Diaziridine compound substrate as well as preparation method and application thereof

Abstract

The invention discloses a bis-aziridine compound substrate as well as a preparation method and application thereof, and relates to the technical field of microarray fixing compounds. The bis-aziridine compound is used for modifying the polylysine substrate to obtain the bis-aziridine compound substrate, and the bis-aziridine compound substrate is simple in synthesis step, wide in application range and high in yield. The method can be applied to the field of high-throughput biochip preparation, research on interaction between biomolecules, biomolecule activity evaluation, disease diagnosis, high-throughput screening, drug screening, fluorescent array imaging, surface plasma resonance imaging and the like, and has a wide market prospect; meanwhile, when the biaziridine compound substrate is used for preparing the biochip, the preparation result is more reliable, the prepared biochip is more sensitive in detection result, low in background, good in signal and universal, and the manufacturing method of the biochip has the characteristics of simplicity in operation, flexibility in control, no need of special equipment, low cost and high working efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of microarray fixing compounds, and in particular to a diaziridine compound substrate and a preparation method and application thereof. Background Art

[0002] Microarray is a high-throughput biotechnology used to simultaneously detect the expression level or presence of thousands of biomolecules (such as DNA, RNA, proteins or sugar molecules). It is a flat device that usually uses silicon wafers, glass sheets or nylon membranes as substrates, on which many tiny probe spots are arranged, each of which carries a specific molecular probe (such as oligonucleotides, antibodies or sugar molecules). These probes can specifically bind to target molecules in the sample. It makes it possible to quickly analyze a large number of biomolecules and has a wide range of applications in biomedicine. Effectively fixing various biomolecules on the substrate surface through special surface chemical treatment is a key step in the preparation and application of biochips.

[0003] The modification of traditional substrates (such as ordinary glass slides) involves the activation of hydroxyl groups on the glass surface, the covalent fixation of molecules with amino groups or other active groups on the glass surface, and the reaction of the fixed molecules with the substrate. The chemical reaction between the molecules and the substrate surface may affect their activity. The operation steps are complicated and the cost is high. The number of molecular probes fixed on the substrate is limited, and these molecular probes can only be arranged on the two-dimensional plane of the substrate surface, which limits the recognition and interaction of proteins or other molecules in three-dimensional space.

[0004] Based on the above background technology, the inventors believe that how to design a microarray chip substrate with a simple production method, a high molecular probe fixation density, a high molecular probe curing efficiency, and good detection sensitivity is a technical problem that technicians in this field urgently need to solve.

[0005] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0006] In view of the above technical problems, the embodiments of the present invention provide a diaziridine compound substrate and a preparation method and application thereof to solve the problems raised in the above background technology.

[0007] A diazirine compound substrate comprises a polylysine substrate modified with a diazirine compound; preferably, the thickness of the diazirine compound layer ranges from 0.01 to 1000 nm.

[0008] A method for preparing the above-mentioned diaziridine compound substrate comprises the following steps: Adding a condensing agent, a diaziridine compound and an organic base into a solvent, stirring until dissolved to obtain a first mixed solution; The poly-lysine substrate is immersed in the first mixed solution, and reacted in the absence of light under the protection of an inert gas to obtain a diaziridine compound substrate; Preferably, in the above preparation method, the final concentration of the diaziridine compound in the mixed solution is selected from 1.5-5 mM; the final concentration of HATU in the mixed solution is selected from 2-7.5 mM.

[0009] In the above preparation method, the inert gas is argon or nitrogen.

[0010] In the above preparation method, the time of the light-proof reaction is selected from 1-48h.

[0011] In the above preparation method, the substrate in the poly-lysine substrate can be selected from ordinary glass substrate, quartz glass substrate, silicon substrate, gold / gold-plated substrate, silver / silver-plated substrate.

[0012] Preferably, the volume ratio of the organic base to the solvent is 1:1-1:200; and the molar ratio of the condensing agent to the diaziridine compound is 1:0.1-1:1.

