142] Step S11, baking the glass slide with the biological tissue.

[0143] In some specific embodiments, step S11 includes:

[0144] S111, heating the heating device to a predetermined temperature.

[0145] Specifically, the heating device includes but is not limited to a heating plate or an oven, etc. In a specific example, the heating plate can be set to a set temperature, such as but not limited to 60° C., and wait until the set temperature is reached.

[0146] S112, placing the biological tissue on a glass slide, and then placing it in a heating device for baking for a predetermined time.

[0147] Specifically, the biological tissue to be tested, such as FFPE tissue, is placed on a glass slide, and then placed on a heating device, such as a hot plate, and baked at a predetermined temperature for 1 hour.

[0148] In the embodiments of the present application, baking can make FFPE tissues less likely to detach.

[0149] Step S12: dewaxing the biological tissue on the glass slide and assembling the microfluidic chip.

[0150] In some specific embodiments, step S12 includes:

[0151] A. Soak the slide glass in the first dissolving solution to dissolve the wax.

[0152] Specifically, the slide can be immersed in the wax dissolving liquid for 3 cycles, each cycle lasting 5-15 minutes, to dissolve the wax using the first dissolving liquid, which includes but is not limited to xylene or xylene substitutes such as HistoClear, xylene-based dewaxing agents, etc.

[0153] B. Soak the slide glass in the second dissolving solution to dissolve the first dissolving solution.

[0154] In some specific embodiments, the second dissolving liquid includes but is not limited to ethanol, etc. Specifically, when HistoClear is used as the first dissolving liquid and ethanol is used as the second dissolving liquid, the immersed slide can be immersed in ethanol for a predetermined time to dissolve the remaining HistoClear with ethanol.

[0155] C. Air-dry the slides;

[0156] D. Assemble into a microfluidic chip.

[0157] In some specific embodiments, assembling a microfluidic chip comprises:

[0158] Step D1, installing the cover glass on the frame;

[0159] Step D2, mounting an optical sealing material on the cover glass to form a resealable flow chamber;

[0160] Step D3, removing the film at the bottom of the cover glass;

[0161] Step D4, placing the slide on the frame;

[0162] Step D5: Install the clips to complete the assembly of the microfluidic chip.

[0163] Specifically, the optical sealing material includes but is not limited to an optical silicone layer.

[0164] Furthermore, during the assembly of the microfluidic chip, the operator needs to only hold the frame with the cover glass and not touch the cover glass to prevent the cover glass from breaking.

[0165] E. Rehydrate using decreasing concentrations of the second dissolving solution and deionized water.

[0166] Specifically, when HistoClear is used as the first dissolving liquid and ethanol is used as the second dissolving liquid, first, 100% ethanol, 90% ethanol, 70% ethanol, and 50% ethanol may be used for washing in sequence, twice in each step;

[0167] Then, the microfluidic chip can be rinsed with deionized water: rinse 3 times to remove the ethanol in the microfluidic chip.

[0168] In some other embodiments, when biological tissue (such as FFPE tissue) is processed, a glass slide carrying the biological tissue can be directly made into a microfluidic chip, and the baking and dewaxing process of the biological tissue can be directly performed on the microfluidic chip, which specifically includes the following steps:

[0169] Step S11 ′, obtaining a glass slide.

[0170] Specifically, step S11 ′ includes: placing the biological tissue on a glass slide.

[0171] Step S12', assembling the microfluidic chip.

[0172] In some specific embodiments, assembling a microfluidic chip comprises:

[0173] Step S121', installing the cover glass on the frame;

[0174] Step S122', installing the optical sealing material on the cover glass to form a resealable flow chamber;

[0175] Step S123', removing the film at the bottom of the cover glass;

[0176] Step S124', placing the slide on the frame;

[0177] Step S125': install the buckle to complete the assembly of the microfluidic chip.

[0178] Specifically, the optical sealing material includes but is not limited to an optical silicone layer.

[0179] Furthermore, during the assembly of the microfluidic chip, the operator needs to only hold the frame with the cover glass and not touch the cover glass to prevent the cover glass from breaking.

[0180] Step S13', baking the microfluidic chip.

[0181] In some specific examples, the microfluidic chip can be baked at a predetermined temperature for 1 hour. Specifically, the electric heating plate can be set to a set temperature in advance, such as but not limited to 60° C., and wait until the set temperature is reached.

[0182] In the embodiment of the present application, baking can prevent the tissue from peeling off easily.

[0183] Step S14', dewaxing the biological tissue in the microfluidic chip.

[0184] Specifically, step S14' includes the following steps:

[0185] a. Soak the slide glass in the first dissolving solution to dissolve the wax.

