A bis-aziridine compound substrate and methods of making and using the same

Abstract

The application discloses a kind of diazirine compound base and preparation method and application thereof, it is related to microarray immobilization compound technical field.The diazirine compound base is obtained by using diazirine compound to modify polylysine base, it is simple in synthesis step, wide in application range, can be applied to high-throughput biochip preparation field, biological molecule interaction research, biological molecule activity evaluation, disease diagnosis, high-throughput screening, drug screening, fluorescence array imaging and surface plasmon resonance imaging etc., with wide market prospect;Meanwhile, when preparing biochip by diazirine compound base, preparation result is more reliable, and the detection result of prepared biochip is more sensitive, with low background and good signal, with universality, and the preparation method of biochip has the characteristics of simple operation, flexible control, without special equipment, low cost, high work efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microarray immobilization compounds, in particular to a kind of diazirine compound substrate and its preparation method and application. BACKGROUND

[0002] Microarray is a kind of high-throughput biological technology, which is used to detect the expression level or existence of thousands of biological molecules (such as DNA, RNA, protein or sugar molecules) at the same time. It is a kind of panel device, usually using silicon chip, glass sheet or nylon film as substrate, and a lot of micro probe points are arranged on it, each point carries specific molecular probe (such as oligonucleotide, antibody or sugar molecule). These probes can specifically bind to target molecules in the sample. It makes it possible to analyze a large number of biological molecules quickly, and has a wide range of applications in biomedical field. Through special surface chemical treatment, various biological molecules are effectively fixed on the substrate surface, which is the key step of biochip preparation and application.

[0003] The modification of traditional matrix (such as ordinary glass slide) involves the activation of glass surface hydroxyl group, the reaction of covalently fixing the molecules with amino group or other active groups on the glass surface, and the reaction of the fixed molecules with the matrix. The chemical reaction of molecules with the surface of the matrix may affect its activity. Its operation steps are complex and the cost is high. The number of molecular probes fixed on the matrix is limited, and these molecular probes can only be arranged on the two-dimensional plane of the matrix 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 kind of microarray chip substrate with simple manufacturing method, large molecular probe fixing density, high molecular probe fixing efficiency and good detection sensitivity is a technical problem that needs to be solved by those skilled in the art.

[0005] The information disclosed in this background section is intended only to increase an understanding of the general context of the present application, and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is already widely known in the art. SUMMARY

[0006] In order to solve the above-mentioned problems in the background art, the embodiments of the present application provide a kind of diazirine compound substrate and its preparation method and application.

[0007] A kind of diazirine compound substrate, including the polylysine substrate modified with diazirine compound;Preferably, the thickness of diazirine compound layer is in the range of 0.01-1000 nm.

[0008] A preparation method of a diazirine compound substrate as described above, comprising the following steps:

[0009] adding a condensing agent, a diazirine compound and an organic base into a solvent, stirring to dissolve to obtain a first mixed solution;

[0010] immersing a polylysine substrate into the first mixed solution, and reacting under the protection of an inert gas and in the dark to obtain a diazirine compound substrate;

[0011] Preferably, in the above preparation method, the final concentration of the diazirine compound in the mixed solution is selected from 1.5-5 mM; and the final concentration of the HATU in the mixed solution is selected from 2-7.5 mM.

[0012] In the above preparation method, the inert gas is argon or nitrogen.

[0013] In the above preparation method, the time of the light-avoiding reaction is selected from 1-48 h.

[0014] In the above preparation method, the substrate in the polylysine substrate can be selected from common glass substrate, quartz glass substrate, silicon substrate, gold / plated gold substrate, silver / plated silver substrate.

[0015] 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 diazirine compound is 1:0.1-1:1.

[0016] Preferably, the volume ratio of the organic base to the solvent is 1:9; and the molar ratio of the condensing agent to the diazirine compound is 1:0.8.

[0017] Preferably, the solvent includes one of N, N-dimethylformamide, DMF; the condensing agent includes one of N, N, N', N'-tetramethylurea hexafluorophosphate, HATU; and the organic base includes one of N, N-diisopropyl ethylamine, DIPEA.

