Chip for biomarker detection and preparation method and application thereof

Abstract

The invention relates to a chip for biomarker detection and a preparation method and application thereof. The chip comprises a conductive layer and an insulating polymer layer which are stacked in sequence, a plurality of micropores are formed in the insulating polymer layer, and each micropore penetrates through the insulating layer; or a semiconductor layer is also arranged between the conductive layer and the insulating polymer layer; the semiconductor layer comprises a semiconductor modified by a silane coupling agent and / or a titanate coupling agent. The microholes can capture various fixed types of fluorescent dyes at the same time, the chip for biomarker detection is used in immunoassay detection, various different markers can be detected, chemical, optical and electric reactions are carried out under the action of chemical reagents, light and electricity, multi-fluorescence imaging is carried out under multiple laser channels, and the biomarker detection accuracy is improved. The multi-channel biomarker detection based on electrochemical single-molecule immunoassay detection is realized, and the method has the advantages of low cost, high sensitivity, simple detection steps and rapid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay detection, and in particular to a chip for biomarker detection and a preparation method and application thereof. Background Art

[0002] The commonly used methods for detecting protein concentration in hospitals and other medical institutions are enzyme-linked immunosorbent assay (ELISA) and chemiluminescence. The main principle is to use antigen-antibody coupling to quantify the concentration of the substance to be measured by quantifying the collected light intensity. It is a collective response to the overall concentration of all target molecules in the entire target solution. In practical applications, it is often used for detection in the middle and late stages of the disease, that is, at a high level of related protein concentration in the body. For the early stage of the disease, the sampling amount is limited, and the concentration is close to the single molecule level. The above methods are far from the detection ability, and the disease cannot be diagnosed effectively and timely, missing the best window period for the treatment of related diseases. Therefore, the development of an in vitro detection method based on single molecule level detection is of great significance for early disease diagnosis.

[0003] The most sensitive commercial detection method is the single molecule array (SiMoA), which is derived from the traditional enzyme-linked immunosorbent assay and is a "sandwich" double antibody sandwich detection method based on microbeads. The main principle is to use a microwell array to capture a single magnetic bead, and after incubation, the ratio of the positive signal to the total magnetic beads is counted to achieve the purpose of quantitative detection. First, the magnetic beads coated with excess capture antibodies far exceeding the amount of the analyte are used to capture the analyte. Based on the principle of Poisson distribution, each magnetic bead captures one or zero analytes, that is, immune complexes, and then all the magnetic beads are loaded into the microwell array. The size of a microwell can contain only one magnetic bead, which means that each microwell will contain one or no magnetic bead. Finally, a fluorescent signal is formed by means of signal amplification. The highly localized fluorescent signal will indicate the result. The ratio of the number of microwells that generate fluorescence to the total number of microwells is statistically calculated by mathematical methods to quantify the concentration of the analyte. However, this type of single-molecule microsphere amplification mechanism represented by SiMoA generally has problems such as high price and complicated detection steps, which limits its application in actual clinical practice. Summary of the invention

[0004] Based on this, the present invention provides a high-throughput, multi-channel chip for biomarker detection, which can be used in immunoassay detection, especially multi-channel biomarker detection based on electrochemical single molecule immunoassay detection, and has the advantages of low cost, high sensitivity, simple detection steps and rapid detection.

[0005] The first aspect of the present invention provides a chip for biomarker detection, and the technical solution is as follows:

[0006] A chip for biomarker detection, comprising:

[0007] Conductive layers and insulating layers are stacked in sequence;

[0008] The insulating layer contains a plurality of micropores, and each of the micropores penetrates the insulating layer.

[0009] In one of the embodiments, a semiconductor layer is further provided between the conductive layer and the insulating layer;

[0010] The semiconductor layer includes a semiconductor modified by a silane coupling agent and / or a titanate coupling agent.

[0011] In one embodiment, the material of the semiconductor layer is selected from at least one of silicon carbide, germanium, silicon, selenium, boron, tellurium, antimony, gallium arsenide, indium phosphide, bismuth telluride and cadmium sulfide.

[0012] In one embodiment, the thickness of the semiconductor layer is 0.1 nm to 500 nm.

[0013] In one embodiment, the silane coupling agent is selected from at least one of an aminosilane coupling agent, a mercaptosilane coupling agent, an alkoxysilane coupling agent and a halogen silane coupling agent.

[0014] In one embodiment, the silane coupling agent is selected from at least one of 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-glycidyloxypropyltrimethoxysilane, vinyltrichlorosilane, vinyltrimethoxysilane, vinyltriethoxysilane and phenylaminopropyltrimethoxysilane.

[0015] In one embodiment, the titanate coupling agent is selected from at least one of a monoalkoxy titanate coupling agent, a monoalkoxy phosphate titanate coupling agent, an integrated titanate coupling agent and a ligand titanate coupling agent.

[0016] In one embodiment, the titanate coupling agent is selected from at least one of isopropyl triisophthaloyl titanate coupling agent, isopropyl trioleophthaloyl titanate coupling agent, isopropyl tri(dodecylbenzenesulfonyl) titanate coupling agent, isopropyl tri(dioctyl pyrophosphoryloxy) titanate coupling agent, isopropyl tri(dioctyl pyrophosphoryl) titanate coupling agent, isopropyl tri(dioctyl pyrophosphoryl) titanate coupling agent, bis(dioctyl pyrophosphate) glycolate titanate coupling agent and dicarboxylic ethylene diethylene titanate coupling agent.

[0017] In one embodiment, the material of the insulating layer includes at least one of an insulating polymer material and an inorganic insulating material.

[0018] In one embodiment, the insulating polymer material is selected from at least one of polydimethylsiloxane, epoxy resin, phenolic resin, polyester, polyurethane, silicone resin, polyimide alkyd resin, polystyrene, polypropylene, polycarbonate, polytetrafluoroethylene, polyimide, polyamide, silicone rubber, polybutadiene and fluorine-containing resin.

[0019] In one embodiment, the inorganic insulating material is selected from at least one of silicon dioxide, silicon nitride, aluminum oxide, boron nitride, hafnium dioxide, zinc oxide, titanium dioxide, zirconium dioxide and yttrium trioxide.

[0020] In one embodiment, the insulating layer is a silicone rubber layer.

[0021] In one embodiment, the material of the silicone rubber layer is polydimethylsiloxane.

[0022] In one embodiment, the thickness of the insulating layer is ≥100 nm.

[0023] In one embodiment, the diameter of a single micropore is ≥ 100 nm.

[0024] In one embodiment, the cross-sectional shape of the micropores includes at least one of a triangle, a square, a rectangle, a circle, an ellipse, a rhombus, a hexagon, an octagon, a pentagon, a trapezoid, a parallelogram, a heart, a pentagram, a heptagon and a semicircle.

[0025] In one embodiment, the material of the conductive layer includes at least one of indium tin oxide, aluminum-doped zinc oxide, gallium-doped zinc oxide, fluorine-doped tin oxide, graphene, metal nanowires, carbon nanotubes, conductive polymers, gold, silver, platinum, copper and aluminum.

[0026] In one embodiment, the thickness of the conductive layer is ≥ 1 nm.

[0027] A second aspect of the present invention provides a method for preparing a chip for biomarker detection as described above, comprising the following steps:

[0028] An insulating layer is prepared on the conductive layer, and a plurality of micropores are prepared in the insulating layer.

[0029] In one embodiment, a method for preparing a chip for biomarker detection as described above comprises the following steps:

[0030] preparing a semiconductor layer on the surface of the conductive layer;

[0031] Modifying the semiconductor layer by using a silane coupling agent and / or a titanate coupling agent;

[0032] An insulating layer is prepared on the modified semiconductor layer, and a plurality of micropores are prepared in the insulating layer.

[0033] In one embodiment, the method for preparing the conductive layer is selected from at least one of magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition.

[0034] In one embodiment, the method for preparing the semiconductor layer is selected from at least one of magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition.

[0035] In one embodiment, the modifying of the semiconductor layer by using a silane coupling agent and / or a titanate coupling agent comprises the following steps:

[0036] The semiconductor layer is immersed in a solution containing the silane coupling agent and / or titanate coupling agent for ≥0.5h, washed, and heat treated at 30°C to 120°C for ≥5min.

[0037] In one embodiment, the modifying of the semiconductor layer by using a silane coupling agent and / or a titanate coupling agent comprises the following steps:

[0038] The semiconductor layer and the solution containing the silane coupling agent and / or titanate coupling agent are placed in the same closed space for ≥0.5h, washed, and heat-treated at 30°C to 120°C for ≥5min.

[0039] In one embodiment, the solution containing the silane coupling agent and / or titanate coupling agent includes 1% to 20% of the silane coupling agent and / or titanate coupling agent and 80% to 99% of the solvent, calculated by volume percentage.

[0040] In one embodiment, the solvent is selected from at least one of ethanol, toluene, ethylbenzene and n-hexane.

[0041] In one embodiment, the method for preparing the insulating layer is selected from at least one of magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition.

[0042] The third aspect of the present invention provides a kit, which includes the chip for biomarker detection as described above.

[0043] The fourth aspect of the present invention provides an immunoassay detection method, the technical solution is as follows:

[0044] An immunoassay detection method comprises the following steps:

[0045] contacting the chip for biomarker detection as described above with an electrolyte;

[0046] Applying the capture agent and the biomarker to be detected with a label into the microwell as described above;

[0047] Connecting the chip to an electrochemical workstation via a working electrode, a reference electrode and a counter electrode;

[0048] Immunoassay detection is performed by combining electrochemical regulation with fluorescence detection imaging.

[0049] In one embodiment, the marker includes a first fluorescent dye and a second fluorescent dye;

[0050] At least one of the first fluorescent dye and the second fluorescent dye contains an aromatic carboxyl group in its structure.

[0051] In one embodiment, the difference between the optimal excitation wavelengths of the first fluorescent dye and the second fluorescent dye is 60 nm to 100 nm.

[0052] In one embodiment, the structure of the aromatic carboxyl group is as shown in formula (I) or formula (II):

[0053]

[0054] Each X is independently selected from O, Si or N;

[0055] Each R is independently selected from hydrogen, deuterium, halogen, alkyl, alkoxy, haloalkyl, silyl, carbonyl, ester, hydroxyl, amino, nitro, carboxyl, sulfonic acid, alkenyl, carbamoyl, haloformyl, formyl, isocyanate, isocyanate, thiocyanate, isothiocyanate, aromatic or heteroaromatic.