[0186] Specifically, the wax dissolving liquid is injected into the microfluidic chip and incubated for 5-15 minutes for 3 cycles to dissolve the wax using a first dissolving liquid, wherein the first dissolving liquid includes but is not limited to xylene or a xylene substitute such as HistoClear, a xylene-based dewaxing agent, and the like.

[0187] b. Soak the slide glass in the second dissolving solution to dissolve the first dissolving solution.

[0188] In some specific embodiments, the second dissolving liquid includes but is not limited to ethanol, etc. Specifically, when HistoClear is used as the first dissolving liquid, ethanol can be injected into the microfluidic chip and incubated for 5-15 minutes to dissolve the remaining HistoClear with ethanol.

[0189] c. Use the second dissolving solution with decreasing concentration and deionized water for rehydration, and rinse the microfluidic chip to dissolve the first solution.

[0190] Specifically, when HistoClear is used as the first dissolving liquid and ethanol is used as the second dissolving liquid, first, the microfluidic chip can be rinsed in sequence of 100% ethanol, 90% ethanol, 70% ethanol, and 50% ethanol, twice in each step; then, the microfluidic chip can be rinsed with deionized water: rinse 3 times to use deionized water to wash away the remaining ethanol in the microfluidic chip.

[0191] Step S2: pre-treating the biological tissue in the microfluidic chip.

[0192] In some embodiments, pre-treating the biological tissue in the microfluidic chip includes:

[0193] Step S21, injecting antigen repair solution, incubating the biological tissue for antigen repair under heating conditions, and cooling.

[0194] In some specific embodiments, step S21 includes:

[0195] Step S211, injecting antigen repair solution into the microfluidic chip, and incubating the microfluidic chip under heating conditions.

[0196] Specifically, antigen repair solution can be injected into the microfluidic chip, and then the microfluidic chip can be placed in a heating device and incubated at a set temperature for a suitable time, such as 30-40 minutes. The heating device includes but is not limited to a hot plate, an oven, etc.

[0197] Step S212, cooling the microfluidic chip to room temperature.

[0198] Step S213: rinse with a washing solution.

[0199] Specifically, the washing solution may be used for rinsing each time, generally 3 times.

[0200] In some specific embodiments, step S21 further includes:

[0201] Step S210: heating a heating device for heating the microfluidic chip to a predetermined temperature.

[0202] Specifically, the heating device includes but is not limited to a heating plate, etc. In some specific examples, the heating plate for heating the microfluidic chip can be set to a required temperature range, such as 85-105°C, and wait for the set temperature to be reached, but is not limited thereto.

[0203] Step S22: inject peroxidase and incubate the biological tissue at room temperature to block endogenous peroxidase activity.

[0204] In some specific embodiments, step S22 includes:

[0205] Step S221: incubate the microfluidic chip with peroxidase.

[0206] Specifically, peroxidase can be injected into the microfluidic chip and incubated at room temperature for 5 minutes. The peroxidase used can be hydrogen peroxide of a certain concentration, but is not limited thereto.

[0207] Step S222, flushing the microfluidic chip.

[0208] Specifically, the washing solution or deionized water may be used to rinse three times.

[0209] In some specific examples, the washing solution may be a buffer solution containing a surfactant, such as phosphate buffered saline (PBS) containing Tween 20, but is not limited thereto.

[0210] Step S23: injecting antibody blocking buffer and incubating the biological tissue at room temperature to block non-specific binding.

[0211] In some specific embodiments, step S23 includes:

[0212] Step S231, incubate the microfluidic chip with an antibody blocking buffer.

[0213] Specifically, an antibody blocking buffer can be injected into the microfluidic chip and incubated at room temperature for a certain period of time, such as 1 hour.

[0214] Step S232: flush the microfluidic chip.

[0215] Specifically, the washing solution may be used for 3 times.

[0216] In some specific examples, the washing solution may be a buffer solution containing a surfactant, such as phosphate buffered saline (PBS) containing Tween 20, but is not limited thereto.

[0217] Step S3: labeling and imaging biological tissues in the microfluidic chip based on the complex with the shearable marker.

[0218] In some specific embodiments, the complex with a cleavable marker includes a molecular group that can bind to a protein tissue in the biological tissue under the catalysis of an enzyme, a marker, and a molecular linker connecting the molecular group and the marker;

[0219] The molecular link can be effectively cut in a cellular environment without damaging biological tissues, and does not affect the effective binding of the complex with protein tissues in the biological tissues.

[0220] In some specific embodiments, the enzyme may be horseradish peroxidase (HRP for short), but is not limited thereto.

[0221] In some specific embodiments, the marker includes but is not limited to detection labels such as fluorescent groups, and may also be other detectable molecules, including but not limited to micron-sized beads.