[0013] Preferably, the volume ratio of the organic base to the solvent is 1:9; and the molar ratio of the condensing agent to the diaziridine compound is 1:0.8.

[0014] Preferably, the solvent includes one of N, N-dimethylformamide and DMF; the condensing agent includes one of N, N, N', N'-tetramethyluronium hexafluorophosphate and HATU; and the organic base includes one of N, N-diisopropylethylamine and DIPEA.

[0015] Preferably, the diaziridine compound is 1,2-diazaspiro[2.3]hex-1-ene-5-carboxylic acid, 5-(prop-2-yn-1-yl)-1,2-diazaspiro[2.3]hex-1-ene-5-carboxylic acid, 1,2-diazaspiro[2.4]hept-1-ene-5-carboxylic acid, 1,2-diazaspiro[2.5]octane-1-ene-6-carboxylic acid, 1 , 2-diazaspiro[2.3]hex-1-ene-5-carboxylic acid methyl ester, 1,2-diazaspiro[2.5]oct-1-ene-6-carboxylic acid methyl ester, 3-methyl-3H-diaziridine-3-propionic acid, 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]benzoic acid, 3H-diaziridine-3,3-dicarboxylic acid dipotassium, methyl-diaziridine-alanine, 3-Methyl-3H-diaziridine-3-propionyl chloride, acid-diaziridine-propionic acid methyl ester, 2-(3-butynylaziridin-3-yl)acetic acid, 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]-D-phenylalanine, 3-(4-(3-(trifluoromethyl)-3H-diaziridine-3-yl)phenyl)propionic acid, 2-(prop-2-ynyl- One of: 1-(3-(trifluoromethyl)-3H-naphthyridin-3-yl)benzoic acid, 3-(3-(trifluoromethyl)-3H-naphthyridin-3-yl)benzoic acid, 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]benzoic acid and 3-(3-(but-3-yn-1-yl)-3H-naphthyridin-3-yl)propanoic acid.

[0016] A microarray chip prepared with the diazirine compound substrate as described above, wherein molecular probes are fixed on the diazirine compound substrate; the diazirine compound substrate is activated to form carbene which reacts and connects with the molecular layer to be fixed.

[0017] Preferably, the molecular probe comprises one of small molecule active compounds, amino acids, polypeptides, proteins, glycoproteins, sugars, derived sugar compounds, DNA, RNA, and lipids.

[0018] A method for preparing a microarray chip prepared on the above-mentioned diaziridine compound substrate comprises the following steps: A diaziridine compound substrate is subjected to a blocking reaction using a compound solution having an activated ester, and after the reaction is completed, the substrate is cleaned and dried to obtain a target diaziridine compound substrate; specifically, the compound having an activated ester is such as butyrolactone, valerolactone, succinimide-activated carboxylic acid, etc.; Preparing a molecular probe solution of a certain concentration; The prepared molecular probe solution is spotted and fixed on the surface of the target diaziridine compound substrate to prepare a microarray chip.

[0019] Preferably, the compound with activated ester is preferably valerolactone, the concentration of valerolactone in the compound solution with activated ester is 10% (v / v), and the concentration of PBS is 20 mM; wherein, the diaziridine compound substrate is subjected to a blocking reaction using the compound solution with activated ester, the purpose of which is to acylate the unreacted amino groups on the polylysine substrate with the activated ester to form amino acid esters. In this way, the chemical properties of the substrate can be further modified and the functionality of the substrate can be improved.

[0020] Preferably, in the step of spotting and fixing the prepared molecular probe solution on the surface of the target diaziridine compound substrate, the molecular probe fixing method includes: a first fixing method and a second fixing method; The first fixing method includes: placing the spotted target diaziridine compound substrate into a sealed container, irradiating it with an ultraviolet lamp for a certain period of time after draining it, washing it and then drying it, thereby obtaining a microarray chip; The second fixing method includes: directly irradiating the target diaziridine compound substrate with ultraviolet light for a certain period of time, washing and drying, and obtaining a microarray chip.