[0056] In one embodiment, the first fluorescent dye and the second fluorescent dye are independently selected from at least one of cyanine, rhodamine, FITC and dylight fluorescent dyes.

[0057] In one embodiment, the electrolyte includes at least one of an organic electrolyte, an ionic liquid electrolyte and a lithium salt electrolyte.

[0058] In one embodiment, the organic electrolyte includes but is not limited to at least one of tetrabutylammonium trifluoromethanesulfonate, tetrabutylammonium hexafluorophosphate, tetraethylammonium tetrafluoroborate, tetraethylammonium bromide, tetraethylammonium iodide, tetra-n-butylammonium bromide and tetra-n-butylammonium iodide.

[0059] In one embodiment, the ionic liquid electrolyte includes a cationic liquid electrolyte and an anionic liquid electrolyte.

[0060] In one embodiment, the cationic liquid electrolyte includes but is not limited to at least one of cationic liquid electrolytes containing imidazole, pyrrolidine, pyridine, morpholine, piperidine, quaternary ammonium, quaternary phosphonium and guanidine cations.

[0061] In one embodiment, the anionic liquid electrolyte includes but is not limited to a halogen salt (such as Cl - Br-, I-, Br 3- ), alkali salts (such as OH - , CO 3 2- , HCO 3 - ) and halides (such as FeCl 4 - 、AlCl 4 - 、ZnCl 3 - ) type anion in at least one of the anionic liquid electrolytes.

[0062] In one embodiment, the lithium salt electrolyte includes but is not limited to at least one of lithium perchlorate, lithium nitrate, lithium hexafluorophosphate, lithium tetrafluoroborate and lithium hexafluoroarsenate.

[0063] In one embodiment, the concentration of the electrolyte in the electrolyte solution is 0.001 mol / L to 1 mol / L.

[0064] In one embodiment, the concentration of the electrolyte in the electrolyte solution is 0.1 mol / L.

[0065] In one embodiment, the solvent in the electrolyte is an organic solvent.

[0066] In one embodiment, the solvent in the electrolyte is selected from at least one of dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone, ethanol, ether and acetone.

[0067] In one embodiment, the solvent in the electrolyte is water, and the electrolyte also includes sodium borohydride.

[0068] In one embodiment, in the electrolyte, the concentration of sodium borohydride is 10 -6 mol / L~1mol / L.

[0069] In one embodiment, the capture agent is selected from at least one of an antibody or a derivative thereof, a nucleic acid, a protein, an inorganic molecule and a polymer.

[0070] In one embodiment, the concentration of the capture agent is 1 mol / L to 10 -8 mol / L.

[0071] In one embodiment, the biomarker to be detected is selected from at least one of proteins, DNA, RNA, enzymes, peptides, carbohydrates, lipids, nucleic acids, polymers and inorganic molecules.

[0072] In one embodiment, the concentration of the biomarker to be detected is 10 -6 mol / L~10 -18 mol / L.

[0073] In one embodiment, the concentration of the biomarker to be detected is 1 nmol / L to 1 amol / L.

[0074] In one embodiment, the material of the working electrode is selected from at least one of indium tin oxide, aluminum-doped zinc oxide, gallium-doped zinc oxide, fluorine-doped tin oxide, graphene, metal nanowires, carbon nanotubes, conductive polymers, gold, silver, platinum, copper and aluminum.

[0075] In one embodiment, the material of the counter electrode is selected from at least one of silver, platinum and glassy carbon.

[0076] In one embodiment, the material of the reference electrode is selected from at least one of silver / silver chloride, silver, silver oxide, calomel, mercury / mercurous sulfate, hydrogen electrode, mercury / mercurous oxide, and a conductive polymer reference electrode.

[0077] In one embodiment, the imaging system in the fluorescence detection imaging method is selected from at least one of flow cytometry, wide field, confocal scanning, two-photon microscopy and total internal reflection fluorescence microscopy fluorescence detection techniques.

[0078] In one embodiment, the electrochemical regulation method is selected from at least one of controlled current step, controlled potential step method, constant current method, constant potential method, linear voltammetry, cyclic voltammetry and AC impedance method.

[0079] The present invention has at least the following beneficial effects:

[0080] The chip for biomarker detection provided by the present invention comprises a conductive layer and an insulating layer stacked in sequence, and the insulating layer contains a plurality of micropores. The micropores in the insulating layer can simultaneously capture multiple fixed types of fluorescent dyes. The chip for biomarker detection provided by the present invention is used in immunoassay detection, and can detect a variety of different markers. Under the action of chemical reagents, light, and electricity, chemical, light, and electricity reactions occur, and multi-fluorescence imaging is performed under multiple laser channels to realize multi-channel biomarker detection based on electrochemical single-molecule immunoassay detection, which has the advantages of low cost, high sensitivity, simple detection steps, and rapid detection.

[0081] Furthermore, a semiconductor layer modified with a silane coupling agent and / or a titanate coupling agent is arranged between the conductive layer and the insulating layer. The semiconductor layer modified with the coupling agent can form a chemical coupling with the capture agent and the biomarker to be detected. Any marker includes a fluorescent dye, and up to 7 different markers can be detected simultaneously. Under the action of chemical reagents, light and electricity, chemical, optical and electrical reactions occur, and multi-fluorescence imaging is performed under multiple laser channels to realize multi-channel biomarker detection based on electrochemical single molecule immunoassay detection, which has the advantages of low cost, high sensitivity, simple detection steps and rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 The schematic diagram of the structure of a chip for biomarker detection prepared in one embodiment of the present invention; Figure 1 A is a cross-sectional diagram. Figure 1 B is a top view; layer 100 is an indium tin oxide layer, and layer 200 is a polydimethylsiloxane layer;

[0083] Figure 2 The schematic diagram of the structure of a chip for biomarker detection prepared in one embodiment of the present invention; Figure 2 A is a cross-sectional diagram. Figure 2 B is a top view; wherein layer 100 is an indium tin oxide layer, layer 200 is a silicon carbide layer, and layer 300 is a polydimethylsiloxane layer;

[0084] Figure 3 A schematic diagram of the working principle of a chip for biomarker detection prepared according to an embodiment of the present invention;

[0085] Figure 4 The diagram is a switching mechanism diagram of Cy2, Alexa Fluor 488, Cy3, ATTO565, Alexa Fluor546, Cy5 and Si-rhodamine in Example 1 of the present invention;

[0086] Figure 5The switching responses of Cy2, Alexa Fluor 488, Cy3, ATTO565, Alexa Fluor546, Cy5, and Si-rhodamine at specific voltages in Example 1 of the present invention;

[0087] Figure 6 The photoelectric response results of Cy3 (red) and ATTO 565 (blue) under the 561nm laser channel, where (a) is a schematic diagram of the signal intensity (ordinate)-potential (abscissa) response of two different fluorescent dye molecules in the same fluorescence channel, (b) is a schematic diagram of single-molecule fluorescence collected at a specific potential, and (c) is a schematic diagram of single-molecule fluorescence after differential processing to achieve signal separation;

[0088] Figure 7 The results of the detection of 6 target molecules after randomly mixing Cy2, Alexa Fluor 488, Cy3, ATTO565, Cy5, and Si-rhodamine. Among them, 4 Cy2 molecules (red) and 1 Alexa Fluor488 molecule (blue) were detected in the 488nm channel; 1 Cy3 molecule (red) and 2 ATTO565 molecules (blue) were detected in the 561nm channel; 1 Cy5 molecule (red) and 1 Si-rhodamine molecule (blue) were detected in the 640nm channel;

[0089] Figure 8 Graph showing the switching response of Cy3 at a specific voltage on indium tin oxide conductive glass without a semiconductor layer. DETAILED DESCRIPTION

[0090] The present invention will be further described below in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the protection scope of the appended claims of the present invention.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0092] Where “including,” “having,” and “comprising” are used herein, it is intended to cover a non-exclusive inclusion, and another component may also be added unless explicit limiting terms such as “only,” “consisting of,” etc. are used.

[0093] The words "preferably", "more preferably", "preferably", "better", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the statement of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention. That is, in the present invention, "preferably", "more preferably", "preferably", "better", etc. are only used to describe implementation methods or examples with better effects, but do not constitute a limitation on the scope of protection of the present invention.

[0094] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0095] In the present invention, "at least one" means more than one, such as one, two and more than two. "Multiple" or "several" means at least two, such as two, three, etc., and "multilayer" means at least two layers, such as two layers, three layers, etc., unless otherwise clearly and specifically defined. In the description of the present invention, "several" means at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0096] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0097] If not otherwise specified, all steps of the present invention may be performed sequentially or randomly. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0098] Unless mentioned to the contrary, terms in the singular may include plural forms and should not be construed as being one in number.

[0099] In the present invention, "above" or "below" includes the number itself. For example, "below 1" includes 1.

[0100] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0101] In the present invention, the number of atoms described by a numerical range includes both integer endpoints of the numerical range and each integer between the two endpoints. For example, "C1-C10 alkyl" means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0102] In the present invention, When R is selected from a single bond, express It means that the connection point of the substituent R to the benzene ring is not limited.

[0103] In the present invention, "*" indicates a linking site.

[0104] In the present invention, "halogen" or "halo" refers to -F, -Cl, -Br or -I.

[0105] In the present invention, the term "alkyl" refers to a monovalent residue formed by the loss of a hydrogen atom from a saturated hydrocarbon containing a primary (normal) carbon atom, a secondary carbon atom, a tertiary carbon atom, a quaternary carbon atom, or a combination thereof. Phrases containing this term, for example, "C1-C10 alkyl" refers to an alkyl group containing 1 to 10 carbon atoms, and each occurrence can be independently C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl or C10 alkyl. Suitable examples include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH (CH 3 ) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH(CH3 ) 2 ), 2-butyl (s-Bu, s-butyl, -CH (CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl-2-butyl (-C(CH 3 ) 2 CH 2 CH 3 ), 3-methyl-2-butyl (-CH(CH 3 )CH(CH 3 ) 2 ), 3-methyl-1-butyl (-CH 2 CH 2 CH(CH 3 ) 2 ), 2-methyl-1-butyl (-CH 2 CH(CH 3 )CH 2 CH 3 )、1-hexyl (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-hexyl (-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 )、3-hexyl(-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 ))、2-methyl-2-pentyl (-C(CH 3 ) 2 CH 2 CH 2 CH3 ), 3-methyl-2-pentyl (-CH(CH 3 )CH(CH 3 )CH 2 CH 3 ), 4-methyl-2-pentyl (-CH(CH 3 )CH 2 CH(CH 3 ) 2 ), 3-methyl-3-pentyl (-C(CH 3 )(CH 2 CH 3 ) 2 ), 2-methyl-3-pentyl (-CH(CH 2 CH 3 )CH(CH 3 ) 2 )、2,3-dimethyl-2-butyl (-C(CH 3 ) 2 CH(CH 3 ) 2 )、3,3-dimethyl-2-butyl (-CH(CH 3 )C(CH 3 ) 3 and octyl (-(CH 2 ) 7 CH 3 ).