[0018] Preferably, the bisacrylidine compound is 1,2-diazaspiro[2.3]hex-1-en-5-carboxylic acid, 5-(prop-2-yn-1-yl)-1,2-diazaspiro[2.3]hex-1-en-5-carboxylic acid, 1,2-diazaspiro[2.4]hept-1-en-5-carboxylic acid, 1,2-diazaspiro[2.5]octane-1-en-6-carboxylic acid, 1 2-Dazaspiro[2.3]hex-1-en-5-carboxylic acid methyl ester, 1,2-Dazaspiro[2.5]oct-1-en-6-carboxylic acid methyl ester, 3-methyl-3H-bisacrididin-3-propionic acid, 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzoic acid, 3H-bisacrididin-3,3-dicarboxylic acid dipotassium, methyl-bisacrididin-alanine, 3-Methyl-3H-bisacrididin-3-propionyl chloride, methyl bisacrididin-propionate, 2-(3-butynylazadiazidin-3-yl)acetic acid, 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]-D-phenylalanine, 3-(4-(3-(trifluoromethyl)-3H-diazacyclopropen-3-yl)phenyl)propionic acid, 2-(prop-2-yne- One of 1-yloxy)-4-(3-(trifluoromethyl)-3H-diazanaphth-3-yl)benzoic acid, 3-(3-(trifluoromethyl)-3H-diazanaphth-3-yl)benzoic acid, 4-[3-(trifluoromethyl)-3H-bisacrylidine-3-yl]benzoic acid and 3-(3-(but-3-yn-1-yl)-3H-diazanaphth-3-yl)propionic acid.

[0019] A microarray chip prepared from a bisacrididine-based compound substrate as described above, wherein molecular probes are immobilized on the bisacrididine-based compound substrate; after activation, the bisacrididine-based compound substrate forms carbenes that react and connect with the molecular layer to be immobilized.

[0020] Preferably, the molecular probe comprises one of the following: small molecule active compounds, amino acids, peptides, proteins, glycoproteins, sugars, derived sugar compounds, DNA, RNA, and lipids.

[0021] A method for fabricating a microarray chip based on a bisacrylidine compound substrate as described above includes the following steps:

[0022] A bisacrylidine-based compound substrate was subjected to a blocking reaction using a solution of a compound containing an activated ester. After the reaction, the substrate was washed and dried to obtain the target bisacrylidine-based compound substrate. Specifically, the activated ester compounds include butyrolactone, valproic acid, and succinimide-activated carboxylic acids.

[0023] Prepare a molecular probe solution of a certain concentration;

[0024] The prepared molecular probe solution was spotted and immobilized on the surface of the target bisacrylidine-based compound substrate to prepare a microarray chip.

[0025] Preferably, the compound containing the activated ester is valproic acid, and the concentration of valproic acid in the compound solution containing the activated ester is 10% (v / v), and the concentration of PBS is 20 mM. The purpose of using the solution of the activated ester to block the bisacrylidine compound substrate is to acylate the unreacted amino groups on the polylysine substrate with the activated ester to form an amino acid ester. In this way, the chemical properties of the substrate can be further modified and the functionality of the substrate can be improved.

[0026] Preferably, in the step of spotting and immobilizing the prepared molecular probe solution onto the surface of the target bisacrylidine compound substrate, the immobilization method of the molecular probe includes: a first immobilization method and a second immobilization method;

[0027] The first fixation method includes: placing the dotted target bisacrylidine compound substrate into a sealed container, drying it, irradiating it with an ultraviolet lamp for a certain period of time, cleaning it, and then drying it to obtain a microarray chip;

[0028] The second fixation method includes: directly irradiating the target bisacrylidine compound substrate with ultraviolet light for a certain period of time, cleaning and drying it to obtain a microarray chip.

[0029] The present invention provides a bisacrylidine compound substrate, its preparation method, and its application, which have the following beneficial effects:

[0030] 1. This invention utilizes bisacrylidine compounds to modify polylysine substrates. The synthesis steps are simple, and the application range is wide. It can be applied to the fields of high-throughput biochip fabrication, biomolecular interaction research, biomolecular activity evaluation, disease diagnosis, high-throughput screening, drug screening, fluorescence array imaging, and surface plasmon resonance imaging, etc., and has broad market prospects.

[0031] 2. The prepared biacrylidine compounds have good substrate stability. When preparing biochips, biomolecules can be fixed by light irradiation without the need for chemical modification of conjugates such as proteins or sugars, making the preparation results more reliable.