[0021] The embodiment of the present invention provides a diaziridine compound substrate and a preparation method and application thereof, which have the following beneficial effects: 1. The present invention utilizes diazirine compounds to modify polylysine substrates, and the synthesis steps are simple and the application range is wide. It can be applied to the field of high-throughput biochip preparation, research on interactions between biomolecules, evaluation of biomolecule activity, disease diagnosis, high-throughput screening, drug screening, fluorescence array imaging, and surface plasmon resonance imaging, etc., and has broad market prospects; 2. The prepared diaziridine compound substrate has good stability. When preparing biochips, biological molecules can be fixed by light irradiation, and there is no need to chemically modify conjugates such as proteins or sugars, making the preparation results more reliable; 3. Compared with the prior art, the biochip prepared by the diaziridine compound substrate developed by the present invention has more sensitive detection results, low background and good signal, and has universal applicability; 4. In addition, the method for making the biochip in the present invention has the characteristics of simple operation, flexible control, no need for special equipment, low cost and high work efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scanning diagram of the screening effect of modifying different photosensitive molecules (chain diaziridine) on a poly-lysine substrate; Figure 2 This is a comparison chart of the screening results of modifying different photosensitive molecules (chain diaziridine) on poly-lysine substrates; Figure 3 This is a scanning diagram of the screening effect of modifying different photosensitive molecules (chain and cyclic diaziridine) on poly-lysine substrate; Figure 4 This is a comparison chart of the screening results of modifying different photosensitive molecules (chain and cyclic diaziridine) on poly-lysine substrates; Figure 5 The figure is a comparison result of the diaziridine substrate chip and the NHS activated ester substrate chip; Figure 6 This is the image of the sugar chip fluorescence array; Figure 7 This is a protein chip fluorescence array imaging image; Figure 8 This is a scanned image comparing the effects of two-dimensional and three-dimensional chips; Fig. 9 This is a data comparison chart of the comparison results of two-dimensional and three-dimensional chips. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0024] In view of the above technical problems, the embodiments of the present invention provide a diaziridine compound substrate and a preparation method and application thereof to solve the problems raised in the above background technology.

[0025] 1. Design concept of the present invention Polylysine slides are a widely used matrix in microarray technology, mainly used to enhance the fixation of biological molecules (such as proteins, DNA, RNA, etc.) on the surface of the slide. Polylysine has a positive charge and can bind to negatively charged molecules (such as nucleic acids or certain proteins) through electrostatic interaction, thereby achieving stable fixation of molecules. The present invention uses a polylysine glass slide as a starting material, and uses the polylysine on its surface to provide a space perpendicular to the glass surface to construct a 3D microarray; this form of modifying polylysine on the glass surface to construct a 3D microarray can fully utilize the three-dimensional space perpendicular to the substrate surface, significantly increasing the molecular fixation density; and this improvement greatly improves the molecular fixation efficiency of the microarray chip technology, thereby significantly improving the sensitivity of analytical detection; At the same time, diaziridine compounds are a common photosensitive compound. Under light, they can be activated into active carbenes, which undergo addition reactions with hydroxyl groups, amino groups, and carbon-hydrogen bonds on the molecules to be fixed, thereby fixing sugars or other molecules on the microarray. The production method is simple.

[0026] 2. Experimental part The materials used in the present invention are as follows: Polylysine substrate was purchased from Nantong Bestec Co., Ltd. Mannan was purchased from Sigma, USA 200KD dextran and amino-modified 200KD dextran were purchased from the National Standard Material Platform. NHS activated ester chip substrate was purchased from Qiyue Biotechnology. Novel coronavirus protein receptor binding region: RBD, donated by Chengdu Maikekang Biotechnology Co., Ltd.

[0027] Meanwhile, other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels.

