Universal biochip as well as preparation method and application thereof

Abstract

The invention discloses a universal biochip as well as a preparation method and application thereof. The preparation method of the universal biochip comprises the following steps: S1, soaking a substrate in a solution consisting of a silane reagent A, an alkaline reagent and a buffer salt solution for reaction, and curing to form a silicon dioxide film on the surface of the substrate; the material of the substrate is selected from any one of glass, metal, plastic, resin and a film material; s2, soaking the substrate treated in the step S1 in a solution of a silane reagent B connected with an active group for reaction so as to modify the active group on the surface of the silicon dioxide film; and S3, carrying out a coupling reaction on the surface active group of the substrate treated in the step S2 and a DNA probe or a group in protein to obtain the universal biochip. The method is suitable for various metal and non-metal material substrates, the preparation process is simple, the condition is mild, the biological chip can be processed into the required shape according to the material, and the application range of the biological chip is further expanded.

Description

Technical Field

[0001] The present invention relates to the field of biochips, and in particular to a universal biochip and a preparation method and application thereof. Background Art

[0002] Biochips originated from gene chips. The main technology is to fix a large number of probe molecules (DNA chains) on a support. They are usually used for hybridization with fluorescently labeled DNA or other sample molecules (such as proteins, factors or small molecules). The number and sequence information of sample molecules are obtained by detecting the hybridization signal intensity of each probe molecule. At present, biochips have been widely used in the operation of various biological macromolecules, cells, and tissues, the detection of biochemical reactions, as well as a variety of major diseases and public safety situations. Biochips also include protein chips, which can use the interaction between proteins and DNA to capture specific DNA chains, and can also use enzyme catalytic activity to catalyze specific reactions.

[0003] At present, most biochips use glass slides as solid phase carriers, and the main advantages are as follows: 1. The glass surface is flat and smooth, which is convenient for coupling a large number of probes; 2. Glass has high light transmittance, which is convenient for fluorescence-based chip applications; 3. Glass has stable properties and good heat resistance, acid and alkali resistance, and water resistance. For example, in Chinese invention patent CN101486532A, a biochip aldehyde-modified slide is disclosed. This biochip uses aminosilane to modify the amino group on the slide surface, and is modified with glutaraldehyde to form a biochip aldehyde-modified slide. Glass solid phase carriers have problems such as easy breakage, difficulty in injection molding and processing, and narrow application range, which also limits the wider application of biochips.

[0004] Compared with glass, metal and plastic materials also have the characteristics of good stability, strong tolerance, low price, and easy processing and shaping. However, the composition and properties of various metals and plastic materials are different, so different methods and processes are required for probe coupling, and probe modification is difficult. There is a lack of a universal, mild and easy-to-operate method for preparing biochips. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a universal biochip and its preparation method and application, which can prepare DNA synthesis biochips based on various metal and non-metal material substrates and carry out biological DNA synthesis applications.

[0006] To achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a universal biochip, comprising the following steps: S1, immersing the substrate in a solution consisting of a silane reagent A, an alkaline reagent and a buffered salt solution to react, and forming a silicon dioxide film on the surface of the substrate after curing; The material of the substrate is selected from any one of glass, metal, plastic, resin, and film material; S2, immersing the substrate treated in step S1 in a solution of a silane reagent B connected with active groups to carry out a reaction, so as to modify the active groups on the surface of the silicon dioxide film; The active group is selected from any one of an azido group, an aminopropyl group, a mercaptopropyl group, a glycidyloxypropyl group, and an isopropionate propyl group; S3, allowing the active groups on the surface of the substrate treated in step S2 to undergo coupling reaction with the groups in the DNA probe or protein to obtain the universal biochip; The group in the DNA probe is a terminal alkynyl group, dibenzocyclooctyne, amino group or maleimide modified at the 5' or 3' end; The groups in the protein are free amino groups.

[0007] Based on the above technical scheme, the present invention forms a silicon dioxide film on the surface of the substrate by immersing the substrate in a solution composed of a silane reagent A, an alkaline reagent and a buffered salt solution and solidifying it, and then further modifies the surface of the silicon dioxide film with active groups such as an azide group and an isopropionate propyl group, and finally couples a DNA probe containing an alkynyl group or a protein containing a free amino group with the active group, thereby fixing the DNA probe or protein on the surface of the substrate. The method of the present invention forms a silicon dioxide film on the surface of the substrate and then introduces active groups to couple DNA probes or proteins. It is suitable for substrates of various materials such as glass, metal, plastic, resin, and membrane materials, and has mild preparation conditions and simple operation.