[0032] 3. Compared with the prior art, the biochip prepared from the bisacrylidine compound substrate developed in this invention has more sensitive detection results, lower background and better signal, and is more universal;

[0033] 4. In addition, the biochip fabrication method of the present invention has the characteristics of simple operation, flexible control, no need for special equipment, low cost and high work efficiency. Attached Figure Description

[0034] Figure 1Scanning results showing the screening effect of different photosensitive molecules (chain-like bipropidines) modified on a polylysine substrate;

[0035] Figure 2 This is a comparison chart of screening results for different photosensitive molecules (chain-like bisacrylidines) modified on a polylysine substrate;

[0036] Figure 3 Scanning images showing the screening effects of different photosensitive molecules (chain and cyclic biacrylidines) modified on a polylysine substrate;

[0037] Figure 4 A comparison of screening results for different photosensitive molecules (chain-like and cyclic bisacrylidines) modified on a polylysine substrate;

[0038] Figure 5 This is a comparison of the results between the bisacrylamide substrate chip and the NHS activated ester substrate chip;

[0039] Figure 6 Image of a glucose chip fluorescence array;

[0040] Figure 7 This is an image of a protein chip fluorescence array.

[0041] Figure 8 Scan images showing the comparison between two-dimensional and three-dimensional chips;

[0042] Figure 9 This is a comparison chart of the data results between two-dimensional and three-dimensional chips. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] To address the aforementioned technical problems, this invention provides a bisacrylidine compound substrate, its preparation method, and its application, thereby resolving the issues raised in the background section.

[0045] I. Design Concept of the Invention

[0046] Poly-Lysine slides are a widely used matrix in microarray technology, mainly used to enhance the fixation of biomolecules (such as proteins, DNA, RNA, etc.) on the slide surface; poly-Lysine carries a positive charge and can bind to negatively charged molecules (such as nucleic acids or certain proteins) through electrostatic interactions, thereby achieving stable molecular fixation.

[0047] This invention utilizes poly-L-lysine glass slides as starting materials, leveraging the poly-L-lysine on the slides to provide space perpendicular to the glass surface, thereby constructing a 3D microarray. This method of modifying the glass surface with poly-L-lysine to construct a 3D microarray can fully utilize the three-dimensional space perpendicular to the matrix surface, significantly increasing the molecular immobilization density. Furthermore, this improvement greatly enhances the molecular immobilization efficiency of microarray chip technology, thereby significantly improving the sensitivity of analytical detection.

[0048] Meanwhile, bisacrylidine compounds are a common type of photosensitive compound. Under light, they can be activated into active carbene compounds, which can then undergo addition reactions with hydroxyl, amino, and C-H bonds on the molecules to be immobilized, thereby immobilizing sugars or other molecules on the microarray. The preparation method is simple.

[0049] II. Experimental Section

[0050] The materials used in this invention are as follows:

[0051] Polylysine substrate, purchased from Nantong Besttech Co., Ltd. Mannan, purchased from Sigma-Aldrich (USA). 200KD dextran and amino-modified 200KD dextran, purchased from the National Standard Material Platform. NHS activated ester chip substrate, purchased from Qiyue Biotechnology. Novel coronavirus protein receptor binding region: RBD, donated by Chengdu Maikekang Biotechnology Co., Ltd.

[0052] Meanwhile, all other materials used in this invention, unless otherwise stated, are available through commercial channels.

[0053] This invention provides a method for preparing a bisacrididine compound substrate, which involves modifying a polylysine substrate with a bisacrididine compound layer, including the following specific steps:

[0054] Mix 10-20 mL of N,N-diisopropylethylamine and 100-110 mL of N,N-dimethylformamide (volume ratio 1:5-1:11, preferably 1:9); then add a diazinonide compound and HATU to make their final concentrations 1.5-5 mM and 2-7.5 mM, respectively; stir until fully dissolved, then immerse the poly-lysine substrate in the above mixed solution, and react for 12 h in the dark under the protection of an inert gas to modify the substrate with the diazinonide compound, thus obtaining the diazinonide compound substrate.

[0055] Example 1 - Screening of bipropidine-based photosensitive molecules:

[0056] (1) Mix 10 mL of N,N-diisopropylethylamine and 90 mL of N,N-dimethylformamide (volume ratio 1:9); then add bisacrylidine compounds and HATU to make their final concentrations 1.5 mM and 2 mM, respectively; stir until fully dissolved, and then soak the polylysine substrate in the above mixed solution. Under the protection of argon, react in the dark for 12 h to obtain the bisacrylidine compound substrate.

[0057] The above-mentioned diazidopropylidine compounds are selected from 3-methyl-3H-diazidopropyl-3-propionic acid, 4-[3-(trifluoromethyl)-3H-diazidopropyl-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.

