Chip preparation method and product and application thereof

Abstract

The invention discloses a chip preparation method and a product and application thereof. The chip is prepared by adopting a fixed-point sample application modification scheme, a hydrophobic surface is constructed, and the fixed sample amount of the chip can be increased to 4000 samples per square centimeter from 500 samples per square centimeter; the surface of the gold film is modified by methods such as incubation / soaking, and the method can be suitable for surface modification of chips with various areas; an analyte is fixed by adopting a carbene free radical reaction, so that the method is wider in application range and is particularly suitable for library building of small molecular compounds without specific groups (such as carboxyl, amino, hydroxyl and sulfydryl).

Description

Technical Field

[0001] This article relates to biological detection technology, in particular to a method for preparing a chip for SPRi detection, its products, and uses. Background Art

[0002] As the source of new drug research and development, drug screening technology has always been valued as a core technology by research institutes and pharmaceutical companies, and biomolecular interaction technology plays a key role in drug screening. Currently, biomolecular interaction technology based on surface plasmon resonance (SPR) mainly focuses on solutions that modify bare gold slides with dextran. This is mainly based on the surface chemistry construction of Biacore CM series chips and alternatives, and achieves covalent / non-covalent connection of analytes by modifying dextran on the gold substrate. These solutions have the disadvantages of low spot density and a narrow range of application. They are mainly suitable for use in scenarios where SPR instruments modify the entire gold film and then press it to form microfluidics. They are not suitable for use in scenarios where high-density connection of analytes is required. Summary of the Invention

[0003] Based on this, the present application provides a chip preparation method, comprising:

[0004] 1) Spotting a mixed solution containing compound 1 and compound 2 on a gold film, incubating to form a dot matrix on the gold film, washing and drying to obtain a substrate chip, wherein:

[0005] The general formula of compound 1 is:

[0006] Here, n is an integer from 1 to 15;

[0007] The general formula of compound 2 is:

[0008] Here, m or n is each independently an integer from 1 to 15;

[0009] And wherein the molar ratio of compound 1 to compound 2 is 1:100-100:1;

[0010] 2) adding a solution containing compound 3 to the surface of the substrate chip obtained in step 1) so that compound 3 is connected to compound 1 on the surface of the substrate chip, washing and drying to obtain a photosensitive chip, wherein:

[0011] The general formula of compound 3 is:

[0012] Here, m or n is an integer of 1 to 15, each independently.

[0013] On the other hand, the chip preparation method provided by the present application further includes:

[0014] 3) Spotting the analyte onto the same dot array as in step 1) on the photosensitive chip, and irradiating the chip with light to cause a carbene radical reaction between the analyte and compound 3, thereby connecting the analyte to compound 3 to obtain a finished chip.

[0015] On the other hand, the present application also provides a photosensitive chip prepared by the preparation method described herein.

[0016] On the other hand, the present application also provides a finished chip prepared by the preparation method described herein.

[0017] In another aspect, the present application also provides the use of the photosensitive chip described herein and the finished chip described herein in SPRi detection.

[0018] On the other hand, the present application also provides the use of the photosensitive chip described herein and the finished chip described herein in drug screening.

[0019] Compared with the existing technology, the technical advantages of this application include:

[0020] 1. This application uses a fixed-point sampling method to construct a hydrophobic surface, which can increase the sample volume fixed on the biochip from 500 samples / square centimeter to 4000 samples / square centimeter;

[0021] 2. This application mainly uses incubation / immersion methods to modify the surface of bare gold glass slides, which can be applied to the surface modification of chips of various areas, providing the possibility of large-scale sample fixation;

[0022] 3. This application uses a carbene free radical reaction to connect the chip surface to the analyte. Unlike schemes based on specific methods to connect analytes (such as amide bond covalent connection, electrostatic adsorption connection, biotin-avidin system connection, etc.), this application has a wider range of applications and is particularly suitable for building libraries of small molecule compounds without specific groups (such as carboxyl, amino, hydroxyl, thiol, etc.).

[0023] Other features and advantages of the present application will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present application. Other advantages of the present application can be realized and obtained by the solutions described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0025] Figure 1 The present invention is a flowchart of a chip preparation method according to one embodiment of the present application.

[0026] Figure 2 This is a schematic diagram of compound 1 and compound 2 being fixed on a gold film surface in one embodiment of the present application.

[0027] Figure 3 This is a schematic diagram of the connection between compound 3 and compound 1 in one embodiment of the present application.