[0222] In some specific embodiments, the molecular linker includes an azido-based linker or an allyl-based linker or a structure that is cleaved by an enzyme, a nucleophile, an electrophile, a reducing agent, an oxidizing agent, light irradiation or a metal catalysis. For details, please refer to the prior art and will not be repeated here.

[0223] In some embodiments, the chemical formula of the complex using an azide-linked cleavable marker is as follows:

[0224]

[0225] Among them, T is a molecular group that binds to protein tissue in biological tissue under the catalysis of enzyme, and R is a marker.

[0226] In some specific embodiments, the molecular group that can bind to protein tissue in biological tissue under the catalysis of an enzyme includes tyramine.

[0227] In some specific embodiments, the chemical structure of the complex with a cleavable marker using an azide-type linker that can bind to protein tissues in biological tissues under the catalysis of an enzyme is as follows:

[0228]

[0229] Among them, R is a marker.

[0230] In some specific examples, the label includes a Cy5 fluorescent group, a tyramide that can bind to protein tissue in biological tissue under the catalysis of an enzyme, and a cleavable Cy5 fluorescent group. The chemical structure of the complex with the cleavable label is as follows:

[0231]

[0232] In some specific embodiments, the molecular group that can bind to protein tissues in biological tissues under the catalysis of an enzyme includes phenylacetamide.

[0233] In some specific embodiments, the chemical structure of the complex having a cleavable marker is as shown below:

[0234]

[0235] In the above formula (3), (4) or (5), Linker is a cleavable molecular link, R is a marker; X is hydrogen, and each group R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 can independently be hydrogen (H), alkyl, alkenyl, alkynyl, halogen, carboxyl, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxyl, phosphoryl, sulfonyl, carbonyl, boroyl, aryl or heteroaryl.

[0236] In some specific embodiments, step S3 includes:

[0237] Step S30', staining the cell nuclei of biological tissues.

[0238] In some specific embodiments, step S30' includes:

[0239] Step S301', diluting the cell nucleus marker solution (DAPI) and phosphate buffer saline (PBS).

[0240] Specifically, the marker labeling solution (DAPI) can be diluted into a phosphate buffered saline (PBS) solution. The specific ratio can be referred to in the prior art and will not be described in detail here.

[0241] Step S302', incubate the microfluidic chip with the marker solution DAPI.

[0242] Specifically, a cell nucleus marker solution (DAPI) can be injected into the microfluidic chip and incubated at room temperature for a certain period of time, such as 1-5 minutes.

[0243] Step S303', flushing the microfluidic chip.

[0244] Specifically, the microfluidic chip can be rinsed with the washing solution for 3 times. In some specific examples, the washing solution can be a buffer solution with a surfactant, such as phosphate buffered saline (PBS) containing Tween 20, but is not limited thereto.

[0245] Step S31 ′: perform background marker imaging.

[0246] In some specific embodiments, step S31' includes:

[0247] Scan and save the image on the microscope, check the background signal of the selected fluorescent channel, and save the microscope settings for imaging. The specific operation can refer to the existing technology and will not be described here.

[0248] Step S32 ′: using the complex with the shearable marker to perform multiple rounds of marker labeling and imaging on the biological tissue in the microfluidic chip.

[0249] In some embodiments, step S32' comprises:

[0250] Step S31 : labeling biological tissue with a marker based on a staining solution containing a complex of an active enzyme marker and a cleavable marker.

[0251] In some specific embodiments, step S31 includes:

[0252] Step S311: Use a specific primary antibody to identify the corresponding antigen on the tissue.

[0253] Specifically, step S311 includes:

[0254] Step S3111, injecting a primary antibody into the microfluidic chip for incubation, so that the primary antibody binds to the antigen.

[0255] Specifically, a primary antibody of appropriate concentration can be injected into the microfluidic chip and incubated at room temperature for 1 hour.

[0256] Step S3112, flushing the microfluidic chip.

[0257] Specifically, the microfluidic chip can be rinsed with a washing solution for 3 times. In some specific examples, the washing solution can be a buffer solution with a surfactant, such as phosphate buffered saline (PBS) containing Tween 20, but is not limited thereto.

[0258] In some specific embodiments, step S311 further includes:

[0259] Step S3110, preparing a primary antibody solution of appropriate concentration.

[0260] In some embodiments, the primary antibody is a protein that can specifically bind to a non-antibody antigen (specific antigen).

[0261] Specifically, a mixture of primary antibody and antibody blocking buffer is prepared in a container (e.g., in a 1.5 mL PCR tube). Antibody blocking buffer can reduce non-specific recognition of antibodies.

[0262] Step S312: using the secondary antibody with enzyme to identify the primary antibody.