[0028] The present invention provides a method for preparing a diaziridine compound substrate, which is to modify a diaziridine compound layer on a polylysine substrate, comprising the following specific steps: Mix 10-20 mL N, N-diisopropylethylamine and 100-110 mL N, N-dimethylformamide (volume ratio of 1:5-1:11, preferably 1:9); then add diazirine compounds and HATU to make their final concentrations 1.5-5 mM and 2-7.5 mM, respectively; stir until fully dissolved, and then immerse the polylysine substrate in the above mixed solution, react under the protection of inert gas and avoid light for 12 hours, modify the diazirine compounds on the substrate, and obtain a diazirine compound substrate.

[0029] Example 1-Screening of diaziridine-based photosensitizing molecules: (1) Mix 10 mL of N, N-diisopropylethylamine and 90 mL of N, N-dimethylformamide (volume ratio of 1:9); then add diazirine compounds and HATU to a final concentration of 1.5 mM and 2 mM, respectively; stir until fully dissolved, and then immerse the poly-lysine substrate in the above mixed solution, and react under argon protection and in the dark for 12 h to obtain a diazirine compound substrate.

[0030] The above-mentioned diaziridine compound is selected from 3-methyl-3H-diaziridine-3-propionic acid, 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]benzoic acid, 4-azido-2,3,5,6-tetrafluorobenzoic acid, 1,2-diazaspiro[2.5]octane-1-ene-6-carboxylic acid and 1,2-diazaspiro[2.3]hex-1-ene-5-carboxylic acid.

[0031] (2) The diaziridine compound substrate was blocked with a mixture of valerolactone and PBS for 12 h, washed and blown dry; the final concentration of valerolactone in the mixture was 10% (v / v), and the final concentration of PBS in the mixture was 20 mM.

[0032] (3) Prepare sugar (mannan) into an aqueous solution with a concentration of 10 mg / mL.

[0033] (4) Using a spotter, spot the mannan solution in step (3) onto the surface of the diaziridine compound substrate sealed in step (2) to form a micro-dot array, place it in a sealed container, drain it, irradiate it with a 365 nm ultraviolet lamp for 12 min, wash it, and blow it dry to obtain a sugar chip.

[0034] (5) The sugar chip was incubated with a solution of FITC-labeled Concanavalin A lectin (CCANA) at room temperature for 30 min (the concentration of CCANA was 100 μg / mL, dissolved in 20 mM Hepes buffer solution with a pH of 7.40 and containing 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, 150 mM NaCl, and 0.1% Tween 20), washed three times with H2O, and dried with nitrogen. Imaging was performed using an Innoscan 710 dual-color fluorescence chip scanner with an excitation light of 635 nm.

[0035] Filter results such as Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown: Figure 1-2 The results showed that among the diazirine-based photosensitizing molecules, 3-methyl-3H-diazirine-3-propionic acid had the best effect; Figure 3-4 The results showed that among the diaziridine photosensitive molecules, 1,2-diazaspiro[2.3]hex-1-ene-5-carboxylic acid had the best effect, and the effect of cyclic diaziridine molecules was significantly better than that of chain diaziridine molecules, and the four-membered ring diaziridine molecules had the best effect.

[0036] Example 2-Comparison with the substrate chip in the prior art: (1) The 3-methyl-3H-diaziridine-3-propionic acid substrate in Example 1 was blocked with a mixture of valerolactone and PBS for 12 h, washed and blown dry; wherein the final concentration of valerolactone in the mixture was 10% (v / v), and the final concentration of PBS in the mixture was 20 mM.

[0037] (2) 200KD dextran and amino-modified 200KD dextran were prepared into aqueous solutions with concentrations of 10 mg / mL and 1 mg / mL, respectively.

[0038] (3) Using a spotter, the amino-modified 200KD dextran solution was spotted onto the surface of the diaziridine compound substrate blocked in step (1) and the commercially available NHS activated ester chip substrate to form a micro-dot array. The micro-dot array was placed in a sealed container, drained, and irradiated with a 365 nm ultraviolet lamp for 12 min. The micro-dot array was then cleaned and blown dry to obtain four sugar chips.