[0106] In the present invention, "haloalkyl" refers to an alkyl group substituted with one or more halogen (chlorine, fluorine, bromine or iodine) atoms. Polyhaloalkyl groups have the same or mixed types of halogen atoms. "Perhaloalkyl" refers to an alkyl group in which each hydrogen atom is replaced by a halogen atom. A haloalkyl group in which a particular carbon atom is "fully halogenated" means that all hydrogen atoms attached to that carbon are replaced by halogen atoms. Representative mono-, di- and trihaloalkyl groups include: chloromethyl, chloroethyl, bromomethyl, bromoethyl, iodomethyl, iodoethyl, chloropropyl, bromopropyl, iodopropyl, 1,1-dichloromethyl, 1,1-dibromomethyl, 1,1-dichloropropyl, 1,2-dibromopropyl, 2,3-dibromopropyl, 1-chloro-2-bromoethyl, 2-chloro-3-bromopropyl, trifluoromethyl, trichloromethyl, and the like.

[0107] In the present invention, "cycloalkyl" refers to a non-aromatic hydrocarbon containing ring carbon atoms, which can be a monocyclic alkyl, a spirocyclic alkyl, or a bridged cycloalkyl. Phrases containing this term, for example, "C3-C10 cycloalkyl" refers to a cycloalkyl containing 3 to 10 carbon atoms, each occurrence of which can be independently C3 cycloalkyl, C4 cycloalkyl, C5 cycloalkyl, C6 cycloalkyl, C7 cycloalkyl, C8 cycloalkyl, C9 cycloalkyl or C10 cycloalkyl. Suitable examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. In addition, "cycloalkyl" may also contain one or more double bonds, and representative examples of cycloalkyl containing double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl and cyclobutadienyl.

[0108] In the present invention, the "number of ring atoms" refers to the number of atoms in the atoms constituting the ring itself of a structural compound (e.g., a monocyclic compound, a condensed ring compound, a cross-linked compound, a carbocyclic compound, a heterocyclic compound) formed by atoms bonding to form a ring. When the ring is substituted by a substituent, the atoms contained in the substituent are not included in the ring atoms. The same is true for the "number of ring atoms" described below unless otherwise specified. For example, the number of ring atoms of a benzene ring is 6, the number of ring atoms of a naphthalene ring is 10, and the number of ring atoms of a biphenyl is 12.

[0109] In the present invention, "aryl, aromatic group or aromatic group" refers to a hydrocarbon group containing at least one aromatic ring. "Heteroaromatic group or heteroaromatic group" refers to an aromatic hydrocarbon group containing at least one heteroatom. The heteroatom is preferably selected from Si, N, P, O, S and / or Ge, and is particularly preferably selected from Si, N, P, O and / or S. A condensed ring aromatic group refers to an aromatic group whose ring may have two or more rings, wherein two carbon atoms are shared by two adjacent rings, i.e., a condensed ring. A condensed heterocyclic aromatic group refers to a condensed ring aromatic hydrocarbon group containing at least one heteroatom. For the purposes of the present invention, an aromatic group or heteroaromatic group includes not only a system of aromatic rings, but also a non-aromatic ring system. Therefore, systems such as pyridine, thiophene, pyrrole, pyrazole, triazole, imidazole, oxazole, oxadiazole, thiazole, tetrazole, pyrazine, pyridazine, pyrimidine, triazine, carbene, etc. are also considered to be aromatic groups or heterocyclic aromatic groups for the purpose of this invention. For the purposes of the present invention, fused aromatic or fused heteroaromatic ring systems include not only systems of aromatic or heteroaromatic groups, but also systems in which multiple aromatic or heteroaromatic groups may be interrupted by short non-aromatic units (<10% non-H atoms, preferably less than 5% non-H atoms, such as C, N or O atoms). Thus, systems such as 9,9'-spirobifluorene, 9,9-diarylfluorene, triarylamines, diaryl ethers, etc. are also considered fused aromatic ring systems for the purposes of this invention.

[0110] In a preferred embodiment, the aromatic group is selected from the group consisting of benzene, naphthalene, anthracene, fluoranthene, phenanthrene, triphenylene, perylene, tetracene, pyrene, benzopyrene, acenaphthene, fluorene, and derivatives thereof; the heteroaromatic group is selected from the group consisting of triazine, pyridine, pyrimidine, imidazole, furan, thiophene, benzofuran, benzothiophene, indole, carbazole, pyrroloimidazole, pyrrolopyrrole, thienopyrrole, thienothiophene, furanopyrrole, furanofuran, thienofuran, benzisoxazole, benzisothiazole, benzimidazole, quinoline, isoquinoline, o-naphthylidene, quinoxaline, phenanthridine, primary idine, quinazoline, quinazolinone, dibenzothiophene, dibenzofuran, carbazole and derivatives thereof.

[0111] In the present invention, the term "arylene" refers to an aromatic hydrocarbon group derived from an aromatic ring compound by removing two hydrogen atoms, which may be a monocyclic arylene, a condensed ring arylene, or a polycyclic arylene. For polycyclic rings, at least one is an aromatic ring system. For example, "C6-C10 arylene" refers to an arylene containing 6 to 10 carbon atoms, and each occurrence may be independently C6 arylene, C7 arylene, C8 arylene, C9 arylene or C10 arylene. Suitable examples include, but are not limited to, phenylene, biphenylene, naphthalene, anthracene, phenanthrene, perylene, triphenylene and their derivatives.

[0112] In the present invention, the term "cycloalkylene" refers to a hydrocarbon group having two monovalent group centers derived from a cycloalkyl group by removing two hydrogen atoms, and may be a monocycloalkylene group, a spirocycloalkylene group, or a bridged cycloalkylene group. For example, "C3-C10 cycloalkylene" refers to a cycloalkylene group containing 3 to 9 carbon atoms, and each occurrence may be independently C3 cycloalkylene, C4 cycloalkylene, C5 cycloalkylene, C6 cycloalkylene, C7 cycloalkylene, C8 cycloalkylene or C9 cycloalkylene. Suitable examples include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene and cycloheptylene. In addition, "cycloalkylene" may also contain one or more double bonds, and representative examples of cycloalkylene groups containing double bonds include cyclopentenylene, cyclohexenylene, cyclohexadienylene and cyclobutadienylene.

[0113] In the present invention, "A and B are independently selected from x, y or z" means that A and B are independent events, and event A does not affect the occurrence of event B. Therefore, when A is selected from x, B can be selected from any of x, y or z, when A is selected from y, B can be selected from any of x, y or z, and when A is selected from z, B can be selected from any of x, y or z. Similarly, in the present invention, when the same substituent appears multiple times, it can be independently selected from different groups. For example, if the general formula contains multiple R 1 , then R 1 Can be independently selected from different groups.

[0114] In the present invention, "substituted" means that a hydrogen atom in a substituted group is replaced by a substituent.

[0115] Biomarkers usually refer to indicators that can be objectively measured and evaluated, reflecting physiological or pathological processes, and producing biological effects on exposure or therapeutic interventions. Biomarkers are mostly derived from human tissues or body fluids, and can cover changes at the physiological, biochemical, immune, cellular and molecular levels. The detection of biomarkers can be widely used in the fields of disease screening, diagnosis, clinical research, drug guidance, prognosis, etc. The detection methods currently used for ultra-sensitive biomarkers have certain limitations. Specifically as follows:

[0116] Enzyme-linked immunosorbent assay (ELISA) is one of the most widely used and mature technologies. ELISA technology is mainly divided into direct method, indirect method, double antibody sandwich method and competitive method. The above four methods mainly label the analyte through antigen-antibody interaction, and all have problems such as long reaction time and many detection steps. At the same time, in all existing ELISA methods, multi-fluorescence channel detection under the same laser channel cannot be achieved, and multi-fluorescence channel detection under multiple laser channels is rarely achieved. In the direct ELISA method, it is necessary to prepare a primary antibody that specifically binds to the analyte. Different primary antibodies need to be prepared for different analytes. The detection cost is high, and there is no secondary antibody amplification system, and the detection sensitivity is low. In the indirect ELISA method, there is the possibility of direct binding of the secondary antibody to the antigen, but the accuracy of the detection is reduced. In the double antibody sandwich ELISA method, the requirements for the antigen are extremely high, and antigens at multiple sites need to be selected for detection, and the antigen is not universal. In the competitive ELISA method, there are problems such as poor overall sensitivity and specificity.

[0117] The most sensitive commercial detection method is the single molecule array (SiMoA), which is derived from the traditional enzyme-linked immunosorbent assay and is a "sandwich" double antibody sandwich detection method based on microbeads. The main principle is to use a microwell array to capture a single magnetic bead, and after incubation, the ratio of the positive signal to the total magnetic beads is counted to achieve the purpose of quantitative detection. First, the magnetic beads coated with excess capture antibodies far exceeding the amount of the analyte are used to capture the analyte. Based on the principle of Poisson distribution, each magnetic bead captures one or zero analytes, that is, immune complexes, and then all the magnetic beads are loaded into the microwell array. The size of a microwell can only contain one magnetic bead, which means that each microwell will contain one or no magnetic bead. Finally, a fluorescent signal is formed by means of signal amplification. The highly localized fluorescent signal will indicate the result. The ratio of the number of microwells that generate fluorescence to the total number of microwells is statistically calculated by mathematical methods to quantify the concentration of the analyte. However, up to 500,000 magnetic beads can be used in one SiMoA test, but the sampling efficiency is only about 5%, which is low and greatly increases the cost of testing. At the same time, the detection cycle required for a single test using SiMoA is long, making it difficult to achieve detection at rapid medical points, and it is impossible to achieve multi-fluorescence channel detection under the same laser channel, and the abundance in a single test needs to be improved. Because this type of single-molecule microsphere amplification mechanism represented by SiMoA generally has problems such as high prices for consumables (a large number of magnetic beads), complex detection steps, and long detection time, its application in actual clinical practice is limited.