[0263] In some specific embodiments, the secondary antibody is an antibody prepared in other hosts that can bind to the primary antibody or a fragment of the primary antibody, and is usually labeled with an enzyme (such as horseradish peroxidase) or fluorescein.

[0264] Specifically, step S312 includes:

[0265] Step S3121, injecting a secondary antibody with an enzyme into the microfluidic chip for incubation, so that the secondary antibody binds to the primary antibody.

[0266] Specifically, the enzyme-labeled secondary antibody can be injected into the microfluidic chip and incubated at room temperature for a certain period of time, such as 1 hour.

[0267] Step S3123, flushing the microfluidic chip.

[0268] Specifically, the washing solution may be used for 3 times.

[0269] In some specific examples, the washing solution may be a buffer solution containing a surfactant, such as phosphate buffered saline (PBS) containing Tween 20, but is not limited thereto.

[0270] In some specific embodiments, step S312 further includes:

[0271] Step S3120, preparing a mixture of enzyme-labeled secondary antibody and antibody blocking buffer.

[0272] Specifically, an enzyme-labeled secondary antibody-antibody blocking buffer can be prepared in a container (such as in a 1.5 mL PCR tube). The antibody blocking solution can reduce non-specific recognition of the antibody.

[0273] Step S313: Use a staining solution containing a complex of a shearable marker to label the enzyme-labeled antigen region.

[0274] Specifically, step S313 includes:

[0275] Step S3131: inject a staining solution containing a complex of a shearable marker into the microfluidic chip for incubation to label the marker antigen with the marker.

[0276] Specifically, the complex with the shearable marker can be injected into the microfluidic chip and incubated at room temperature for a certain period of time, such as 8-15 minutes (the microfluidic chip is placed incubated under light-proof conditions);

[0277] Step S3132, flushing the microfluidic chip.

[0278] Specifically, the washing solution may be used for rinsing 3 times. In some specific examples, the washing solution may be a buffer solution with a surfactant, such as phosphate buffered saline (PBS) containing Tween 20, but is not limited thereto.

[0279] In some specific embodiments, step S313 further includes:

[0280] Step S3130, preparing a staining solution containing a complex having a cleavable marker.

[0281] Specifically, the complex with the cleavable marker and the staining solution can be mixed in a container (such as a 1.5 mL PCR tube). The specific operation can refer to the prior art.

[0282] Step S32: performing marker imaging on the biological tissue marked with the marker.

[0283] Apply the same image settings as in step 'S31", scan and save the image (eg using a Zeiss AxioObserver microscope).

[0284] Step S33: removing active enzyme markers.

[0285] In some specific embodiments, step S33 includes:

[0286] Remove both primary and secondary antibodies, remove only the secondary antibody, or inactivate the enzyme.

[0287] In some embodiments, the primary antibody and the secondary antibody are removed simultaneously, comprising:

[0288] Step S331, heating the heating device of the microfluidic chip to a predetermined temperature.

[0289] Specifically, a heating device (such as a heating plate) for heating the microfluidic chip may be set to a certain temperature, such as 85-105°C.

[0290] Step S332, injecting antigen repair solution into the microfluidic chip, placing the microfluidic chip in a heating device, and incubating the microfluidic chip under heating conditions to cut the primary antibody that recognizes the biological tissue and the secondary antibody that binds to the primary antibody.

[0291] In some specific embodiments, step S332 includes:

[0292] injecting antigen retrieval solution into the microfluidic chip;

[0293] The microfluidic chip is placed in a heating device for incubation at a certain temperature (such as 85-105°C) for a certain time (such as 20-40 minutes) or until the set temperature is reached, and the primary antibody and the secondary antibody bound to the primary antibody are removed through the antigen retrieval process.

[0294] Step S333, cooling the microfluidic chip.

[0295] Specifically, the microfluidic chip heating device, such as a hot plate, can be turned off, and the microfluidic chip can be removed from the hot plate for a certain period of time (eg, 5-15 minutes) or until it cools to room temperature.

[0296] Step S334, flushing the microfluidic chip.

[0297] Specifically, the washing solution may be used for 3 times. In some specific examples, the washing solution may be a buffer solution with a surfactant, such as phosphate buffered saline (PBS) containing Tween 20, but is not limited thereto.

[0298] Regarding the scheme for deactivating the enzyme, specific reference may be made to the prior art and will not be described in detail here.

[0299] Regarding the solution of removing only the secondary antibody, I will not go into details here.

[0300] Step S34, cutting out the markers marking the biological tissue.

[0301] In some specific embodiments, step S34 includes:

[0302] Step S341: inject shearing fluid into the microfluidic chip, and incubate the biological tissue in the microfluidic chip under heating conditions to shear the markers marking the biological tissue.