[0008] In some embodiments of the present invention, before the soaking step in step S1, the process further includes: plasma cleaning the substrate; the working gas for the plasma cleaning is selected from any one of nitrogen, oxygen, argon and air; the flow rate of the working gas for the plasma cleaning is 10 to 100 ml / h, such as 10 ml / h, 50 ml / h, 80 ml / h, 30 ml / h, 90 ml / h; in the plasma cleaning step, the power of the plasma cleaning machine is 30 to 120 W, such as 50 W, 80 W, 100 W, 60 W; the time for the plasma cleaning is 2 to 60 min, such as 3 min, 5 min, 15 min. The plasma cleaning step can not only decontaminate the substrate, but also facilitate the formation of the silicon dioxide film on the substrate.

[0009] In some embodiments of the present invention, the metal includes any one of iron, copper, aluminum, and stainless steel; The plastic includes any one of polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), and polystyrene (PS); The resin includes any one of acrylonitrile-butadiene-styrene copolymer (ABS), polyimide (PI), polyetheretherketone (PEEK), polycarbonate (PC), polymethyl acrylate (PMMA), polyethylene terephthalate (PET), and polyurethane (PU); The membrane material includes any one of polyvinylidene fluoride (PVDF), polyethylene (PP), nylon membrane (Nylon), and polyethersulfone (PES).

[0010] In some embodiments of the present invention, the silane reagent A is selected from one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrabutyl orthosilicate, sodium silicate, trimethylethoxysilane and polydiethoxysiloxane; the silane reagent A can form a silicon dioxide film with a suitable thickness and deposition density on the surface of the substrate, which is more conducive to the subsequent modification of active groups; The mass ratio of the silane reagent A, the alkaline reagent and the buffered salt solution is 1:(0.01-1):(1-10); The alkaline agent is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, ammonia water, ammonium bicarbonate, sodium bicarbonate, ethanolamine, triethylamine, and ethylenediamine; The buffered saline solution is an ammonium chloride solution containing a surfactant, the concentration of the ammonium chloride in the buffered saline solution is 1 to 100 mM, and the volume concentration of the surfactant in the buffered saline solution is 1 to 5%; The surfactant is Tween 80; The reaction time in step S1 is 1 to 24 hours, such as 2 hours, 8 hours, 5 hours, 12 hours, and 16 hours; The curing temperature in step S1 is 60-120° C., such as 80° C., 100° C., or 60° C., and the curing time is 0.5 h to 5.0 h, such as 3 h or 1 h.

[0011] In some embodiments of the present invention, the silane reagent B connected with the active group is at least one of azidopropyl triethoxysilane, azidotrimethylsilane, aminopropyl trimethoxysilane, mercaptopropyl trimethoxysilane, glycidyloxypropyl trimethoxysilane, and isocyanate propyl triethoxysilane; the silane reagent B is more conducive to modifying the active group on the surface of the silicon dioxide film; In the solution of the silane reagent B connected with an active group, the solvent is at least one of N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile and dioxane; The mass concentration of the solution of the silane reagent B connected with the active group is 1% to 5%, such as 1%, 1.5%, 5%; The reaction time in step S2 is 12 to 48 hours, such as 12 hours.

[0012] In some embodiments of the present invention, the group in the DNA probe is a 5' end modification; The sequence of the DNA probe is as follows: 5′-TTTTTTTTTTTTTTTTTTGACATGCACTAGGAATT-3′; In step S3, the coupling reaction is carried out by dropping a solution containing 0.1 to 100 pmol of the DNA probe on the surface of the substrate treated in step S2; wherein the size of the substrate is not limited, for example, when the substrate area is 4 mm*4 mm, a solution containing 1 pmol of the DNA probe is dropped on the surface of the substrate treated in step S2 to carry out the coupling reaction; The solvent in the solution of the DNA probe is a buffer solution containing or not containing monovalent copper ions; the buffer solution can be a sodium chloride solution or PBS; as an example, the monovalent copper ions are produced by reducing copper sulfate with sodium ascorbate, such as 4mM copper sulfate + 1.5 mM sodium ascorbate; as an example, the buffer solution is a 0.1M sodium chloride solution or a 1*PBS buffer. The monovalent copper ions can be used as a catalyst for the reaction of the alkyne group with the azide, and whether to add them can be selected according to the types of the modified groups on the DNA probe and the surface active groups of the silica film.