[0118] With the change of technology, Professor David Wart of Harvard University developed a dropcast single molecule array method (dSiMoA) based on SiMoA, which is one of the most sensitive technologies currently developed in the laboratory. Compared with SiMoA, dSiMoA has a technological change and abandoned the microwell array, but still uses excess magnetic beads to capture the analyte to form an immune complex. Similar to SiMoA, an excess of magnetic beads are first used to capture the analyte. After incubation, all magnetic beads are randomly sprinkled on the surface of the slide. Among them, all magnetic beads are activated and positioned using a 488nm laser, and the total number of magnetic beads is counted; then a 640nm laser is used to excite the immune complex, and the number of all immune complexes is counted. The concentration of the analyte is quantified by counting the proportion of immune complexes to the total number of magnetic beads. Compared with SiMoA, dSiMoA improves the utilization rate of magnetic beads and reduces the number of magnetic beads, but in one test, up to 100,000 magnetic beads are still required, and the sampling efficiency is about 50%-60%. The detection cost is still high and the detection time is long, which limits its widespread use in clinical practice. In addition, dSiMoA cannot reduce the impact of background fluorescence, and there are still false positive results that interfere with the accuracy of detection.

[0119] Macdonald et al. developed a repeated sampling direct counting method using single-molecule total internal reflection fluorescence microscopy (SM-TIRFM). This method selected streptavidin and biotin, which are known to have the strongest affinity, as the model system, and found that repeated sampling after the first sampling can improve the detection ability of the analyte. After surface background correction, the sample concentration is positively correlated with the number of samplings and the detection limit reaches the femtomolar level, proving the rationality of multiple sampling. However, in actual detection, due to the batch nature of different sample preparations, it is difficult to ensure that the results are exactly the same, and it is impossible to eliminate interference such as background noise. The accuracy of the experimental results needs to be further improved.

[0120] In 2014, Oja et al. proposed a fluorescence switch method based on chemical reaction and electrochemical reaction. Resorufin was selected as the experimental fluorescent dye, and it was found that the fluorescence of resorufin gradually disappeared in the deprotonated glucose molecule solution, and the solution changed from red to colorless. With the application of oxidation potential, the solution changed from colorless to red. In the deprotonated glucose molecule solution, resorufin and glucose molecule solution underwent a reduction reaction, and resorufin was reduced to dihydroresorufin, and the fluorescence disappeared; under the oxidation potential, dihydroresorufin lost electrons and was oxidized to resorufin, and fluorescence was generated. This experiment demonstrated the possibility of electrochemical modulation of fluorescent dye switching and provided a way to monitor the progress of electrochemical reactions optically.

[0121] In 2019, Fan et al. also proposed a method to observe the electrochemical response of Alexa Fluor 647 using total internal reflection fluorescence microscopy. Alexa Fluor 647 was modified on bovine serum albumin (BSA), and BSA-Alexa Fluor 647 was dropped on indium tin oxide conductive glass by nonspecific adsorption. A square wave voltage was applied, and it was found that under negative potential, the fluorescent molecule Alexa Fluor 647 was in a "dark" state, and under positive potential, Alexa Fluor 647 was in a "bright" state, which was reversible and the transition between the "bright" state and the "dark" state was rapid. This experiment further verified the feasibility of electrochemical modulation of fluorescence, and found that this process can be completed instantaneously.

[0122] In 2015, Zhang et al. proposed a spectrally resolved stochastic optical reconstruction microscopy (SR-STORM) method, which achieved the distinction between multiple fluorescent dyes under the same laser excitation. SR-STORM uses dual-channel imaging to simultaneously record the spatial and spectral information of a single fluorescent point, and achieves multi-fluorescence distinction by differential analysis of the coupling of spatial and spectral information of different fluorescent molecules. However, SR-STORM has extremely high requirements for equipment, high cost, and difficulty in achieving quantitative analysis, which limits its application.

[0123] Based on this, the present invention provides a high-throughput, multi-channel biomarker chip, which can be used in immunoassay detection, especially the detection of multi-channel biomarkers based on electrochemical single molecule immunoassay detection, and has the advantages of low cost, high sensitivity, simple detection steps and rapid detection.

[0124] The technical solution is as follows:

[0125] A chip for biomarker detection, comprising:

[0126] Conductive layers and insulating layers are stacked in sequence;

[0127] The insulating layer contains a plurality of micropores, and each of the micropores penetrates the insulating layer.

[0128] The chip for biomarker detection includes a conductive layer and an insulating layer stacked in sequence, and the insulating layer contains micropores. The micropores in the insulating layer can simultaneously capture multiple fixed types of fluorescent dyes. The chip is used in immunoassay detection to detect multiple different markers. Under the action of chemical reagents, light, and electricity, chemical, light, and electricity reactions occur, and multi-fluorescence imaging is performed under multiple laser channels to achieve multi-channel biomarker detection based on electrochemical single-molecule immunoassay detection, which has the advantages of low cost, high sensitivity, simple detection steps, and rapid detection.

[0129] See also Figure 2 , a biomarker chip provided by one embodiment of the present invention comprises:

[0130] A conductive layer, a semiconductor layer and an insulating layer are stacked in sequence;

[0131] The semiconductor layer includes a semiconductor modified by a silane coupling agent and / or a titanate coupling agent;

[0132] The insulating layer contains a plurality of micropores, and each of the micropores penetrates the insulating layer.

[0133] The chip for biomarker detection includes a conductive layer, a semiconductor layer and an insulating layer stacked in sequence, and the insulating layer contains micropores. Among them, the micropores in the insulating layer can capture multiple fixed types of fluorescent dyes at the same time, and the semiconductor modified by a silane coupling agent and / or a titanate coupling agent can form a chemical coupling with the capture agent and the biomarker to be detected. Any marker includes a fluorescent dye, and up to 7 different markers can be detected at the same time. Under the action of chemical reagents, light, and electricity, chemical, light, and electricity reactions occur, and fluorescence imaging is performed under multiple laser channels to realize the detection of multi-channel biomarkers based on electrochemical single molecule immunoassay detection, which has the advantages of low cost, high sensitivity, simple detection steps, and rapid detection.

[0134] Specifically, refer to Figure 3 In any micropore of the insulating layer, multiple fixed types of fluorescent dyes described in the present invention are captured simultaneously, for example, Cy2, Alexa Fluor 488 in the 488nm channel; Cy3, ATTO 565, Alexa Fluor 546 in the 561nm channel; and Cy5, Si-rhodamine in the 640nm channel can be selected simultaneously. The electrochemical cell shown above is constructed, and after applying a fixed voltage, after data processing, the above 7 dyes can be distinguished at the same time, thereby determining the 7 biomarkers coupled to the 7 dyes. The principle of separation is that 488nm, 561nm, and 640nm are three different laser channels, and the excitation wavelengths thereof differ by more than 60nm. The dyes selected to be excited by the three lasers will not be excited by the lasers in other laser channels, that is, Cy2, Alexa Fluor 488 in the 488nm channel; Cy3, ATTO565, Alexa Fluor 546 in the 561nm channel and Cy5, Si-rhodamine in the 640nm channel can be distinguished in a single channel. For fluorescent dyes in the same channel, such as the 561nm channel, the present invention has a preliminary experiment on the change of the fluorescence intensity of these three dyes with voltage. For any unknown target molecule, by determining the change of its fluorescence intensity with potential, it is possible to determine which fluorescent molecule it is. If the molecule is not within the above three judgments, it is interference and is excluded. Through the above method, the distinction of 7 different fluorescent molecules under 3 laser channels is achieved, that is, multi-channel. In particular, the present invention focuses on the distinction of different fluorescent dyes under the same laser channel, such as the identification of Cy3, ATTO 565, and Alexa Fluor 546 under 561nm laser.

[0135] In one embodiment, the material of the conductive layer includes at least one of indium tin oxide, aluminum-doped zinc oxide, gallium-doped zinc oxide, fluorine-doped tin oxide, graphene, metal nanowires, carbon nanotubes, conductive polymers, gold, silver, platinum, copper and aluminum. Preferably, the material of the conductive layer is indium tin oxide.

[0136] In one embodiment, the thickness of the conductive layer is ≥ 1 nm, including but not limited to 1 nm, 2 nm, 5 nm, 8 nm.

[0137] m, 10nm, 12nm, 13nm, 15nm, 17nm, 18nm, 20nm, 30nm, 40nm, 5nm, 60nm, 70n

[0138] m, 80nm, 90nm or 100nm. Preferably, the thickness of the conductive layer is ≥1nm. Further, the thickness of the conductive layer is 20mm-100mm.

[0139] In one embodiment, the material of the semiconductor layer is selected from at least one of silicon carbide, germanium, silicon, selenium, boron, tellurium, antimony, gallium arsenide, indium phosphide, bismuth telluride and cadmium sulfide. Preferably, the material of the semiconductor layer is silicon carbide.

[0140] In one embodiment, the thickness of the semiconductor layer is 0.1nm to 500nm, which is conducive to synergistic conductivity and coupling. Including but not limited to 0.1nm, 0.5nm, 1nm, 1.5nm, 5nm, 8nm, 10nm, 12nm, 15nm,

[0141] Preferably, the semiconductor layer has a thickness of 1 nm to 60 nm.

[0142] In one embodiment, the silane coupling agent is selected from at least one of an aminosilane coupling agent, a mercaptosilane coupling agent, an alkoxysilane coupling agent and a halogen silane coupling agent.

[0143] In one embodiment, the silane coupling agent is selected from at least one of 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-glycidyloxypropyltrimethoxysilane, vinyltrichlorosilane, vinyltrimethoxysilane, vinyltriethoxysilane and phenylaminopropyltrimethoxysilane.

[0144] In one embodiment, the titanate coupling agent is selected from at least one of a monoalkoxy titanate coupling agent, a monoalkoxy phosphate titanate coupling agent, an integrated titanate coupling agent and a ligand titanate coupling agent.

[0145] In one embodiment, the titanate coupling agent is selected from at least one of isopropyl triisophthaloyl titanate coupling agent, isopropyl trioleophthaloyl titanate coupling agent, isopropyl tri(dodecylbenzenesulfonyl) titanate coupling agent, isopropyl tri(dioctyl pyrophosphoryloxy) titanate coupling agent, isopropyl tri(dioctyl pyrophosphoryl) titanate coupling agent, isopropyl tri(dioctyl pyrophosphoryl) titanate coupling agent, bis(dioctyl pyrophosphate) glycolate titanate coupling agent and dicarboxylic ethylene diethylene titanate coupling agent.