[0058] (2) The substrate of biacpropidine compound was blocked with a mixture of valproic acid and PBS for 12 h, washed and dried; wherein the final concentration of valproic acid in the mixture was 10% (v / v) and the final concentration of PBS in the mixture was 20 mM.

[0059] (3) Prepare an aqueous solution of sugar (mannan) with a concentration of 10 mg / mL.

[0060] (4) Using a spotting instrument, spot the mannan solution from step (3) onto the surface of the sealed biacrylidine compound substrate from step (2) to form a microarray. Place it in a sealed container, dry it, and then irradiate it with a 365 nm UV lamp for 12 min. After cleaning, blow it dry to obtain the sugar chip.

[0061] (5) The sugar chip was incubated with a fluorescein-labeled concanavalin A solution at room temperature for 30 min (the concentration of the fluorescein-labeled concanavalin A solution was 100 μg / mL, dissolved in 20 mM Hepes buffer solution at 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. Imaging was performed using an Innoscan 710 dual-color fluorescence chip scanner under an excitation light of 635 nm.

[0062] The filtering results are as follows Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown: Figures 1-2 The results showed that among the biacrylidine-based photosensitizing molecules, 3-methyl-3H-bisacrylidine-3-propionic acid had the best effect; Figures 3-4The results showed that among the bisacrylidine photosensitive molecules, 1,2-diazaspiro[2.3]hex-1-ene-5-carboxylic acid had the best effect, and the cyclic bisacrylidine molecules were significantly better than the chain bisacrylidine molecules, with the four-membered ring bisacrylidine molecules having the best effect.

[0063] Example 2 - Comparison with substrate chips in the prior art:

[0064] (1) The 3-methyl-3H-bisacrylidine-3-propionic acid substrate in Example 1 was blocked with a mixture of valproic acid and PBS for 12 h, washed and dried; wherein the final concentration of valproic acid in the mixture was 10% (v / v) and the final concentration of PBS in the mixture was 20 mM.

[0065] (2) Prepare aqueous solutions of 200KD glucan and amino-modified 200KD glucan with concentrations of 10 mg / mL and 1 mg / mL, respectively.

[0066] (3) Using a spotting instrument, the amino-modified 200KD dextran solution was spotted onto the surface of the biacrimidine compound substrate blocked in step (1) and the commercially available NHS activated ester chip substrate to form a microarray. The microarray was placed in a sealed container, dried, and then irradiated with a 365 nm UV lamp for 12 min. After cleaning, it was dried to obtain four types of sugar chips.

[0067] (4) The above-mentioned sugar chip was incubated with a fluorescein isothiocyanate-labeled concanavalin A solution for 30 min at room temperature (the concentration of the fluorescein isothiocyanate-labeled concanavalin A solution was 100 μg / mL, dissolved in 20 mM Hepes buffer solution with pH 7.40 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 an excitation light of 635 nm.

[0068] The test results are as follows Figure 5 As shown: Figure 5 The results showed that the binding affinity of the bisacrylidine-based chip was significantly stronger than that of the NHS-activated ester-based chip. Furthermore, the preparation process of the sugar chip was simpler, requiring no modification of the sugar molecules, and the results were more reliable.

[0069] Example 3 - Application Example:

[0070] 1. Application of sugar chips

[0071] The steps for preparing the sugar chip are as follows:

[0072] (1) The 3-methyl-3H-bisacrylidine-3-propionic acid substrate from Example 1 was blocked with a mixture of valerolactone and PBS for 12 h, washed, and then 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.

[0073] (2) Prepare a series of aqueous solutions of mannan with concentrations of (0.009, 0.019, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 mg / mL).

[0074] (3) Using a spotting instrument, the mannan solution is spotted onto the surface of the bisacrylidine-based compound substrate sealed in step (1) to form a microarray. The substrate is placed in a sealed container, dried, and then irradiated with a 365 nm ultraviolet lamp for 12 min. After cleaning, it is dried to obtain the sugar chip.

[0075] (4) The sugar chip was incubated with concanavalin A solution at room temperature for 30 min, and then with AF647 NHS activated ester at room temperature for 5 h (concanavalin A solution concentration of 50 μg / mL, AF647 NHS activated ester solution concentration of 20 μg / mL, all reagents were dissolved in 20 mM pH 7.40 Hepes buffer solution containing 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, 150 mM NaCl and 0.1% Tween 20), and then dried under vacuum. Imaging was performed using an Innoscan 710 dual-color fluorescence chip scanner under excitation light of 635 nm.