[0303] In some specific embodiments, the shearing liquid can be tri(2-carboxyethyl)phosphine (TCEP), terephthalic acid (PTA), or a mixture of tri(2-carboxyethyl)phosphine (TCEP) and terephthalic acid (PTA).

[0304] In some embodiments, the fluorescent molecule shear fluid may be dissolved first.

[0305] Specifically, a 1.5 mL PCR tube can be used to dissolve the fluorescent molecule shearing solution into the corresponding compatible solution.

[0306] Furthermore, a fluorescent molecule shearing solution may be injected into the microfluidic chip and incubated at a certain temperature (eg, 35-45° C.) for a suitable time (eg, 10-30 minutes) to shear the markers of the labeled biological tissue.

[0307] Step S3432, flushing the microfluidic chip.

[0308] Specifically, the washing solution may be used for 3 times. In some specific examples, the washing solution may be a buffer solution with a surfactant, such as phosphate buffered saline (PBS) containing Tween 20, but is not limited thereto.

[0309] In some specific embodiments, step S34 further includes:

[0310] Step S340: preheating a heating device for heating the microfluidic chip to a predetermined temperature.

[0311] Specifically, the heating device for heating the microfluidic chip includes but is not limited to a heating plate, an oven, etc.; the predetermined temperature may be within a certain range, for example, the predetermined temperature of the heating plate for heating the microfluidic chip may be set to 35-45° C. Subsequently, the microfluidic chip may be placed in a heating device that reaches the predetermined temperature, so that the microfluidic chip is incubated under heating conditions.

[0312] Step S35, repeat steps S31-S34 for the biological tissue to be tested until the processing result meets the requirements.

[0313] In some specific embodiments, if all biological tissues on the tissue sections are correctly stained, the processing result is considered to meet the requirements. The judgment criteria for correct staining of biological tissues can be determined by those skilled in the art based on existing technologies and will not be repeated here.

[0314] Step S4: performing hematoxylin and eosin tissue staining and color imaging on the biological tissue that has been marked with the marker and imaged.

[0315] In some specific embodiments, step S4 includes:

[0316] Step S400: performing hematoxylin and eosin tissue staining on the biological tissue that has been marked with the marker and imaged.

[0317] Step S41: inject hematoxylin and eosin (H&E) tissue staining solution into the microfluidic chip and incubate at room temperature for a predetermined time.

[0318] Step S42: rinse the microfluidic chip with deionized water.

[0319] Specifically, the microfluidic chip can be rinsed with deionized water three times each time.

[0320] Step S43: inject Bluing reagent and incubate at room temperature for a predetermined time.

[0321] Specifically, Bluing reagent can be injected and incubated at room temperature for 10-15 seconds.

[0322] Step S44, rinse the microfluidic chip with deionized water.

[0323] Specifically, the microfluidic chip can be rinsed with deionized water three times each time.

[0324] Step S45, flushing the microfluidic chip with ethanol.

[0325] Specifically, the microfluidic chip can be rinsed once each time using deionized water.

[0326] Step S46, connecting the vacuum kit to the port of the microfluidic chip to dry the biological tissue;

[0327] Step S47: injecting eosin Y solution into the microfluidic chip and incubating at room temperature for a predetermined time.

[0328] Specifically, the incubation may be carried out at room temperature for 2-3 minutes.

[0329] Step S48, rinse the microfluidic chip with ethanol.

[0330] Specifically, the microfluidic chip can be rinsed with deionized water three times each time.

[0331] Step S410: performing bright field imaging on the biological tissue after hematoxylin and eosin tissue staining.

[0332] Specifically, the chemical reaction of hematoxylin and eosin staining is a self-luminous process, and the color can be seen under bright field but not under fluorescence. Based on this, imaging can be performed under bright field, using Keyence BZ-X810 or any digital microscope, but not limited to this.

[0333] Specific examples

[0334] Figure 3-Figure 4 An example of using fluorescent groups for fluorescent staining as markers is shown in FIG. Figure 3 , Fluorescence staining and imaging of FFPE tonsils in microfluidic chips: The same FFPE tonsil tissue at the same location was stained for 12 different proteins using CFP and a microfluidic chip, and the cell nuclei were stained with DAPI (scale bar, 250 μm).

[0335] The 12 different stains correspond to 12 different protein markers. DAPI staining is targeted at the cell nucleus, which helps to identify the distribution and location of cells on the slice, and also helps to align markers after multiple staining.

[0336] Figure 4 The H&E staining results of FFPE tonsils after 12 protein stainings are shown. It can be seen that multiple stainings in the microfluidic chip do not have a significant chemical impact on the FFPE slices themselves, and subsequent H&E detection and analysis can still be performed (the complete slice tissue morphology can be seen after hematoxylin and eosin tissue staining, proving that the tissue has not been destroyed after multiple rounds of fluorescent staining). However, the technology of other manufacturers will affect FFPE, making it impossible to perform subsequent H&E detection.