[0146] In one embodiment, the material of the insulating layer includes at least one of an insulating polymer material and an inorganic insulating material.

[0147] In one embodiment, the insulating polymer material is selected from at least one of polydimethylsiloxane, epoxy resin, phenolic resin, polyester, polyurethane, silicone resin, polyimide alkyd resin, polystyrene, polypropylene, polycarbonate, polytetrafluoroethylene, polyimide, polyamide, silicone rubber, polybutadiene and fluorine-containing resin.

[0148] In one embodiment, the inorganic insulating material is selected from at least one of silicon dioxide, silicon nitride, aluminum oxide, boron nitride, hafnium dioxide, zinc oxide, titanium dioxide, zirconium dioxide and yttrium trioxide.

[0149] In one embodiment, the insulating layer is a silicone rubber layer.

[0150] In one embodiment, the material of the silicone rubber layer is polydimethylsiloxane.

[0151] In one embodiment, the thickness of the insulating layer is ≥100nm, including but not limited to 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1500nm, 2000nm, 2500nm, 3000nm, 4000nm, 5000nm, 6000nm, 7000nm, 8000nm or 9000nm. Preferably, the thickness of the insulating layer is 1000nm to 9000nm. Further, the thickness of the insulating layer is 1000nm to 5000nm.

[0152] In one embodiment, the diameter of a single micropore is ≥ 100 nm, including but not limited to 100 nm, 500 nm,

[0153] 1000nm, 2000nm, 3000nm, 4000nm, 5000nm, 10000nm, 50000nm, 100000nm, 500000nm or 1000000nm Preferably, the diameter of a single micropore is 1000nm to 1000000nm. Further preferably, the diameter of a single micropore is 1000000nm.

[0154] In one embodiment, the cross-sectional shape of the micropores includes but is not limited to at least one of a triangle, a square, a rectangle, a circle, an ellipse, a rhombus, a hexagon, an octagon, a pentagon, a trapezoid, a parallelogram, a heart, a five-pointed star, a heptagon and a semicircle.

[0155] It can be understood that in the present invention, the micropores are arranged in the insulating layer and are located on the conductive layer or the semiconductor layer. The micropores mainly serve as containers for electrolyte solutions when the chip is used for biomarker detection.

[0156] The present invention also provides a method for preparing the chip for biomarker detection as described above, comprising the following steps:

[0157] An insulating layer is prepared on the conductive layer, and a plurality of micropores are prepared in the insulating layer.

[0158] In one embodiment, a method for preparing a chip for biomarker detection as described above comprises the following steps:

[0159] preparing a semiconductor layer on the surface of the conductive layer;

[0160] Modifying the semiconductor layer by using a silane coupling agent and / or a titanate coupling agent;

[0161] An insulating layer is prepared on the modified semiconductor layer, and a plurality of micropores are prepared in the insulating layer.

[0162] In one embodiment, a method for preparing a chip for biomarker detection as described above comprises the following steps:

[0163] An insulating polymer layer is prepared on the conductive layer, and a plurality of micropores are prepared in the insulating polymer layer.

[0164] In one embodiment, a method for preparing a chip for biomarker detection as described above comprises the following steps:

[0165] preparing a semiconductor layer on the surface of the conductive layer;

[0166] Modifying the semiconductor layer by using a silane coupling agent and / or a titanate coupling agent;

[0167] An insulating polymer layer is prepared on the modified semiconductor layer, and a plurality of micropores are prepared in the insulating polymer layer.

[0168] In one embodiment, the method for preparing the conductive layer is selected from at least one of magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition.

[0169] In one embodiment, the method for preparing the semiconductor layer is selected from at least one of magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition.

[0170] In one embodiment, the modifying of the semiconductor layer by using a silane coupling agent and / or a titanate coupling agent comprises the following steps:

[0171] The semiconductor layer is immersed in a solution containing the silane coupling agent and / or titanate coupling agent for ≥0.5h, washed, and heat treated at 30°C to 120°C for ≥5min.

[0172] In one embodiment, the modifying of the semiconductor layer by using a silane coupling agent and / or a titanate coupling agent comprises the following steps:

[0173] The semiconductor layer and the solution containing the silane coupling agent and / or titanate coupling agent are placed in the same closed space for ≥0.5h, washed, and heat-treated at 30°C to 120°C for ≥5min.

[0174] It can be understood that when the semiconductor layer and the solution containing the silane coupling agent and / or titanate coupling agent are placed in the same closed space, the silane coupling agent and / or titanate coupling agent will be vaporized and gradually deposited on the surface of the semiconductor layer. Preferably, the deposition process is carried out in a vacuum environment, and the vaporization diffusion rate of the silane coupling agent and / or titanate coupling agent is accelerated by vacuuming.

[0175] In one embodiment, the solvent in the solution containing the silane coupling agent and / or titanate coupling agent is selected from at least one of ethanol, toluene, ethylbenzene and n-hexane. Preferably, the solvent is toluene.

[0176] In one embodiment, the solution containing the silane coupling agent and / or titanate coupling agent includes 1% to 20% of the silane coupling agent and / or titanate coupling agent and 80% to 99% of the solvent in terms of volume percentage. It is understood that the total volume percentage of the coupling agent (the silane coupling agent and / or titanate coupling agent) includes but is not limited to 1%, 2%, 5%, 10%, 15%, 18% or 20%, and the volume percentage of the solvent includes but is not limited to 99%, 98%, 95%, 90%, 85%, 82% or 80%.

[0177] In one embodiment, the solution containing the silane coupling agent includes 1% to 20% of the silane coupling agent and 80% to 99% of the solvent by volume. The volume percentage of the silane coupling agent includes but is not limited to 1%, 2%, 5%, 10%, 15%, 18% or 20%, and the volume percentage of the solvent includes but is not limited to 99%, 98%, 95%, 90%, 85%, 82% or 80%.

[0178] In one embodiment, the solution containing the titanate coupling agent includes 1% to 20% of the titanate coupling agent and 80% to 99% of the solvent by volume. The volume percentage of the titanate coupling agent includes but is not limited to 1%, 2%, 5%, 10%, 15%, 18% or 20%, and the volume percentage of the solvent includes but is not limited to 99%, 98%, 95%, 90%, 85%, 82% or 80%.

[0179] It can be understood that the types of silane coupling agents and titanate coupling agents are the same as those described above for the chip for biomarker detection, and will not be repeated here.

[0180] In one embodiment, the method for preparing the insulating layer is selected from at least one of magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition.

[0181] In the present invention, the method of preparing micro-holes includes but is not limited to at least one of punching holes in the insulating layer using a puncher, laser punching, and cutting corresponding sizes with a knife. In a specific example, the method of preparing micro-holes is punching holes with a puncher.

[0182] In one of the embodiments, a semiconductor layer with a thickness of 40 nm is deposited on the surface of the indium tin oxide conductive layer by at least one deposition technique including but not limited to magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition to achieve the purpose of surface modification of the indium tin oxide conductive layer, the substrate is immersed in HPLC pure toluene to dissolve silane coupling agent and / or titanate coupling agent solvent for 3 hours, taken out and rinsed with toluene, heated at 75 degrees Celsius for 15 minutes, and a polydimethylsiloxane film prefabricated with micropores is covered and bonded to the surface of the semiconductor layer (the preparation method of the micropores is punching with a puncher) to prepare a chip for biomarker detection having a conductive layer, a semiconductor layer and an insulating layer stacked in sequence, the insulating layer containing a plurality of micropores, each of the micropores penetrating the insulating layer, and the semiconductor layer comprising a semiconductor modified by a silane coupling agent and / or a titanate coupling agent.

[0183] The present invention also provides a kit, which comprises the chip for biomarker detection as described above.

[0184] The present invention also provides an immunoassay detection method, and the technical solution is as follows:

[0185] An immunoassay detection method comprises the following steps:

[0186] contacting the chip for biomarker detection as described above with an electrolyte;

[0187] Applying the capture agent and the biomarker to be detected with a label to the microwell as described above;

[0188] Connecting the chip to an electrochemical workstation via a working electrode, a reference electrode and a counter electrode;

[0189] Immunoassay detection is performed by combining electrochemical regulation with fluorescence detection imaging.

[0190] In one embodiment, the solvent in the electrolyte is an organic solvent. Optionally, the organic solvent is selected from at least one of dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone, ethanol, ether and acetone.

[0191] In one embodiment, the solvent in the electrolyte is water, and the electrolyte also includes sodium borohydride. Further, in the electrolyte, the concentration of sodium borohydride is 10 -6 mol / L~1mol / L.

[0192] In one embodiment, the electrolyte includes at least one of an organic electrolyte, an ionic liquid electrolyte and a lithium salt electrolyte.

[0193] In one embodiment, the organic electrolyte includes at least one of tetrabutylammonium trifluoromethanesulfonate, tetrabutylammonium hexafluorophosphate, tetraethylammonium tetrafluoroborate, tetraethylammonium bromide, tetraethylammonium iodide, tetra-n-butylammonium bromide and tetra-n-butylammonium iodide.

[0194] In one embodiment, the ionic liquid electrolyte includes a cationic liquid electrolyte and an anionic liquid electrolyte.

[0195] In one embodiment, the cationic liquid electrolyte includes at least one of imidazole, pyrrolidine, pyridine, morpholine, piperidine, quaternary ammonium, quaternary phosphonium and guanidine cationic liquid electrolytes.

[0196] In one embodiment, the anionic liquid electrolyte includes a halogen salt (such as Cl - Br - ,I - Br - ), alkali salts (such as OH - , CO 3 2- , HCO 3 - ) and halides (FeCl 4 - 、AlCl 4 - 、ZnCl 3 - ) type of anionic liquid electrolyte.

[0197] In one embodiment, the lithium salt electrolyte includes at least one of lithium perchlorate, lithium nitrate, lithium hexafluorophosphate, lithium tetrafluoroborate and lithium hexafluoroarsenate.

[0198] Preferably, the electrolyte is selected from lithium perchlorate.

[0199] In one embodiment, the concentration of the electrolyte in the electrolyte solution is 0.001 mol / L to 1 mol / L.

[0200] Fluorescent electrochemical switching is caused by the different electrochemical responses of different fluorescent dyes at different potentials. In the present invention, the fluorescent dye can be selected from but not limited to all commercially available, reported and unreported fluorescent dyes that respond to electrochemical reactions.