[0039] (4) The above sugar chip was incubated with FITC-labeled Concanavalin A lectin solution at room temperature for 30 min (FITC-labeled Concanavalin A lectin solution concentration was 100 μg / mL, dissolved in 20 mM Hepes buffer solution with a pH of 7.40 and containing 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, 150 mM NaCl and 0.1% Tween 20), washed 3 times with H2O, and dried with nitrogen. Imaging was performed using an Innoscan 710 dual-color fluorescence chip scanner under the condition of excitation light of 635 nm.

[0040] The test results are as follows Figure 5 As shown: Figure 5 The results showed that the binding ability of the diazirine-based chip was significantly stronger than that of the NHS-activated ester-based chip. In addition, the process of preparing the sugar chip was simpler, and no modification of sugar molecules was required, so the results obtained were more reliable.

[0041] Embodiment 3-Application example: 1. Sugar chip application To prepare the sugar chip, the steps are as follows: (1) The 3-methyl-3H-diaziridine-3-propionic acid substrate in Example 1 was blocked with a mixture of valerolactone and PBS for 12 h, washed and dried. The final concentration of valerolactone in the mixture was 10% (v / v), and the final concentration of PBS in the mixture was 20 mM.

[0042] (2) Mannan was prepared into aqueous solutions with a series of concentrations (0.009, 0.019, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, and 10 mg / mL).

[0043] (3) Using a spotter, the mannan solution is spotted onto the surface of the diaziridine compound substrate sealed in step (1) to form a micro-dot array, which is then placed in a sealed container. After being drained, the solution is irradiated with a 365 nm ultraviolet lamp for 12 min, washed, and blown dry to obtain a sugar chip.

[0044] (4) The sugar chip was incubated with the Concanavalin A lectin solution at room temperature for 30 min, and then incubated with AF647 NHS activated ester at room temperature for 5 h (the concentration of Concanavalin A lectin solution was 50 μg / mL, and the concentration of AF647 NHS activated ester solution was 20 μg / mL. The above reagents were dissolved in 20 mM Hepes buffer solution with a pH of 7.40 and containing 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, 150 mM NaCl and 0.1% Tween 20), and then vacuum-dried. The images were taken using an Innoscan 710 dual-color fluorescence chip scanner under the condition of excitation light of 635 nm.

[0045] The imaging results are as follows Figure 6 As shown: Figure 6 The results showed that the mannan sample points with a series of concentrations on the sugar chip had fluorescent signals. The existing technology adds sugar under alkaline conditions, which will affect the structure of the sugar. However, the diazirine-based chip uses light reaction to couple sugars, which will not change the structure of the sugar. Compared with existing chips, the results we obtained are more reliable.

[0046] 2. Protein chip application The steps for preparing protein microarray are as follows: (1) The 3-methyl-3H-diaziridine-3-propionic acid substrate in Example 1 was blocked with a mixture of valerolactone and PBS for 12 h, washed and dried. The final concentration of valerolactone in the mixture was 10% (v / v), and the final concentration of PBS in the mixture was 20 mM.

[0047] (2) The protein (novel coronavirus protein receptor binding domain: RBD) was prepared into aqueous solutions with a series of concentrations (1.5 and 0.15 mg / mL).

[0048] (3) Using a spotter, spot the protein solution onto the surface of the diaziridine compound substrate sealed in step (1) to form a micro-dot array. Place the micro-dot array in a sealed container, irradiate with a 365 nm ultraviolet lamp in a humid environment for 12 min, wash and blow dry to obtain a protein chip.

[0049] (4) Incubate the protein chip with 1% (v / v) BSA (solvent: 50 mM Tris-HCl, 150 mM NaCl) at room temperature for 1 h.

[0050] (5) The protein chip was incubated with mouse serum at room temperature for 5 h (mouse serum was diluted 1:100 and dissolved in a buffer solution of 50 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20).