[0076] Imaging results as follows Figure 6 As shown: Figure 6 The results showed that mannan sample spots of various concentrations on the sugar chip exhibited fluorescent signals. Existing techniques, which involve loading sugars under alkaline conditions, can affect the sugar structure. However, the biacaridine-based chip utilizes a photo-induced reaction for sugar coupling, thus preserving the sugar structure. Our results are more reliable compared to existing chips.

[0077] 2. Protein chip applications

[0078] The steps for preparing a protein chip are as follows:

[0079] (1) The 3-methyl-3H-bisacrylidine-3-propionic acid substrate from Example 1 was blocked with a mixture of valerolactone and PBS for 12 h, washed, and then 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.

[0080] (2) Prepare aqueous solutions of the protein (receptor binding region of the novel coronavirus protein: RBD) at a series of concentrations (1.5 and 0.15 mg / mL).

[0081] (3) Using a spotting instrument, the protein solution is spotted onto the surface of the bipropidine-based compound substrate sealed in step (1) to form a microarray. The microarray is placed in a sealed container and irradiated with a 365 nm UV lamp in a humid environment for 12 min. After cleaning, it is dried to obtain the protein chip.

[0082] (4) Incubate the protein chip with 1% (v / v) BSA (solvents are 50 mM Tris-HCl and 150 mM NaCl) at room temperature for 1 h.

[0083] (5) The protein chip was incubated with mouse serum at room temperature for 5 h (the mouse serum was diluted 1:100 and dissolved in 50 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20 buffer solution).

[0084] (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 then dried under vacuum. Imaging was performed using an Innoscan 710 dual-color fluorescence chip scanner at an excitation light of 635 nm.

[0085] Imaging results as follows Figure 7 As shown: Figure 7 The results showed that the protein chip could exhibit a fluorescent signal after incubation with a fluorescent antibody.

[0086] Current protein microarrays are expensive and complex to operate, and their coupling conditions are subject to many limitations due to the instability of proteins. Bisacrididine-based microarrays, however, immobilize proteins on the chip via photocoupling, which is rapid and widely applicable. A comparison with natural adsorption methods shows that photosensitive molecular coupling is more effective.

[0087] Example 4 - Comparison of commercially available substrates:

[0088] (1) Cleaning and activation of glass substrate

[0089] The glass substrate was ultrasonically cleaned three times with acetone and H2O for 5 minutes each time, and then dried with N2. It was then immersed in H2SO4 / H2O2 cleaning solution (v / v=3 / 1) at 70℃ for 30 minutes, cleaned three times with H2O, and then dried with N2.

[0090] (2) Aminosilanization of glass substrate surface

[0091] 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 washed with ethanol and H2O and then dried with N2.

[0092] The surface of the ordinary amino substrate (two-dimensional) and the commercially available polylysine substrate (three-dimensional) from step (2) was modified with 3-methyl-3H-bisacrylidine-3-propionic acid: N,N-diisopropylethylamine and N,N-dimethylformamide in a volume ratio of 1:9. Bisacrylidine with a final concentration of 1.5 mM and HATU with a final concentration of 2 mM were added and stirred until fully dissolved. The commercially available polylysine substrate was immersed in the mixed solution and reacted in the dark under the protection of an inert gas until the bisacrylidine compound was successfully modified onto the substrate, thus obtaining the substrate modified with the bisacrylidine compound.

[0093] The 3-methyl-3H-bisacrylidine-3-propionic acid substrate was blocked for 12 h using a mixture of 10% (v / v) valproic acid and 20 mM PBS, and then washed and dried.

[0094] (5) Prepare aqueous solutions of sugar (mannan) with concentrations of 10 mg / mL and 1 mg / mL.

[0095] (6) Using a spotting instrument, spot the biomolecular compound solution from step (5) onto the substrate surface modified by the closed diazinon compound from step (4) to form a microarray. Place it in a sealed container, dry it, and then irradiate it with a 365 nm UV lamp for 12 min. After cleaning, blow it dry to obtain the sugar chip.

[0096] (7) The sugar chip was incubated with a fluorescein isothiocyanate-labeled concanavalin A solution at room temperature for 30 min (the concentration of the fluorescein isothiocyanate-labeled concanavalin A solution was 100 μg / mL, dissolved in 20 mM pH 7.40 Hepes buffer solution 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 fluorescent chip scanner under an excitation light of 635 nm.

[0097] See Figure 8and Figure 9 , Figures 8-9 The results showed that the three-dimensional chip performed better, indicating that the commercially available substrate for photosensitive molecule coupling should be a polylysine substrate.