[0201] In one embodiment, the label includes a first fluorescent dye and a second fluorescent dye;

[0202] At least one of the first fluorescent dye and the second fluorescent dye contains an aromatic carboxyl group in its structure.

[0203] In one embodiment, the difference between the optimal excitation wavelengths of the first fluorescent dye and the second fluorescent dye is 60 nm to 100 nm.

[0204] In one embodiment, the structure of the aromatic carboxyl group is as shown in formula (I) or formula (II):

[0205]

[0206] Each X is independently selected from O, Si or N;

[0207] Each R is independently selected from hydrogen, deuterium, halogen, alkyl, alkoxy, haloalkyl, silyl, carbonyl, ester, hydroxyl, amino, nitro, carboxyl, sulfonic acid, alkenyl, carbamoyl, haloformyl, formyl, isocyanate, isocyanate, thiocyanate, isothiocyanate, aromatic or heteroaromatic.

[0208] Further, each R is independently selected from hydrogen, deuterium, halogen, C 1 -C 20 Alkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkylsilyl, hydroxyl C 1 -C 20 Alkyl, amino C 1 -C 20 Alkyl, aldehyde C 1 -C 20 Alkyl, mercapto C 1 -C 20 Alkyl, halogenated C 1 -C 20 Alkyl, acyloxy C 1 -C 20 Alkyl, amino, nitro, carboxyl C 1 -C 20 An alkyl group, an aromatic group having 6 to 40 ring atoms, or a heteroaromatic group having 6 to 40 ring atoms.

[0209] In one embodiment, the first fluorescent dye and the second fluorescent dye are independently selected from at least one of cyanine, rhodamine, FITC and dylight fluorescent dyes. Further, the cyanine series is selected from at least one of Cy2, Cy3 and Cy5; the rhodamine series is selected from at least one of rhodamine B, rhodamine 101, rhodamine 6G and Atto series, and optionally, the Atto series is selected from at least one of Atto488, Atto565 and Atto640; the Alexa Fluor series is selected from at least one of Alexa Fluor488, Alexa Fluor555 and Alexa Fluor633; the DyLight series is selected from at least one of DyLight405, DyLight488 and DyLight650; the FITC-like series is selected from at least one of FITC and Eosin-5.

[0210] In one embodiment, the capture agent is selected from at least one of an antibody or a derivative thereof, a nucleic acid, a protein, an inorganic molecule, and a polymer. It is understood that the antibody solution includes but is not limited to rabbit IgG or other primary antibody solutions.

[0211] In one embodiment, the concentration of the capture agent is 1 mol / L to 10 -8 mol / L.

[0212] In one embodiment, the biomarker to be detected is selected from at least one of proteins, DNA, RNA, enzymes, peptides, carbohydrates, lipids, nucleic acids, polymers and inorganic molecules.

[0213] In one embodiment, the concentration of the biomarker to be detected is 10 -6 mol / L~10 -18 mol / L.

[0214] In one embodiment, the concentration of the biomarker to be detected is 1 nmol / L to 1 amol / L.

[0215] It can be understood that in the present invention, the working electrode, reference electrode and counter electrode can be homemade or purchased.

[0216] In one embodiment, the material of the working electrode is selected from at least one of indium tin oxide, aluminum-doped zinc oxide, gallium-doped zinc oxide, fluorine-doped tin oxide, graphene, metal nanowires, carbon nanotubes, conductive polymers, gold, silver, platinum, copper and aluminum.

[0217] In one embodiment, a transparent conductive film is prepared on the surface of a cover glass as an electrochemical working electrode by using at least one deposition technique including but not limited to magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition, and then a layer of semiconductor with a thickness of 40 nm is deposited on the surface of the electrode by using at least one deposition technique including but not limited to magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition to modify the surface of indium tin oxide, and the substrate is immersed in HPLC pure toluene to dissolve silane coupling agent and / or titanate coupling agent solvent for 3 hours, and then rinsed with toluene after being taken out, and heated at 75 degrees Celsius for 15 minutes, and a polydimethylsiloxane film prefabricated with small holes is covered on the surface of the substrate, and a silver wire is connected to the conductive film as a working electrode and connected to the electrochemical workstation.

[0218] In one embodiment, the material of the counter electrode is selected from at least one of silver, platinum and glassy carbon.

[0219] In one embodiment, a transparent conductive film is prepared on the surface of a cover glass as an electrochemical working electrode using at least one deposition technology including but not limited to magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition, and the indium tin oxide conductive glass is immersed in 3-aminopropyltriethoxysilane dissolved in HPLC pure ethanol, rinsed with ethanol after being taken out, heated at 120 degrees Celsius for 30 minutes, and immersed in NHS-Biotin solution dissolved in HPLC pure DMF again, rinsed with DMF after being taken out, and a polydimethylsiloxane film prefabricated with small holes is covered on the surface of the substrate, and a silver wire is connected to the conductive film as a working electrode connected to the electrochemical workstation.

[0220] In one embodiment, the material of the reference electrode is selected from at least one of silver / silver chloride, silver, silver oxide, calomel, mercury / mercurous sulfate, hydrogen electrode, mercury / mercurous oxide, and a conductive polymer reference electrode.

[0221] It is understandable that in the immunoassay detection of the present invention, the fluorescent molecules are regulated by electrochemical methods, including but not limited to at least one of two-electrode or three-electrode current step, controlled potential step method, constant current method, constant potential method, linear voltammetry, cyclic voltammetry and AC impedance method.

[0222] The immunoassay detection of the present invention can realize specific multi-channels, including but not limited to specific channels, multi-fluorescent dye imaging under any single channel such as 488nm, 561nm, 640nm, etc.; including but not limited to simultaneous imaging of multiple fluorescent channels such as 488nm, 561nm, 640nm, etc. without spectral crosstalk; including but not limited to microscopy imaging under multiple laser channels and multiple fluorescent imaging in the same channel.

[0223] In one embodiment, the immunoassay detection of the present invention utilizes a series of illumination methods including but not limited to ordinary laser illumination, waveguide illumination, and prism illumination to provide an imaging basis for large field of view imaging.

[0224] In one embodiment, the immunoassay detection of the present invention utilizes at least one of the fluorescence detection technologies selected from flow, wide field, confocal scanning, two-photon microscopy and total internal reflection fluorescence microscopy as a fluorescence detection imaging system to achieve high-sensitivity wide-field signal rapid detection, and combines super-resolution positioning technology to achieve high-throughput positioning observation of the object to be tested. Further, at least one of a fluorescence microscope, a confocal microscope, a total internal reflection fluorescence microscope and a two-photon microscope can be used as a fluorescence detection imaging system to achieve high-sensitivity wide-field signal rapid detection.

[0225] In one embodiment, the immunoassay detection of the present invention utilizes post-processing methods including but not limited to AI recognition, background subtraction, frame subtraction / division and other processing methods.

[0226] Specific embodiments are listed below to illustrate the present invention.

[0227] Example 1

[0228] This embodiment provides a chip for biomarker detection and a preparation method and application thereof, as follows:

[0229] (1) Plating 40nm silicon carbide on the surface of indium tin oxide:

[0230] A 40 nm silicon carbide film is plated on the surface of the indium tin oxide conductive glass by magnetron sputtering, the indium tin oxide conductive glass plated with the 40 nm silicon carbide film is taken out, and immersed in a 2% by volume HPLC toluene solution of 3-glycidoxypropyltrimethoxysilane, a magnetron is added, and the solution is treated at 800 rpm at room temperature for 3 hours, and the indium tin oxide conductive glass plated with silicon carbide is taken out, and the glass is rinsed with toluene and set aside for use;

[0231] A hole with a diameter of 4 mm was punched in the polydimethylsiloxane film, and the polydimethylsiloxane film was covered on the silicon carbide-plated surface to prepare a biomarker chip.

[0232] (2) Use high-temperature tape to lead a silver wire from the conductive surface of the indium tin oxide conductive glass (not bonded with the polydimethylsiloxane film) and connect it to the electrochemical workstation as a working electrode;

[0233] (3) Add a solution containing rabbit IgG to the pores of the polydimethylsiloxane film, let it stand for 1 hour, and then rinse with water;

[0234] Then add bovine serum albumin blocking solution, let stand for 1 hour, and rinse with water;

[0235] Then, add the solution of the test substance labeled with specific fluorescent dyes, let it stand for 1 hour, and rinse with water. The specific fluorescent dyes are Cy3, ATTO 565, and Alexa Fluor 546;

[0236] (4) Add 10 uL of DMSO to dissolve a 100 mM lithium perchlorate solution as the electrolyte, insert a platinum wire electrode as the counter electrode and an Ag / AgCl electrode as the reference electrode and connect them to the electrochemical workstation;

[0237] (5) Cyclic voltammetry is used to detect the object to be tested. At the same time, a high-speed camera is used on a total internal reflection fluorescence microscope to observe and record the imaging in real time. The laser wavelength is 561 nm.