[0013] The coupling reaction of the DNA probe is carried out at a temperature of 25° C. and a humidity of 80% for 12 to 48 hours, such as 14 hours or 24 hours.

[0014] In some embodiments of the present invention, the protein is bovine serum albumin, as a representative of protein molecules; In step S3, the coupling reaction is carried out by immersing the substrate treated in step S2 in a protein solution having a mass concentration of 0.5% to 5% (e.g., 1%); The solvent in the protein solution is a sodium carbonate solution with a pH of 8; The protein coupling reaction time is 1 to 48 hours, such as 12 hours.

[0015] In some embodiments of the present invention, the preparation method further comprises: performing quality control on the universal biochip obtained in step S3; Specifically, when the biochip is a chip that fixes the DNA probe, the quality control step includes: immersing the DNA chip in a solution containing a fluorescent complementary chain for hybridization, and observing the fluorescence image after hybridization of the fluorescent complementary chain through a fluorescence microscope. More specifically, the fluorescent complementary chain may be a DNA complementary sequence with fluorescent probes such as Cy3 and FITC, such as the sequence of the fluorescent complementary chain is 5'-CCTAGTGCATGTCAA-3', with 3' modified Cy3. The molar dosage ratio of the DNA probe to the fluorescent complementary chain may be 20:1. The hybridization includes a solvent, and the solvent may be a mixed solution of an SSC buffer solution and a PBS buffer solution in a volume ratio of 1:1; the hybridization temperature may be 25-100°C, and the time may be 10-60 minutes, such as hybridization at 25°C for 30 minutes; Specifically, when the biochip is a chip for fixing the protein, the quality control step includes: testing the difference in protein loading in the supernatant solution of the protein chip before and after the protein is fixed, and quantifying it using an enzyme marker.

[0016] In a second aspect, the present invention provides a universal biochip prepared by any of the methods described above.

[0017] In a third aspect, the present invention provides the use of the universal biochip in biological DNA synthesis or protein immobilization.

[0018] The present invention has the following beneficial effects: The preparation method of the universal biochip of the present invention is applicable to various metal and non-metal substrates, such as glass, metal, plastic, membrane materials, etc. The preparation process is simple and the conditions are mild. The material can be processed into a desired shape, further expanding the application scope of the biochip, solving the problems of easy breakage, difficulty in injection molding and processing of the current material glass as a biochip substrate, and laying a foundation for further development of biological DNA synthesis or immobilized protein applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a flowchart for preparing a fixed universal biochip in accordance with the present invention 1.

[0020] Figure 2 This is a fluorescence image after the PP sheet is hybridized with the fluorescent complementary chain in Example 1 of the present invention.

[0021] Figure 3 This is a fluorescence image after the aluminum sheet is hybridized with the fluorescent complementary chain in Example 2 of the present invention.

[0022] Figure 4 This is a fluorescence image after the PVDF membrane is hybridized with the fluorescent complementary chain in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0024] The methods used in the following examples, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0025] The operations in the following examples were all carried out at room temperature unless otherwise specified.

[0026] The modified DNA probes described in the following examples are purchased from a DNA probe synthesis service company, such as Sangon Biotech (Shanghai) Co., Ltd.

[0027] The sequence of the base probe is as follows: SEQ ID NO: 1: 5'-TTTTTTTTTTTTTTTTTGACATGCACTAGGAATT-3', 5' modified terminal alkynyl, dibenzocyclooctyne (DBCO), maleimide or amino group.

[0028] The sequence of the quality control probe is as follows: SEQ ID NO:2: 5'-CCTAGTGCATGTCAA-3', 3' modified Cy3.

[0029] The buffer solutions in the following examples were purchased from Beijing Solebow Technology Co., Ltd.: SSC buffer solution is 20*SSC buffer; The PBS buffer solution is 1*PBS buffer with pH=7.4; Each buffer in the following embodiments is prepared by oneself: Ammonium chloride solution: 1-100 mM ammonium chloride solution containing 1-5% Tween 80 by volume.