[0337] Comparative Example

[0338] In some embodiments, see Figure 5-Figure 6 The example shown is a fluorescent staining of a thyroid tissue section using a fluorescent group as a marker. Figure 6 The results of fluorescent staining of thyroid tissue sections in a microfluidic chip based on the complex with a shearable marker of the present application are shown. Figure 5 The results of fluorescent staining using traditional staining methods are shown. It can be seen that compared with traditional staining methods, after fluorescent staining of thyroid tissue sections in the microfluidic chip, the staining effect is more obvious, the signal intensity is higher, the signal-to-noise ratio is better, and the distribution is more obvious, which is conducive to the subsequent identification of the distribution of different single cells.

[0339] In this embodiment, biological tissues are labeled with multiple markers using a staining solution of a complex with shearable markers in a resealable microfluidic chip, so that the biological tissues and the staining solution of the complex with shearable markers are mixed more evenly, the multiple marker labeling effect is better, and there is no significant chemical impact on the biological tissues themselves after the multiple marker labeling; because the microfluidic chip is resealable, that is, the microfluidic chip can be reused, the glass slide containing the biological tissue that has been tested can be disassembled for subsequent storage and can be used for other biological tests (such as multiple RNA tests, hematoxylin and eosin tests, etc.), so as to obtain maximized biological detection diagnostic information on the same piece of biological tissue.

[0340] Some other embodiments of the present application provide a biomolecule detection system, the biomolecule detection system comprising:

[0341] A microfluidic chip comprising a resealable flow cell and a biological tissue, the biological tissue corresponding to the flow cell;

[0342] a microfluidic chip processing unit configured to pre-process biological tissue in the microfluidic chip;

[0343] A staining processing unit, which is configured to: perform marker marking and imaging processing on biological tissues in the microfluidic chip based on a complex having a shearable marker;

[0344] The hematoxylin and eosin tissue staining unit is configured to perform hematoxylin and eosin tissue staining and color imaging on biological tissues that have been marked with markers.

[0345] In some specific embodiments, the preprocessing comprises:

[0346] injecting antigen repair solution, incubating the biological tissue for antigen repair under heating conditions, and cooling;

[0347] Inject peroxidase and incubate the biological tissue at room temperature to block endogenous peroxidase activity;

[0348] Inject antibody blocking buffer and incubate the tissue at room temperature to block nonspecific binding.

[0349] In some specific embodiments, labeling and imaging of biological tissues in a microfluidic chip based on a complex having a cleavable marker includes:

[0350] Marking the cell nuclei of biological tissues with markers;

[0351] Perform background marker imaging;

[0352] Multiple rounds of labeling and imaging of biological tissues in microfluidic chips were performed using complexes with cleavable markers.

[0353] In some specific embodiments, multiple rounds of labeling and imaging of biological tissues in a microfluidic chip using a complex having a cleavable label include:

[0354] Labeling biological tissues with markers based on a staining solution containing a complex of an active enzyme marker and a cleavable marker;

[0355] Performing marker imaging on biological tissues labeled with markers;

[0356] Removal of active enzyme tags;

[0357] Cutting markers to mark biological tissues;

[0358] Repeat the above steps for the biological tissue to be tested until the processing results meet the requirements.

[0359] In some embodiments of the present application, the scheme of the biomolecule detection method embodiment can be combined with the biomolecule detection system embodiment, and the specific implementation of the microfluidic chip, microfluidic chip processing unit, staining processing unit, staining unit, and imaging unit can refer to the biomolecule detection method and will not be repeated here.

[0360] In this embodiment, biological tissues are labeled with multiple markers using a staining solution of a complex with shearable markers in a resealable microfluidic chip, so that the biological tissues and the staining solution of the complex with shearable markers are mixed more evenly, the multiple marker labeling effect is better, and there is no significant chemical impact on the biological tissues themselves after the multiple marker labeling; because the microfluidic chip is resealable, that is, the microfluidic chip can be reused, the glass slide containing the biological tissue that has been tested can be disassembled for subsequent storage and can be used for other biological tests (such as multiple RNA tests, hematoxylin and eosin tests, etc.), so as to obtain maximized biological detection diagnostic information on the same piece of biological tissue.

[0361] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting biomolecules, It is characterized in that The biomolecule detection method comprises: preparing a microfluidic chip, the microfluidic chip comprising a resealable flow unit and a biological tissue to be tested, the biological tissue corresponding to the flow unit; Pre-treat biological tissues in microfluidic chips; Marking and imaging biological tissues in microfluidic chips based on complexes with shearable markers; Perform hematoxylin and eosin tissue staining and color imaging on biological tissues that have been labeled with markers and processed for imaging.