[0238] Figure 4 This is a mechanism diagram of Cy3, ATTO565, and Alexa Fluor546 under the 561nm channel. A series of cyanine dye structures, taking Cy3 as an example, have a rapid photobleaching phenomenon under a specific oxidation potential. Taking cyclic voltammetry, applying a potential of -1V to +3V to -1V, and a three-electrode system as an example, before adding an electrolyte, Cy3 is in a fluorescent state. After adding a specific electrolyte, Cy3 is still in a fluorescent state. Using cyclic voltammetry to apply a potential of -1V to +3V to -1V, at a certain potential, the Cy3 dye immediately changes from a fluorescent state to a non-fluorescent state and cannot change to a fluorescent state again. This process is considered by the present invention to be a rapid photobleaching phenomenon of Cy3 at a specific potential. The mechanism is as follows Figure 4 As shown in Cy3, the responses under different potentials are as follows Figure 5 As shown in Cy3. Taking ATTO565 as an example, the dye changes from the initial fluorescent state to the non-fluorescent state at a specific reduction potential. At a specific oxidation potential, the non-fluorescent dye will quickly turn to the fluorescent state and will quickly turn off the bleaching. Taking cyclic voltammetry, applying a potential of -1V to +3V to -1V, and a three-electrode system as an example, before adding an electrolyte, ATTO 565 is in a fluorescent state. After adding a specific electrolyte, ATTO 565 changes from a fluorescent state to a non-fluorescent state. Using cyclic voltammetry, applying a potential of -1V to +3V to -1V, as shown in Figure 5As shown in ATTO 565, at a certain oxidation potential, the dye in the non-fluorescent state will quickly turn into a fluorescent state, and after reaching a certain potential, it will quickly photobleach and cannot turn into a fluorescent state again. This process is considered to be a chemical reaction between DMSO and ATTO565 molecules, and the ATTO565 molecules undergo an intramolecular helical cyclization reaction, and the fluorescence is turned off. At a specific oxidation potential, the intramolecular helix of the dye in the non-fluorescent state opens to produce fluorescence. Further, at a higher oxidation potential, the ATTO565 molecule is rapidly bleached. The main reason why ATTO 565 is electrochemically regulated is that the carboxyl group on the benzene ring close to the three-membered ring undergoes a cyclization reaction with the three-membered ring, which plays a regulatory role. Among them, the amino groups at both ends of the three-membered ring are characteristic groups. Taking the central oxygen atom as an example but not limited to the oxygen atom, the central oxygen atom can be replaced by a series of similar atoms such as silicon and nitrogen, and all of them have this response. The mechanism is as follows Figure 5 As shown in ATTO565. Taking cyclic voltammetry, applying a potential of -1V to +3V to -1V, and a three-electrode system as an example, before adding electrolyte, Alexa Fluor546 is in a fluorescent state. After adding a specific electrolyte, Alexa Fluor546 changes from a fluorescent state to a weak fluorescent state. Applying a potential of -1V to +3V to -1V in cyclic voltammetry, as shown in Figure 5 As shown in Alexa Fluor546, at a certain oxidation potential, the dye in a weak fluorescent state will quickly turn into a fluorescent state, and after reaching a certain potential, it will quickly photobleach and cannot turn into a fluorescent state again. This process is believed to be a chemical reaction between DMSO and Alexa Fluor546 molecules, and the Alexa Fluor546 molecules undergo an intramolecular helical cyclization reaction. However, due to the greater stability of the piperidine ring of Alexa Fluor 546, it cannot form a heteroanthracene structure, causing the fluorescence to become weak fluorescence. At a specific oxidation potential, the intramolecular helix of the dye in a weak fluorescent state opens to produce strong fluorescence. Furthermore, at a higher oxidation potential, the Alexa Fluor546 molecules are rapidly photobleached. The mechanism is as follows: Figure 4 Alexa Fluor 546 is shown.

[0239] The mechanism of Cy2 and Cy5 is similar to that of Cy3, and the mechanism of Alexa Fluor 488 and Si-rhodamine is similar to that of ATTO565. Figure 4 The response at different potentials and the fluorescence switching at different potentials are shown in Figure 5 shown.

[0240] Example 2

[0241] This embodiment provides a chip for biomarker detection and a preparation method and application thereof, as follows:

[0242] (1) Plating 40nm silicon carbide on the surface of indium tin oxide:

[0243] A 40 nm silicon carbide film is plated on one surface of an indium tin oxide conductive glass by magnetron sputtering, the indium tin oxide conductive glass plated with the 40 nm silicon carbide film is taken out, and immersed in a 2% by volume HPLC toluene solution of 3-glycidoxypropyltrimethoxysilane, a magnetron is added, and the solution is treated at 800 rpm at room temperature for 3 hours, and the indium tin oxide conductive glass plated with silicon carbide is taken out, and the glass is rinsed with toluene and then set aside for use;

[0244] A hole with a diameter of 4 mm was punched in the polydimethylsiloxane film, and the polydimethylsiloxane film was covered on the silicon carbide-plated surface to prepare a biomarker chip.

[0245] (2) Use high-temperature tape to lead a silver wire from the conductive surface of the indium tin oxide conductive glass (not bonded with the polydimethylsiloxane film) and connect it to the electrochemical workstation as a working electrode;

[0246] (3) Add a solution containing rabbit IgG to the pores of the polydimethylsiloxane film, let it stand for 1 hour, and then rinse with water;

[0247] Then add bovine serum albumin blocking solution, let stand for 1 hour, and rinse with water;

[0248] Then, add the solution of the substance to be tested which has been labeled with specific fluorescent dyes, let it stand for 1 hour, and rinse with water. The specific fluorescent dyes are Cy3 and ATTO 565.

[0249] (4) Add 10 μL DMSO to dissolve 100 mM lithium perchlorate solution as the electrolyte, insert a platinum wire electrode as the counter electrode and an Ag / AgCl electrode as the reference electrode and connect them to the electrochemical workstation;

[0250] (5) Cyclic voltammetry is used to detect the object to be tested. At the same time, a high-speed camera is used on a total internal reflection fluorescence microscope to observe and record the imaging in real time. The laser wavelength is 561 nm.

[0251] The traditional optical diffraction limit is 200nm. Two molecules below 200nm are difficult to distinguish under a traditional optical microscope. Rust et al. proposed the STORM mechanism in 2006. By exchanging time for space, all signal molecules can be distinguished in time sequence. Two fluorescent dye molecules separated by less than 200nm are recorded as A and B respectively. At time 1, let the A molecule be in a "bright" state and the B molecule be in a "dark" state, and record the spatial position of the A molecule. At time 2, let the A molecule be in a "dark" state and the B molecule be in a "bright" state, and record the spatial position of the B molecule, so that super-resolution processing can be achieved.

[0252] Based on this principle, the present invention takes ATTO 565 and Cy3 as examples, and the photoelectric response results under the 561nm laser channel are as follows: Figure 6 As shown, red and blue represent Cy3 and ATTO 565, respectively. Figure 6 (a) Response diagram of the fluorescence signal intensity (ordinate) of Cy3 (red) and ATTO565 (blue) as a function of potential (abscissa). Figure 6 (b) shows the brightness and darkness relationship of two fluorescent dyes at different potentials. 1 Cy3 and ATTO 565 are both in the "light" state at the same time. 2 At this moment, Cy3 is in a "dark" state and ATTO 565 is in a "bright" state. By using the difference in fluorescence switching under different potentials and performing differential processing, the signal separation of Cy3 and ATTO 565 can be achieved, that is, the multi-channel signal separation under different electrochemical responses can be achieved. Figure 6 (c) The separated signals are processed by super-resolution positioning to achieve parallel counting of multiple molecules to be detected.

[0253] The first step is to distinguish single molecules from the background. The background includes the entire imaging background and impurity signals. First, the intensity of each pixel is read to effectively distinguish the background signal from the target signal. The background signal intensity is recorded as A. All pixels identified above A are temporarily considered valid data points. Then the intensity of all valid pixels within the electrochemical control time range is read to change with time. If the intensity remains constant or changes within a very small range, it is identified as background impurities. Finally, through machine learning of all the remaining valid pixels, the target molecules can be divided into libraries. For example, the intensity of the Cy3 single molecule can be identified with machine learning. All data is put into the prior database for judgment, which can effectively distinguish all single molecules and their types.

[0254] In the second step, the dense signal molecules are transformed into sparse signal molecules by differential means, such as Figure 6 As shown in (a), red and blue represent Cy3 and ATTO 565 respectively. For Cy3, a single molecule of fluorescence switching within N time can be obtained by subtracting T+N time from T time. For ATTO 565, a single molecule of fluorescence switching within N time can be obtained by subtracting T+N time from T time and T+N time from T time. It is necessary to ensure that the number of molecules obtained by subtracting the two sides have the same relative position. A large number of operations and comparisons are performed on the computer to select the appropriate N time as the phase difference time to obtain the experimental results (here, the processing methods of division, addition and multiplication are the same as the above subtraction).

[0255] The third step is to distinguish all single molecules through three-dimensional reconstruction to obtain the corresponding experimental results. In the second step, the corresponding single molecules and their relative positions are obtained through differential means, and the time dimension is introduced at the same time. On the basis of the original experiment, three-dimensional imaging can be obtained by adding the time dimension, and then the three-dimensional results are coupled into two-dimensional results and compared with the non-differential data to determine the correctness and accuracy of the three-dimensional reconstruction and complete the data processing.

[0256] For multi-fluorescence imaging of a single laser channel, the present invention utilizes electrochemical regulation to achieve differentiation of multiple dyes.

[0257] The difference between multiple fluorescent dyes under multiple laser channels is achieved by different excitation and emission wavelengths, and fluorescence crosstalk will not be achieved, but it is difficult to achieve fluorescence switching under multiple channels. For example, in this example, the exposure time is 50ms, and the imaging requirement is to take 20 frames of pictures in 1s, where the 1st, 4th, 7th and other frames are 488nm channels, the 2nd, 5th, 8th and other frames are 561nm channels, and the 3rd, 6th, 9th and other frames are 640nm channels. In the traditional method of switching filter blocks, since the switching of the filter blocks is mechanical rotation switching, the switching rate is much lower than 20 times per second, and it is difficult to achieve fluorescence switching under multiple channels. The present invention chooses to achieve this goal by switching lasers, selects a four-way filter block, can simultaneously allow 488nm, 561nm and 640nm lasers to pass, and through the signal triggering mode, the laser is switched once every 50ms, so that the frequency of laser switching is equal to the exposure time, that is, fluorescence imaging under multiple laser channels is realized.

[0258] Example 3

[0259] This embodiment provides a chip for biomarker detection and a preparation method and application thereof, as follows:

[0260] (1) Plating 40nm silicon carbide on the surface of indium tin oxide:

[0261] A 40 nm silicon carbide film is plated on one surface of an indium tin oxide conductive glass by magnetron sputtering, the indium tin oxide conductive glass plated with the 40 nm silicon carbide film is taken out, and immersed in a 2% by volume HPLC toluene solution of 3-glycidoxypropyltrimethoxysilane, a magnetron is added, and the solution is treated at 800 rpm at room temperature for 3 hours, and the indium tin oxide conductive glass plated with silicon carbide is taken out, and the glass is rinsed with toluene and then set aside for use;

[0262] A hole with a diameter of 4 mm was punched in the polydimethylsiloxane film, and the polydimethylsiloxane film was covered on the silicon carbide-plated surface to prepare a biomarker chip.

[0263] (2) Use high-temperature tape to lead a silver wire from the conductive surface of the indium tin oxide conductive glass (not bonded with the polydimethylsiloxane film) and connect it to the electrochemical workstation as a working electrode;

[0264] (3) Add a solution containing rabbit IgG to the pores of the polydimethylsiloxane film, let it stand for 1 hour, and then rinse with water;

[0265] Then add bovine serum albumin blocking solution, let stand for 1 hour, and rinse with water;

[0266] Then, add the solution of the substance to be tested which has been labeled with a specific fluorescent dye, let it stand for 1 hour, and rinse with water. The specific fluorescent dyes are Cy2, Alexa Fluor488, Cy3, ATTO 565, Cy5, and Si-rhodamine;

[0267] (4) Add 10 μL DMSO to dissolve 100 mM lithium perchlorate solution as the electrolyte, insert a platinum wire electrode as the counter electrode and an Ag / AgCl electrode as the reference electrode and connect them to the electrochemical workstation;

[0268] (5) Cyclic voltammetry is used to detect the object to be tested. At the same time, a high-speed camera is used on a total internal reflection fluorescence microscope to observe and record the imaging in real time. The laser wavelength is 561 nm.