[0030] Example 1: Preparation of plastic gene chip according to Figure 1 The plastic gene chip is prepared according to the flowchart shown in the figure. The specific steps are as follows: (1) Surface cleaning: Cut the polypropylene (PP) material into 4 mm*4 mm*1 mm thin sheets and put them into the plasma cleaning machine. Turn on the vacuum pump and the oxygen cylinder in turn and control the flow rate to 10 ml / h. Turn on the RF power supply and adjust the power to 50 w. Clean for 3 minutes.

[0031] (2) Silica deposition: Prepare the reaction solution: 3 ml of tetraethyl orthosilicate (TEOS), 1 ml of concentrated ammonia water (25%) and 6 ml of 20 mM ammonium chloride solution (containing 1% Tween 80), mix well and place in a square container.

[0032] The PP sheet cleaned in step (1) was immersed in the reaction solution, placed at the bottom of the container to react for 12 hours, taken out and washed twice with ethanol, blown dry with nitrogen, and placed in an oven at 80°C for 3 hours.

[0033] (3) Surface modification: The PP sheet treated in step (2) was immersed in 100 μL of acetonitrile solution containing 1% (mass concentration) azidopropyltriethoxysilane overnight, and then washed twice with acetonitrile and dried with nitrogen.

[0034] (4) DNA immobilization Drop 1 μl, 1 μmol / L 5'-end modified terminal alkyne base probe solution (solvent is 4 mM copper sulfate + 1.5 mM sodium ascorbate) on the PP sheet treated in step (3) and react for 14 hours at 25°C and 80% humidity. Rinse the PP sheet after the reaction with water three times and blow dry with nitrogen.

[0035] (5) Quality control The PP sheet treated in step (4) was immersed in a well plate containing a quality control probe (100 μl, 0.5 μmol / L, the solvent was a mixed solution of SSC buffer solution and PBS buffer solution in a volume ratio of 1:1) and hybridized at 25°C for 30 minutes. The hybridization solution was aspirated, washed twice with 0.1 mol / L sodium chloride solution, blown dry with nitrogen gas, and observed under a fluorescence microscope.

[0036] Figure 2 This is a fluorescence image after the PP sheet was hybridized with the quality control probe. From the fluorescence image, it can be seen that the DNA was successfully inoculated on the PP sheet, and the universal gene chip made of plastic material was successfully prepared.

[0037] Example 2: Preparation of metal gene chip according to Figure 1 The metal gene chip is prepared according to the flowchart shown in the figure. The specific steps are as follows: (1) Surface cleaning: The aluminum material was cut into 4 mm*4 mm*0.5 mm aluminum sheets and placed in a plasma cleaning machine. The vacuum pump was turned on in sequence, the argon gas bottle was turned on and the flow rate was controlled to 50 ml / h, the RF power supply control was turned on and the power was 80 w, and cleaning was performed for 5 minutes.

[0038] (2) Silica deposition: Prepare the reaction solution: dissolve 1g sodium silicate and 0.59g sodium bicarbonate in 10ml 10mM ammonium chloride solution (containing 2% Tween), disperse evenly and place in a square container.

[0039] The aluminum sheet cleaned in step (1) was immersed in the reaction solution, placed at the bottom of the vessel to react for 14 h, taken out and washed twice with ethanol, blown dry with nitrogen, and placed in an oven at 100° C. for 1 h.

[0040] (3) Surface modification: The aluminum sheet treated in step (2) was immersed in 100 μL of acetonitrile solution containing 1.5% (mass concentration) azidopropyltriethoxysilane overnight, and then washed twice with acetonitrile and dried with nitrogen.

[0041] (4) DNA immobilization Drop 1 μl, 1 μmol / L 5'-end modified DBCO base probe (solvent: PBS solution) on the aluminum sheet treated in step (3) and react for 14 hours at 25°C and 80% humidity. Rinse the aluminum sheet with water three times and blow dry with nitrogen.

[0042] (5) Quality Control The aluminum sheet treated in step (4) was immersed in a well plate containing a quality control probe (same as in Example 1) and hybridized at 25°C for 30 minutes. The hybridization solution was aspirated, washed twice with 0.1 mol / L sodium chloride solution, blown dry with nitrogen, and observed under a fluorescence microscope.

[0043] Figure 3 This is a fluorescence image after the aluminum sheet was hybridized with the quality control probe. From the fluorescence image, it can be seen that the DNA was successfully inoculated on the aluminum sheet, and the universal gene chip made of metal was successfully prepared.