[0028] Figure 4 This is a schematic diagram of the connection between the analyte and compound 3 in one embodiment of the present application.

[0029] Figure 5 It shows that the chip printing quality was inspected by SPRi equipment in Example 1, and the 3240 spotted compounds were evenly covalently fixed within the imaging field of view.

[0030] Figure 6 The diagram shows the experiment of flowing GPX4 protein sequentially through the microarray chip in Example 1.

[0031] Figure 7 The resulting graph shows the signal of each compound in Example 1 at the highest concentration. DETAILED DESCRIPTION

[0032] Unless otherwise indicated, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, without considering whether the range is specifically revealed. Unless otherwise indicated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0033] The terms "about" and "approximately" when used in conjunction with a numerical variable generally mean that the value of that variable and all values ​​of that variable are within experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the stated value, or wider.

[0034] The expression "comprising" or its synonyms "including," "containing," and "having" are open-ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any elements, steps, or ingredients not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or ingredients, plus any optional elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0035] The expression "at least one" or "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0036] Currently, biomolecular interaction technologies based on surface plasmon resonance (SPR) primarily rely on solutions that modify bare gold slides with dextran. This approach is primarily suitable for SPR instrumentation applications where an entire gold film is modified and then pressed to form microfluidics, but is not suitable for applications requiring high-density analyte attachment. This approach, in which dextran is modified on the gold film surface, renders the chip surface relatively hydrophilic. This hydrophilic surface significantly limits the density of biological spotting when high-density spotting is required while preventing adhesion between spots. The dextran modified with this approach is primarily suitable for specific methods of analyte attachment (e.g., amide bond covalent attachment, electrostatic adsorption attachment, and biotin-avidin system attachment). However, this approach cannot broadly attach analytes when the immobilized substrate lacks specific reaction sites.

[0037] In this application, a method for constructing a biochip substrate with high density is proposed. The biochip substrate is prepared by a fixed-point spotting modification scheme to construct a hydrophobic surface, which can greatly increase the sample spotting density in order to construct a large-scale microarray biochip. At the same time, the carbene free radical reaction is selected as the method for cross-linking the analyte and the chip, which can effectively increase the biochip spotting density and expand the scope of application of the analyte.

[0038] This application provides a chip preparation method, comprising:

[0039] 1) Spotting a mixed solution containing compound 1 and compound 2 on a gold film, incubating to form a dot matrix on the gold film, washing and drying to obtain a substrate chip, wherein:

[0040] The general formula of compound 1 is:

[0041] Here, n is an integer from 1 to 15;

[0042] The general formula of compound 2 is:

[0043] Here, m or n is each independently an integer from 1 to 15;

[0044] And wherein the molar ratio of compound 1 to compound 2 is 1:100-100:1;

[0045] 2) adding a solution containing compound 3 to the surface of the substrate chip obtained in step 1) so that compound 3 is connected to compound 1 on the surface of the substrate chip, washing and drying to obtain a photosensitive chip, wherein:

[0046] The general formula of compound 3 is:

[0047] Here, m or n is an integer of 1 to 15, each independently.

[0048] In some embodiments, compound 1 is 23-mercapto-3,6,9,12-tetraoxatricanoic acid, 26-mercapto-3,6,9,12,15-pentaoxahexacanoic acid, 32-mercapto-3,6,9,12,15,18,21-heptaoxatriacontanoic acid, 11-mercaptoundecanoic acid, 12-mercaptododecanoic acid or 16-mercaptohexadecanoic acid; the compound 2 is 2-((11-mercaptodecanoic acid) 2-(2-((6-mercaptohexyl)oxy)ethoxy)ethane-1-ol, 2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethane-1-ol, 2-(2-(2-((6-mercaptohexyl)oxy)ethoxy)ethoxy)ethane-1-ol, 2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethane-1-ol, 18-Mercapto-3,6,9,12-tetraoxaoctadecane-1-ol, 23-Mercapto-3,6,9,12-tetraoxatricosan-1-ol, 24-Mercapto-3,6,9,12,15,18-hexaoxatricosan-1-ol, 26-Mercapto-3,6,9,12,15-pentaoxatricosan-1-ol, 29-Mercapto-3,6,9,12,15,18-hexaoxatricosan-1-ol 1-ol, 2,5,8,11-tetraoxadocosapentadecane-22-thiol, 2,5,8,11,14,17,20-heptaoxatriacontane-31-thiol, 11-mercaptoundecan-1-ol or 16-mercaptohexadecane-1-ol; compound 3 is N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diaziridin-3-yl)benzamide.