[0269] By capturing the intensity of each signal point at different potentials, such as Figure 7 In the 488nm channel, 4 Cy2 molecules (red), 1 Alexa Fluor 488 molecule (blue); 1 Cy3 molecule (red), 2 ATTO 565 molecules (blue); 1 Cy5 molecule (red), 1 Si-rhodamine molecule (blue) were found. This shows that the present invention successfully realizes the recognition of different signal molecules. Similarly, different electrical response molecules can be further added to the channel to achieve simultaneous distinction of more channels.

[0270] Example 4

[0271] This embodiment provides a chip for biomarker detection and a preparation method and application thereof, as follows:

[0272] (1) Soaking the clean indium tin oxide conductive glass in a 2% by volume solution of 3-glycidoxypropyltrimethoxysilane dissolved in HPLC toluene, adding a magnet, treating at 800 rpm at room temperature for 3 hours, taking out the indium tin oxide conductive glass, rinsing it with toluene and setting it aside;

[0273] A hole with a diameter of 4 mm was punched in the polydimethylsiloxane film, and the polydimethylsiloxane film was covered on the silicon carbide-plated surface to prepare a biomarker chip.

[0274] (2) Use high-temperature tape to lead a silver wire from the conductive surface of the indium tin oxide conductive glass (not bonded with the polydimethylsiloxane film) and connect it to the electrochemical workstation as a working electrode;

[0275] (3) Add a solution containing rabbit IgG to the pores of the polydimethylsiloxane film, let it stand for 1 hour, and then rinse with water;

[0276] Then add bovine serum albumin blocking solution, let stand for 1 hour, and rinse with water;

[0277] Then add the solution of the substance to be tested which has been labeled with a specific fluorescent dye, let it stand for 1 hour, and rinse with water. The specific fluorescent dye is Cy3.

[0278] (4) Add 10 μL DMSO to dissolve 100 mM lithium perchlorate solution as the electrolyte, insert a platinum wire electrode as the counter electrode and an Ag / AgCl electrode as the reference electrode and connect them to the electrochemical workstation;

[0279] (5) Cyclic voltammetry is used to detect the object to be tested. At the same time, a high-speed camera is used on a total internal reflection fluorescence microscope to observe and record the imaging in real time. The laser wavelength is 561 nm.

[0280] Figure 8 Schematic diagram of the fluorescence change of Cy3 on ITO conductive glass with potential. Figure 8 It can be seen that Cy3 can also be electrochemically regulated on indium tin oxide conductive glass without a semiconductor layer.

[0281] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0282] The above-described embodiments only express several implementation methods of the present invention, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings may be used to explain the contents of the claims.

Claims

1. A chip for biomarker detection, It is characterized in that include: Conductive layers and insulating layers are stacked in sequence; The insulating layer contains a plurality of micropores, and each of the micropores penetrates the insulating layer; or A semiconductor layer is also provided between the conductive layer and the insulating layer; The semiconductor layer includes a semiconductor modified by a silane coupling agent and / or a titanate coupling agent.

2. The chip for biomarker detection according to claim 1, It is characterized in that Satisfy at least one of the following (1) to (10): (1) The material of the semiconductor layer is at least one selected from silicon carbide, germanium, silicon, selenium, boron, tellurium, antimony, gallium arsenide, indium phosphide, bismuth telluride and cadmium sulfide; (2) The thickness of the semiconductor layer is 0.1 nm to 500 nm; (3) the silane coupling agent is at least one selected from an aminosilane coupling agent, a mercaptosilane coupling agent, an alkoxysilane coupling agent and a halogen silane coupling agent; (4) The titanate coupling agent is selected from at least one of a monoalkoxy titanate coupling agent, a monoalkoxy phosphate titanate coupling agent, an integrated titanate coupling agent and a ligand titanate coupling agent; (5) The material of the insulating layer includes at least one of an insulating polymer material and an inorganic insulating material; (6) The thickness of the insulating layer is ≥100 nm; (7) The diameter of a single micropore is ≥100 nm; (8) The cross-sectional shape of the micropores includes at least one of a triangle, a square, a rectangle, a circle, an ellipse, a rhombus, a hexagon, an octagon, a pentagon, a trapezoid, a parallelogram, a heart, a pentagram, a heptagon and a semicircle; (9) The material of the conductive layer includes at least one of indium tin oxide, aluminum-doped zinc oxide, gallium-doped zinc oxide, fluorine-doped tin oxide, graphene, metal nanowires, carbon nanotubes, conductive polymers, gold, silver, platinum, copper and aluminum; (10) The thickness of the conductive layer is ≥ 1 nm.

3. A method for preparing a chip for biomarker detection according to any one of claims 1 to 2, It is characterized in that The steps include: An insulating layer is prepared on the conductive layer, and a plurality of micropores are prepared in the insulating layer.

4. The method for preparing a chip for biomarker detection according to claim 3, It is characterized in that A semiconductor layer is also provided between the conductive layer and the insulating layer; The semiconductor layer includes a semiconductor modified by a silane coupling agent and / or a titanate coupling agent; The method for preparing the semiconductor layer comprises the following steps: preparing a semiconductor layer on the surface of the conductive layer; The semiconductor layer is modified by using a silane coupling agent and / or a titanate coupling agent.

5. The method for preparing a chip for biomarker detection according to claim 4, It is characterized in that Satisfy at least one of the following (1) to (2): (1) The method for preparing the semiconductor layer is selected from at least one of magnetron sputtering, thermal evaporation, electron beam evaporation, plasma chemical vapor deposition, vacuum evaporation coating, multi-arc ion plating, molecular beam epitaxy, pulsed laser deposition and underpotential deposition; (2) Modifying the semiconductor layer using a silane coupling agent and / or a titanate coupling agent comprises the following steps: Immersing the semiconductor layer in a solution containing the silane coupling agent and / or titanate coupling agent for ≥0.5 h, washing, and heat treating at 30° C. to 120° C. for ≥5 min; or Placing the semiconductor layer and the solution containing the silane coupling agent and / or titanate coupling agent in the same closed space for ≥0.5h, washing, and heat treating at 30°C to 120°C for ≥5min; Calculated by volume percentage, the solution containing a silane coupling agent and / or a titanate coupling agent comprises 1% to 20% of the silane coupling agent and / or the titanate coupling agent and 80% to 99% of a solvent; The solvent is selected from at least one of ethanol, toluene, ethylbenzene and n-hexane.

6. A kit, It is characterized in that A chip for biomarker detection comprising any one of claims 1 to 2.

7. An immunoassay detection method, It is characterized in that The steps include: contacting the chip for biomarker detection according to any one of claims 1 to 2 with an electrolyte; Applying a capture agent and a biomarker to be detected with a label into the microwell according to any one of claims 1 to 2; Connecting the chip to an electrochemical workstation via a working electrode, a reference electrode and a counter electrode; Immunoassay detection is performed by combining electrochemical regulation with fluorescence detection imaging.

8. The immunoassay detection method according to claim 7, It is characterized in that The marker includes a first fluorescent dye and a second fluorescent dye; At least one of the first fluorescent dye and the second fluorescent dye contains an aromatic carboxyl group in its structure; The difference between the optimal excitation wavelengths of the first fluorescent dye and the second fluorescent dye is 60nm to 100nm; The structure of the aromatic carboxyl group is shown in formula (I) or formula (II): Each X is independently selected from O, Si or N; Each R is independently selected from hydrogen, deuterium, halogen, alkyl, alkoxy, haloalkyl, silyl, carbonyl, ester, hydroxyl, amino, nitro, carboxyl, sulfonic acid, alkenyl, carbamoyl, haloformyl, formyl, isocyanate, isocyanate, thiocyanate, isothiocyanate, aromatic or heteroaromatic.

9. The immunoassay detection method according to claim 8, It is characterized in that The first fluorescent dye and the second fluorescent dye are independently selected from at least one of cyanine fluorescent dyes, rhodamine fluorescent dyes, FITC fluorescent dyes and dylight fluorescent dyes.

10. The immunoassay detection method according to any one of claims 7 to 9, It is characterized in that Satisfy at least one of the following (1) to (12): (1) The electrolyte includes at least one of an organic electrolyte, an ionic liquid electrolyte and a lithium salt electrolyte; (2) The concentration of the electrolyte in the electrolyte solution is 0.001 mol / L to 1 mol / L; (3) The solvent in the electrolyte is an organic solvent; or the solvent in the electrolyte is water, and the electrolyte further includes sodium borohydride; (4) the capture agent is selected from at least one of an antibody or its derivative, a nucleic acid, a protein, an inorganic molecule and a polymer; (5) The concentration of the capture agent is 1 mol / L to 10 -8 mol / L; (6) The biomarker to be detected is selected from at least one of proteins, DNA, RNA, enzymes, peptides, carbohydrates, lipids, nucleic acids, polymers and inorganic molecules; (7) The concentration of the biomarker to be detected is 10 -6 mol / L~10 -18 mol / L; (8) The material of the working electrode is selected from at least one of indium tin oxide, aluminum-doped zinc oxide, gallium-doped zinc oxide, fluorine-doped tin oxide, graphene, metal nanowires, carbon nanotubes, conductive polymers, gold, silver, platinum, copper and aluminum; (9) The material of the counter electrode is selected from at least one of silver, platinum and glassy carbon; (10) The material of the reference electrode is at least one selected from the group consisting of silver / silver chloride, silver, silver oxide, calomel, mercury / mercurous sulfate, hydrogen electrode, mercury / mercurous oxide, and a conductive polymer reference electrode; (11) The electrochemical control method is selected from at least one of controlled current step, controlled potential step, constant current method, constant potential method, linear voltammetry, cyclic voltammetry and electrochemical impedance spectroscopy; (12) The imaging system in the fluorescence detection imaging method is selected from at least one of flow cytometry, wide field, confocal scanning, two-photon microscopy and total internal reflection fluorescence microscopy fluorescence detection technologies.

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