2. The biomolecule detection method according to claim 1, Features: Based on the complex with a cleavable marker, the biological tissue in the microfluidic chip is labeled and imaged, including: Marking the cell nuclei of biological tissues with markers; Perform background marker imaging; Multiple rounds of labeling and imaging of biological tissues in microfluidic chips were performed using complexes with cleavable markers.

3. The biomolecule detection method according to claim 2, Features: Multiple rounds of labeling and imaging of biological tissues in microfluidic chips using complexes with cleavable markers, including: Labeling biological tissues with markers based on a staining solution containing a complex of an active enzyme marker and a cleavable marker; Performing marker imaging on biological tissues labeled with markers; Removal of active enzyme tags; Cutting markers to mark biological tissues; Repeat the above steps for the biological tissue to be tested until the processing results meet the requirements.

4. The biomolecule detection method according to claim 3, Features: The staining solution based on active enzyme labeling and complexes with cleavable labels is used to label biological tissues, including: Use specific primary antibodies to recognize the corresponding antigen on the tissue; Use enzyme-linked secondary antibodies to recognize primary antibodies; The region of the antigen labeled with the enzyme is labeled with the marker using a staining solution containing a complex with a cleavable marker.

5. The biomolecule detection method according to claim 4, Features: Use specific primary antibodies to recognize the corresponding antigen on the tissue, including: Injecting a primary antibody into the microfluidic chip for incubation so that the primary antibody binds to the antigen; Rinse the microfluidic chip.

6. The biomolecule detection method according to claim 4, Features: Use an enzyme-linked secondary antibody to recognize the primary antibody, including: Injecting a secondary antibody with an enzyme into the microfluidic chip for incubation so that the secondary antibody binds to the primary antibody; Rinse the microfluidic chip.

7. The biomolecule detection method according to claim 4, Features: Use a staining solution with a cleavable marker complex to label the region of the enzyme-labeled antigen, including: Injecting a staining solution containing a complex of a shearable marker into the microfluidic chip for incubation to label the marker antigen with the marker; Rinse the microfluidic chip.

8. The biomolecule detection method according to claim 3, Features: Active enzyme labels include primary antibodies that recognize corresponding antigens on biological tissues and secondary antibodies that recognize the primary antibodies and are labeled with enzymes; Removal of active enzyme labels, including: simultaneous removal of primary and secondary antibodies, removal of secondary antibodies only, or inactivation of enzyme labels.

9. The biomolecule detection method according to claim 3, Features: Cutting markers for biological tissues, including: Injecting a shearing fluid into the microfluidic chip, and incubating the biological tissue in the microfluidic chip under heating conditions to shear the markers of the biological tissue; Rinse the microfluidic chip.

10. The biomolecule detection method according to claim 1, Features: Complexes with cleavable markers include: Molecular groups, markers, and molecular links connecting the molecular groups and markers that can bind to protein tissues in the biological tissues under the catalysis of enzymes; The molecular link can be effectively cut in a cellular environment without damaging biological tissues, and does not affect the effective binding of the complex with protein tissues in the biological tissues.

11. The biomolecule detection method according to claim 10, Features: The molecular linkage includes an azido linkage or an allyl linkage or a structure that is cleaved by an enzyme, a nucleophile, an electrophile, a reducing agent, an oxidizing agent, light irradiation or metal catalysis.

12. The biomolecule detection method according to claim 11, Features: The chemical formula of the complex with a cleavable marker using an azido-linked compound is as follows: Among them, T is a molecular group that can bind to protein tissue in biological tissue under the catalysis of enzyme, and R is a marker.

13. The biomolecule detection method according to claim 12, Features: The molecular group that can be combined with protein tissue in biological tissue under the catalysis of enzyme includes tyramine.

14. The biomolecule detection method according to claim 13, Features: The chemical formula of the complex with a cleavable marker using an azide-type linker that can bind to protein tissue in biological tissue under enzyme catalysis is as follows: Among them, R is a marker.

15. The biomolecule detection method according to claim 14, Features: The label includes a fluorescent group, an azide-type linker, a tyramide that can bind to protein tissue in biological tissue under the catalysis of an enzyme, and a cleavable Cy5 fluorescent group. The chemical structure of the complex with the cleavable label is as follows:

16. The biomolecule detection method according to claim 10, Features: The molecular groups that can be combined with protein tissues in biological tissues under the catalysis of enzymes include phenylacetamide.