[0049] In some embodiments, Compound 1 can be the following compound:

[0050]

[0051]

[0052] In some embodiments, compound 2 can be the following compound:

[0053]

[0054]

[0055] In some embodiments, compound 3 is preferably N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamide.

[0056] In some embodiments, the molar ratio of Compound 1 to Compound 2 is 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.

[0057] In some embodiments, compound 1 is 3,2-mercapto-3,6,9,12,15,18,21-heptaoxatriacontanoic acid; compound 2 is 2-(2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethoxy)ethan-1-ol; and compound 3 is N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diaziridin-3-yl)benzamide.

[0058] On the other hand, the chip preparation method provided by the present application further includes:

[0059] 3) Spotting the analyte onto the same dot array as in step 1) on the photosensitive chip, and irradiating the chip with light to cause a carbene radical reaction between the analyte and compound 3, thereby connecting the analyte to compound 3 to obtain a finished chip.

[0060] In some embodiments, the analyte may include a small molecule compound without specific groups (eg, carboxyl, amino, hydroxyl, sulfhydryl, etc.).

[0061] In some embodiments, the method comprises:

[0062] 1) Use a plasma cleaner to clean the gold film chip, or use piranha solution to clean the gold film chip, and then use isopropyl alcohol, ethanol, and water to clean it alternately, and dry it;

[0063] 2) using a high-density biospotter, spotting an ethylene glycol solution containing a mixture of 3,2-mercapto-3,6,9,12,15,18,21-heptaoxatriacontanoic acid and 2-(2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethoxy)ethane-1-ol at a molar ratio of 1:10-1:3 onto a gold film, incubating the solution in a constant temperature humidity chamber containing saturated ethanol vapor to form a dot matrix on the gold film, and then washing the solution with isopropyl alcohol, ethanol, and water alternately, followed by drying to obtain a substrate chip;

[0064] 3) PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), DIPEA (N,N-diisopropylethylamine) and DMF (N,N-dimethylformamide) were dissolved and mixed to prepare solution A. N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diaziridin-3-yl)benzamide was dissolved in DMF to prepare solution B. Solution A and solution B were mixed and then dropwise added to the surface of the substrate chip. After incubation in the dark, the chip was rinsed with ethanol. The washed chip was immersed in a mixed solution of ethanolamine and DMF for 1 hour, and then washed alternately with DMF, ethanol, and water, and dried to obtain a photosensitive chip.

[0065] 4) The analyte is spotted onto the photosensitive chip using a biospotting instrument in the same dot array as in step 2). After the solvent is evaporated, the chip is irradiated with UV light under vacuum conditions. The chip is then washed alternately with DMF, ethanol, and water, and dried to obtain a finished chip.

[0066] In some embodiments, the method comprises:

[0067] 1) Clean the gold film chip using a plasma cleaner for 5 minutes, or clean it with piranha solution for 2 minutes, then rinse it three times with isopropyl alcohol, ethanol, and water, and dry it with N2;

[0068] 2) using a high-density biospotting instrument to inkjet print an ethylene glycol solution containing a mixture of 0.15 mol / L 3,2-mercapto-3,6,9,12,15,18,21-heptaoxatriacontanoic acid and 0.85 mol / L 2-(2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethoxy)ethane-1-ol as a substrate, printing the solution into a dot array equal to the number of target analytes. The printed chip was incubated in a 4°C constant temperature humidity chamber containing saturated ethanol vapor for 12 hours, and then rinsed three times alternately with isopropanol, ethanol, and water, and dried with nitrogen to obtain a substrate chip;

[0069] 312 mg of PyBOP, 100 μL of DIPEA, and 2 mL of DMF were dissolved and mixed to prepare Solution A. 216 mg of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diaziridin-3-yl)benzamide was dissolved in 4 mL of DMF to prepare Solution B. Equal amounts of Solution A and Solution B were mixed and added dropwise to the surface of the substrate chip. The chip was incubated in the dark for 4 hours and then rinsed with ethanol. The washed chip was immersed in a mixture of 30 mL of ethanolamine and 500 mL of DMF for 1 hour. The chip was then rinsed three times alternately with DMF, ethanol, and water. The chip was dried under nitrogen to obtain a photosensitive chip and stored in a vacuum-sealed container in the dark.

[0070] 4) The analyte is spotted onto the photosensitive chip using a bio-spotting instrument in the same location as in step 2). After evaporation of the solvent, the chip is irradiated with UV light at 365 nm under vacuum for 15 minutes. The chip is then washed three times alternately with DMF, ethanol, and water, and dried with nitrogen to obtain a finished chip containing the analyte.