[0098] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A bisacrylidine compound substrate, characterized in that, Including polylysine substrates modified with diaziridine compounds; A method for preparing a bisacrylidine-based compound substrate includes the following steps: A condensing agent, a diazinon compound, and an organic base are added to a solvent and stirred until dissolved to obtain a first mixed solution; The polylysine substrate was immersed in the first mixed solution and reacted in the dark under the protection of an inert gas to obtain a bisacrylidine compound substrate. Among them, the bisacrylidine compounds are 1,2-diazaspiro[2.3]hex-1-en-5-carboxylic acid, 5-(prop-2-yn-1-yl)-1,2-diazaspiro[2.3]hex-1-en-5-carboxylic acid, 1,2-diazaspiro[2.4]hept-1-en-5-carboxylic acid, 1,2-diazaspiro[2.5]octane-1-en-6-carboxylic acid, 1 2-Dazaspiro[2.3]hex-1-en-5-carboxylic acid methyl ester, 1,2-Dazaspiro[2.5]oct-1-en-6-carboxylic acid methyl ester, 3-methyl-3H-bisacrididin-3-propionic acid, 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzoic acid, 3H-bisacrididin-3,3-dicarboxylic acid dipotassium, methyl-bisacrididin-alanine, 3-Methyl-3H-bisacrididin-3-propionyl chloride, methyl bisacrididin-propionate, 2-(3-butynylazadiazidin-3-yl)acetic acid, 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]-D-phenylalanine, 3-(4-(3-(trifluoromethyl)-3H-diazacyclopropen-3-yl)phenyl)propionic acid, 2-(prop-2-yne- One of 1-yloxy)-4-(3-(trifluoromethyl)-3H-diazanaphth-3-yl)benzoic acid, 3-(3-(trifluoromethyl)-3H-diazanaphth-3-yl)benzoic acid, 4-[3-(trifluoromethyl)-3H-bisacrylidine-3-yl]benzoic acid and 3-(3-(but-3-yn-1-yl)-3H-diazanaphth-3-yl)propionic acid.

2. The bisacrylidine compound substrate according to claim 1, characterized in that, The volume ratio of organic base to solvent is 1:1 to 1:200; the molar ratio of condensing agent to diazinon-propidine compound is 1:0.1 to 1:

1.

3. The bisacrylidine compound substrate according to claim 1, characterized in that, The volume ratio of organic base to solvent is 1:9; the molar ratio of condensing agent to diazinon compound is 1:0.

8.

4. The bisacrylidine compound substrate according to claim 1, characterized in that, The solvent includes N,N-dimethylformamide, one of DMF; the condensing agent includes N,N,N',N'-tetramethylurea hexafluorophosphate, one of HATU; the organic base includes N,N-diisopropylethylamine, one of DIPEA.

5. A microarray chip prepared on a bisacrididine-based compound substrate as described in claim 1, characterized in that, Molecular probes are immobilized on a substrate of adipropionyl compounds.

6. The microarray chip prepared on the bisacrididine compound substrate according to claim 5, characterized in that, The molecular probes include one of the following: small molecule active compounds, amino acids, peptides, proteins, glycoproteins, sugars, derived sugar compounds, DNA, RNA, and lipids.

7. A method for fabricating a microarray chip based on a bisacrylidine compound substrate as described in claim 5, characterized in that, Includes the following steps: A bisacrylidine-based compound substrate was subjected to a blocking reaction using a solution of a compound containing an activated ester. After the reaction was completed, the substrate was washed and dried to obtain the target bisacrylidine-based compound substrate. Prepare a molecular probe solution of a certain concentration; The prepared molecular probe solution was spotted and immobilized on the surface of the target bisacrylidine-based compound substrate to prepare a microarray chip.

8. The method for fabricating a microarray chip based on a bisacrylidine compound substrate according to claim 7, characterized in that, In the step of spotting and immobilizing the prepared molecular probe solution onto the surface of the target bisacrylidine compound substrate, the immobilization methods of the molecular probe include: a first immobilization method and a second immobilization method; The first fixation method includes: placing the dotted target bisacrylidine compound substrate into a sealed container, drying it, irradiating it with an ultraviolet lamp for a certain period of time, cleaning it, and then drying it to obtain a microarray chip; The second fixation method includes: directly irradiating the target bisacrylidine compound substrate with ultraviolet light for a certain period of time, cleaning and drying it to obtain a microarray chip.

Images