[0051] (6) The protein chip was incubated with Multi-rAb CoraLite® Plus 647-Goat Anti-Mouse Recombinant Secondary Antibody (H+L) for 2 h (diluted 1:1000 in a buffer solution of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 1% BSA) and vacuum-dried. The images were imaged using an Innoscan 710 dual-color fluorescence chip scanner at an excitation light of 635 nm.

[0052] The imaging results are as follows Figure 7 As shown: Figure 7 The results showed that the protein chip could have fluorescent signals through incubation with fluorescent antibodies.

[0053] The current protein chips are expensive and complicated to operate. Due to the instability of proteins, there are many restrictions on their coupling conditions. The diazirine-based chip uses photocoupling to fix proteins on the chip, which is fast and universal. By comparing with the natural adsorption method, it can be seen that the photosensitive molecule coupling effect is better.

[0054] Example 4 - Comparison of commercially available substrates: (1) Glass substrate cleaning and activation The glass substrate was ultrasonically cleaned with acetone and H2O for 3 times, each time for 5 min, and then dried with N2; it was immersed in H2SO4 / H2O2 washing solution (v / v=3 / 1) at 70°C for 30 min, cleaned with H2O for 3 times, and then dried with N2.

[0055] (2) Aminosilanization of glass substrate surface The glass substrate treated in step (1) was immersed in 10% (v / v) 3-aminopropyltrimethoxysilane in ethanol for 3 h. After the reaction was completed, the substrate was cleaned with ethanol and H2O and then dried with N2.

[0056] The common amino substrate (two-dimensional) and the commercially available poly-lysine substrate (three-dimensional) in step (2) are respectively surface-modified with 3-methyl-3H-diaziridine-3-propionic acid: N, N-diisopropylethylamine and N, N-dimethylformamide in a volume ratio of 1:9 are mixed, and then diaziridine with a final concentration of 1.5 mM and HATU with a final concentration of 2 mM are added, and stirred until fully dissolved. The commercially available poly-lysine substrate is immersed in the mixed solution and reacted in the dark under the protection of an inert gas until the diaziridine compound is successfully modified on the substrate, thereby obtaining a substrate modified with the diaziridine compound. The 3-methyl-3H-diaziridine-3-propionic acid substrate was blocked for 12 h using a mixture of 10% (v / v) valerolactone and 20 mM PBS, and then washed and dried. (5) Sugar (mannan) was prepared into aqueous solutions with concentrations of 10 mg / mL and 1 mg / mL.

[0057] (6) Using a spotter, spot the biomolecule compound solution in step (5) onto the surface of the substrate modified with the diaziridine compound blocked in step (4) to form a micro-dot array, place it in a sealed container, drain it, irradiate it with a 365 nm ultraviolet lamp for 12 min, wash it, and blow it dry to obtain a sugar chip.

[0058] (7) The sugar chip was incubated with a solution of FITC-labeled Concanavalin A lectin (CCANA) at room temperature for 30 min (the concentration of CCANA was 100 μg / mL, dissolved in 20 mM Hepes buffer (pH 7.40) containing 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, 150 mM NaCl, and 0.1% Tween 20), washed three times with H2O, and dried with nitrogen. The chips were imaged using an Innoscan 710 dual-color fluorescence chip scanner at an excitation light of 635 nm.

[0059] See also Figure 8 and Fig. 9 , Figure 8-9 The results showed that the three-dimensional chip had better effect, that is, the commercially available substrate for coupling photosensitive molecules should be a poly-lysine substrate.

[0060] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A diaziridine compound substrate, characterized in that: It includes a poly-lysine substrate modified with a diazirine compound.

2. A method for preparing a diaziridine compound substrate as claimed in claim 1, characterized in that: The following steps are involved: Adding a condensing agent, a diaziridine compound and an organic base into a solvent, stirring until dissolved to obtain a first mixed solution; The poly-lysine substrate is immersed in the first mixed solution, and reacted in the protection of an inert gas and in the dark to obtain a diaziridine compound substrate.