17. The biomolecule detection method according to claim 10, Features: The chemical structure of the complex with a cleavable marker is as follows: Wherein, Linker is the cleavable molecular link, R is a marker; X is hydrogen, and each group R1, R2, R3, R4, R5, R6 and R7 can independently be hydrogen, alkyl, alkenyl, alkynyl, halogen, carboxyl, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxyl, phosphoryl, sulfonyl, carbonyl, boroyl, aryl or heteroaryl.

18. The biomolecule detection method according to claim 10, Features: The chemical structure of the complex with a cleavable marker is as follows: Wherein, Linker is the cleavable molecular link, R is a marker; X is hydrogen, and each group R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 can independently be hydrogen, alkyl, alkenyl, alkynyl, halogen, carboxyl, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxyl, phosphoryl, sulfonyl, carbonyl, boroyl, aryl or heteroaryl.

19. The biomolecule detection method according to claim 10, Features: The chemical structure of the complex with a cleavable marker is as follows: Wherein, Linker is a cleavable molecular link, R is a marker; X is hydrogen, and each group R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 can independently be hydrogen, alkyl, alkenyl, alkynyl, halogen, carboxyl, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxyl, phosphoryl, sulfonyl, carbonyl, boroyl, aryl or heteroaryl.

20. The biomolecule detection method according to claim 1, Features: Pre-process biological tissues in microfluidic chips, including: injecting antigen repair solution, incubating the biological tissue for antigen repair under heating conditions, and cooling; Inject peroxidase and incubate the biological tissue at room temperature to block endogenous peroxidase activity; Inject antibody blocking buffer and incubate the tissue at room temperature to block nonspecific binding.

21. The biomolecule detection method according to claim 1, Features: The microfluidic chip comprises: A resealable fluidic device having a resealable flow cell; a glass slide provided with biological tissue, the glass slide being fixedly mounted to a resealable fluidic device; A snap fit is used to securely mount the slide to the resealable fluidic device.

22. The biomolecule detection method according to claim 21, Features: The resealable fluidic device comprises: frame; a cover glass, fixed to the frame as the top of the flow cell, which is provided with fluid inlet and outlet; A layer of optical sealing material is combined with the cover glass to form a resealable flow cell.

23. The biomolecule detection method according to claim 1, Features: Preparation of microfluidic chip includes: Baking slides with biological tissue; Dewaxing biological tissues on glass slides and assembling microfluidic chips; Wherein, assembling the microfluidic chip comprises: Mount the coverslip to the frame; mounting an optical sealing material to a cover glass to form a resealable flow chamber; Remove the film from the bottom of the coverslip; Place the slide in the frame; Install the clips to complete the assembly of the microfluidic chip.

24. The biomolecule detection method according to claim 1, Features: Preparation of microfluidic chip includes: Obtain slides; Assembling microfluidic chips; Baking the microfluidic chip; Dewaxing the biological tissue in the microfluidic chip; Wherein, assembling the microfluidic chip comprises: Mount the coverslip to the frame; mounting an optical sealing material to a cover glass to form a resealable flow chamber; Remove the film from the bottom of the coverslip; Place the slide in the frame; Install the clips to complete the assembly of the microfluidic chip.

25. A biomolecule detection system, It is characterized in that The biomolecule detection system comprises: A microfluidic chip comprising a resealable flow cell and a biological tissue, the biological tissue corresponding to the flow cell; a microfluidic chip processing unit configured to pre-process biological tissue in the microfluidic chip; A staining processing unit, which is configured to: perform marker marking and imaging processing on biological tissues in the microfluidic chip based on a complex having a shearable marker; The hematoxylin and eosin tissue staining unit is configured to perform hematoxylin and eosin tissue staining and color imaging on biological tissues that have been marked with markers.

26. The biomolecule detection system according to claim 25, It is characterized in that The pre-processing comprises: injecting antigen repair solution, incubating the biological tissue for antigen repair under heating conditions, and cooling; Inject peroxidase and incubate the biological tissue at room temperature to block endogenous peroxidase activity; Inject antibody blocking buffer and incubate the tissue at room temperature to block nonspecific binding.

27. The biomolecule detection system according to claim 25, It is characterized in that Based on the complex with a cleavable marker, the biological tissue in the microfluidic chip is labeled and imaged, including: Marking the cell nuclei of biological tissues with markers; Perform background marker imaging; Multiple rounds of labeling and imaging of biological tissues in microfluidic chips were performed using complexes with cleavable markers.

28. The biomolecule detection system according to claim 27, Features: Multiple rounds of labeling and imaging of biological tissues in microfluidic chips using complexes with cleavable markers, including: Labeling biological tissues with markers based on a staining solution containing a complex of an active enzyme marker and a cleavable marker; Performing marker imaging on biological tissues labeled with markers; Removal of active enzyme tags; Cutting markers to mark biological tissues; Repeat the above steps for the biological tissue to be tested until the processing results meet the requirements.

Images