[0044] Example 3: Preparation of membrane gene chip according to Figure 1 The membrane gene chip is prepared according to the flowchart shown in the figure. The specific steps are as follows: (1) Surface cleaning: Cut the PVDF membrane into 4 mm*4 mm, put it into the plasma cleaning machine, turn on the vacuum pump, turn on the nitrogen bottle and control the flow rate to 80 ml / h, turn on the RF power control and the power to 100 W, and clean for 15 min.

[0045] (2) Silica deposition: Prepare the reaction solution: 2 ml of tetramethyl orthosilicate, 1 ml of triethylamine and 7 ml of 10 mM ammonium chloride solution (containing 1% Tween), mix well and place in a square container.

[0046] The PP sheet cleaned in step (1) was immersed in the reaction solution, placed at the bottom of the container to react for 5 h, taken out and washed twice with ethanol, blown dry with nitrogen, and placed in an oven at 60°C for 0.5 h.

[0047] (3) Surface modification: The PVDF membrane after silicon oxide deposition was immersed in 100 μL of 5% (mass concentration) mercaptopropyltrimethylsilane (acetonitrile) solution overnight, washed twice with acetonitrile, and dried with nitrogen gas.

[0048] (4) DNA immobilization: 1 μl, 1 μmol / L 5'-end modified maleimide group base probe solution (solvent: PBS) was dropped onto the PVDF membrane treated in step (3) and reacted for 24 hours at 25°C and 80% humidity. The reacted PP sheet was rinsed three times with water and dried with nitrogen.

[0049] (5) Quality Control The PVDF membrane treated in step (4) was immersed in a well plate containing a quality control probe (same as in Example 1) and hybridized at 25°C for 30 minutes. The hybridization solution was aspirated, washed twice with 0.1 mol / L sodium chloride solution, blown dry with nitrogen, and observed under a fluorescence microscope.

[0050] Figure 4 This is a fluorescence image after the PVDF membrane was hybridized with the quality control probe. From the fluorescence image, it can be seen that the DNA was successfully inoculated on the PVDF membrane, and the universal gene chip of membrane material was successfully prepared.

[0051] Example 4: Preparation of plastic protein / peptide chips according to Figure 1 The flowchart shown is used to prepare the plastic protein / peptide chip. The specific steps are as follows: (1) Surface cleaning: Cut the PE material into 4mm*4mm*1mm PE sheets, put them into the plasma cleaning machine, turn on the vacuum pump in turn, turn on the oxygen cylinder and control the flow rate to 30ml / h, turn on the RF power control and the power to 50w, and clean for 5min.

[0052] (2) Silica deposition: Prepare the reaction solution: 3 ml of tetrabutyl orthosilicate, 2 ml of ethanolamine and 5 ml of 20% ammonium chloride solution (containing 5% Tween), mix well and place in a square container.

[0053] The PE sheet cleaned in step (1) was immersed in the reaction solution, placed at the bottom of the vessel to react for 10 h, taken out and washed twice with ethanol, blown dry with nitrogen, and placed in an oven at 60°C for 1 h.

[0054] (3) Surface modification: The PE sheet treated in step (2) was immersed in 100 μL of 5% (mass concentration) isocyanate propyl triethoxysilane (acetonitrile) solution overnight, washed twice with acetonitrile, and dried with nitrogen.

[0055] (4) Protein immobilization The PE sheet treated in step (3) was immersed in 1% BSA (0.1 M sodium carbonate solution, pH = 8) for 12 h, and then washed three times with 0.1 M sodium carbonate solution.

[0056] (5) Quality Control The protein loading on the protein chip was calculated by the concentration difference before and after the protein solution (quantitative analysis by microplate reader).

[0057] Amount of immobilized protein: 122 ng.

[0058] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for preparing a universal biochip, characterized in that: The steps include: S1, immersing the substrate in a solution consisting of a silane reagent A, an alkaline reagent and a buffered salt solution to react, and forming a silicon dioxide film on the surface of the substrate after curing; The material of the substrate is selected from any one of glass, metal, plastic, resin, and film material; S2, immersing the substrate treated in step S1 in a solution of a silane reagent B connected with active groups to carry out a reaction, so as to modify the active groups on the surface of the silicon dioxide film; The active group is selected from any one of an azido group, an aminopropyl group, a mercaptopropyl group, a glycidyloxypropyl group, and an isopropionate propyl group; S3, allowing the active groups on the surface of the substrate treated in step S2 to undergo coupling reaction with the groups in the DNA probe or protein to obtain the universal biochip; The group in the DNA probe is a terminal alkynyl group, dibenzocyclooctyne, amino group or maleimide modified at the 5' or 3' end; The groups in the protein are free amino groups.