3. The method for preparing a diaziridine compound substrate according to claim 2, characterized in that: The volume ratio of the organic base to the solvent is 1:1-1:200; the molar ratio of the condensing agent to the diaziridine compound is 1:0.1-1:

1.

4. The method for preparing a diaziridine compound substrate according to claim 2, characterized in that: The volume ratio of the organic base to the solvent is 1:9; the molar ratio of the condensing agent to the diaziridine compound is 1:0.

8.

5. The method for preparing a diaziridine compound substrate according to claim 2, characterized in that: The solvent includes one of N,N-dimethylformamide, DMF; the condensing agent includes one of N,N,N',N'-tetramethyluronium hexafluorophosphate, HATU; and the organic base includes one of N, N-diisopropylethylamine, DIPEA.

6. The method for preparing a diaziridine compound substrate according to claim 2, characterized in that: The diaziridine compounds are 1,2-diazaspiro[2.3]hex-1-ene-5-carboxylic acid, 5-(prop-2-yn-1-yl)-1,2-diazaspiro[2.3]hex-1-ene-5-carboxylic acid, 1,2-diazaspiro[2.4]hept-1-ene-5-carboxylic acid, 1,2-diazaspiro[2.5]octane-1-ene-6-carboxylic acid, 1,2- Diazaspiro[2.3]hex-1-ene-5-carboxylic acid methyl ester, 1,2-diazaspiro[2.5]oct-1-ene-6-carboxylic acid methyl ester, 3-methyl-3H-diaziridine-3-propionic acid, 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]benzoic acid, 3H-diaziridine-3,3-dicarboxylic acid dipotassium, methyl-diaziridine-alanine, 3- Methyl-3H-diaziridine-3-propionyl chloride, acid-diaziridine-propionic acid methyl ester, 2-(3-butynylaziridin-3-yl)acetic acid, 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]-D-phenylalanine, 3-(4-(3-(trifluoromethyl)-3H-diaziridine-3-yl)phenyl)propionic acid, 2-(prop-2-yn-1 -yloxy)-4-(3-(trifluoromethyl)-3H-naphthyridin-3-yl)benzoic acid, 3-(3-(trifluoromethyl)-3H-naphthyridin-3-yl)benzoic acid, 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]benzoic acid and 3-(3-(but-3-yn-1-yl)-3H-naphthyridin-3-yl)propanoic acid.

7. A microarray chip prepared using the diaziridine compound substrate as claimed in claim 1 or 2, characterized in that: Molecular probes are fixed on the diaziridine compound substrate.

8. The microarray chip prepared from the diaziridine compound substrate according to claim 7, characterized in that: The molecular probe includes one of small molecule active compounds, amino acids, polypeptides, proteins, glycoproteins, sugars, derived sugar compounds, DNA, RNA, and lipids.

9. A method for preparing a microarray chip prepared on a diaziridine compound substrate as claimed in claim 7, characterized in that: The following steps are involved: Taking a diaziridine compound substrate and performing a blocking reaction with a compound solution containing an activated ester, and washing and drying it after the reaction is completed to obtain a target diaziridine compound substrate; Preparing a molecular probe solution of a certain concentration; The prepared molecular probe solution is spotted and fixed on the surface of the target diaziridine compound substrate to prepare a microarray chip.

10. The method for preparing a microarray chip prepared on a diazirine compound substrate according to claim 9, characterized in that: In the step of spotting and fixing the prepared molecular probe solution on the surface of the target diaziridine compound substrate, the molecular probe fixing method includes: a first fixing method and a second fixing method; The first fixing method includes: placing the spotted target diaziridine compound substrate into a sealed container, irradiating it with an ultraviolet lamp for a certain period of time after draining it, washing it and then drying it, thereby obtaining a microarray chip; The second fixing method includes: directly irradiating the target diaziridine compound substrate with ultraviolet light for a certain period of time, washing and drying, and obtaining a microarray chip.

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