2. The method for preparing a universal biochip according to claim 1, characterized in that: Before the soaking step in step S1, the method further includes: plasma cleaning the substrate; The working gas for plasma cleaning is selected from any one of nitrogen, oxygen, argon and air; The flow rate of the working gas for plasma cleaning is 10-100 ml / h; In the plasma cleaning step, the power of the plasma cleaning machine is 30 to 120 W; The plasma cleaning time is 2 to 60 minutes.

3. The method for preparing a universal biochip according to claim 1, characterized in that: The metal includes any one of iron, copper, aluminum and stainless steel; The plastic includes any one of polyethylene, polypropylene, polyvinyl chloride, and polystyrene; The resin includes any one of acrylonitrile-butadiene-styrene copolymer, polyimide, polyetheretherketone, polycarbonate, polymethyl acrylate, polyethylene terephthalate, and polyurethane; The membrane material includes any one of polyvinylidene fluoride, polyethylene, nylon membrane, and polyethersulfone.

4. The method for preparing a universal biochip according to claim 1, characterized in that: The silane reagent A is selected from one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrabutyl orthosilicate, sodium silicate, trimethylethoxysilane and polydiethoxysiloxane; The mass ratio of the silane reagent A, the alkaline reagent and the buffered salt solution is 1:(0.01-1):(1-10); The alkaline agent is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, ammonia water, ammonium bicarbonate, sodium bicarbonate, ethanolamine, triethylamine, and ethylenediamine; The buffered saline solution is an ammonium chloride solution containing a surfactant, the concentration of the ammonium chloride in the buffered saline solution is 1 to 100 mM, and the volume concentration of the surfactant in the buffered saline solution is 1 to 5%; The surfactant is Tween 80; The reaction time in step S1 is 1 to 24 hours; The curing temperature in step S1 is 60-120° C., and the curing time is 0.5 h to 5.0 h.

5. The method for preparing a universal biochip according to claim 1, characterized in that: The silane reagent B connected with an active group is at least one of azidopropyltriethoxysilane, azidotrimethylsilane, aminopropyltrimethoxysilane, mercaptopropyltrimethoxysilane, glycidyloxypropyltrimethoxysilane, and isocyanatepropyltriethoxysilane; In the solution of the silane reagent B connected with an active group, the solvent is at least one of N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile and dioxane; The mass concentration of the solution of the silane reagent B connected with the active group is 1% to 5%; The reaction time in step S2 is 12 to 48 hours.

6. The method for preparing a universal biochip according to any one of claims 1 to 5, characterized in that: The group in the DNA probe is 5' end modified; The sequence of the DNA probe is as follows: 5′-TTTTTTTTTTTTTTTTTTGACATGCACTAGGAATT-3′; In step S3, the coupling reaction is carried out by dropping a solution containing 0.1 to 100 pmol of the DNA probe onto the surface of the substrate treated in step S2; The solvent in the solution of the DNA probe is a buffer solution containing or not containing monovalent copper ions; The coupling reaction of the DNA probe is carried out for 12 to 48 hours at a temperature of 25° C. and a humidity of 80%.

7. The method for preparing a universal biochip according to any one of claims 1 to 5, characterized in that: The protein is bovine serum albumin; In step S3, the coupling reaction is carried out by immersing the substrate treated in step S2 in a protein solution with a mass concentration of 0.5% to 5%; The solvent in the protein solution is a sodium carbonate solution with a pH of 8; The protein coupling reaction time is 1 to 48 hours.

8. The method for preparing a universal biochip according to claim 1, characterized in that: The preparation method further comprises: performing quality control on the universal biochip obtained in step S3; When the biochip is a chip on which the DNA probe is fixed, the quality control step comprises: immersing the DNA chip in water containing fluorescent complementary chains for hybridization, and observing the fluorescence image after hybridization of the fluorescent complementary chains through a fluorescence microscope; When the biochip is a chip for fixing the protein, the quality control step includes: testing the difference in protein loading in the supernatant solution of the protein chip before and after the protein is fixed, and quantifying it using an enzyme marker.

9. A universal biochip obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the universal biochip according to claim 9 in biological DNA synthesis or protein immobilization.

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