[0071] On the other hand, the present application also provides a photosensitive chip prepared by the preparation method described herein.

[0072] On the other hand, the present application also provides a finished chip prepared by the preparation method described herein.

[0073] On the other hand, the present application also provides the use of the photosensitive chip described herein and the finished chip described herein in SPRi detection.

[0074] On the other hand, the present application also provides the use of the photosensitive chip described herein and the finished chip described herein in drug screening.

[0075] Currently, the throughput of biological interaction technologies used for drug screening, from high to low, mainly includes Fluorescence Resonance Energy Transfer (FRET), Ambient Ionization Mass Spectrometry (AS-MS), Surface Plasmon Resonance (SPR), Biolayer Interferometry (BLI), Nuclear Magnetic Resonance (NMR), and Isothermal Titration Calorimetry (ITC). The chip surface chemical treatment technology involved in this application is mainly used in drug screening based on surface plasmon resonance imaging (SPRi) technology.

[0076] Limited by the area of ​​the optical sensor, the throughput of the SPRi screening method described in this application is slightly lower than that of FRET-based screening methods. However, this technology is unique in its ability to monitor interactions between biomolecules in real time, providing dynamic data that can track real-time changes in reactions. Furthermore, the analyte and the analyte do not require biolabeling, making it more widely applicable.

[0077] Traditional SPR screening technology uses flow channels to fix and analyze samples, and can generally detect no more than 20 samples at the same time. In contrast, BLI screening technology uses an optical fiber head as a detection format, and can generally detect no more than 100 samples at the same time. This application uses a parallel light incidence and imaging method to simultaneously fix and complete the analysis of 100-5000 samples, greatly improving the efficiency of drug screening. It not only achieved a huge breakthrough in throughput, but also has a broader application prospect in the field of drug screening through real-time monitoring and the characteristics of no labeling.

[0078] The following table shows some characteristics of each method:

[0079]

[0080] Figure 1A flow chart of a chip preparation method according to an embodiment of the present application is shown. The bare gold chip is cleaned by plasma cleaning and then solvent cleaning for subsequent base layer spotting of compound 1 and compound 2. After the fixed-point spotting is completed, constant temperature and humidity incubation is performed so that compound 1 and compound 2 are attached to the bare gold chip in a certain proportion in the form of a dot matrix to obtain a base chip. Subsequently, a photocrosslinker (compound 3) is condensed on the base chip, and then surface sealing is performed. After cleaning with a solvent, a photosensitive chip is obtained. The obtained photosensitive chip can then be used for library construction of various compounds. The specific method is to spot the compound to be analyzed in the dot matrix of the photosensitive chip, vacuum dry it, and then a carbene radical reaction occurs under ultraviolet light, so that the compound to be analyzed is fixedly cross-linked on compound 3. After cleaning with a solvent, a finished compound library chip is obtained.

[0081] Figure 2 It shows that Compound 1 and Compound 2 are fixed on the gold film surface in a certain ratio.

[0082] Figure 3 It shows that compound 3 reacts with compound 1 through condensation and is then fixed on the gold film surface.

[0083] Figure 4 It shows that the compound library to be analyzed is printed on the gold film at a fixed point, and the compound to be analyzed reacts with compound 3 under ultraviolet light to produce a carbene radical reaction, and is then fixed on the gold film at a fixed point.

[0084] This application describes multiple embodiments, but this description is exemplary rather than restrictive, and it will be apparent to those skilled in the art that more embodiments and implementations may be included within the scope of the embodiments described herein. Although many possible feature combinations are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

[0085] This application includes and contemplates combinations of features known to those of ordinary skill in the art. The embodiments and features disclosed in this application may also be combined with any conventional features to form a unique inventive solution defined by the claims. Any feature of any embodiment may also be combined with features from other inventive solutions to form another unique inventive solution defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in this application may be implemented individually or in any appropriate combination. Therefore, the embodiments are not subject to other limitations except as provided in the appended claims and their equivalents. In addition, various modifications and changes may be made within the scope of protection of the appended claims.

[0086] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps described herein, the method or process should not be limited to the steps in the specific order described. As will be understood by those skilled in the art, other orders of steps are also possible. Therefore, the specific order of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, the claims for the method and / or process should not be limited to performing their steps in the order written, and those skilled in the art can readily understand that these orders can be changed and still remain within the spirit and scope of the embodiments of the present application.

[0087] The experimental methods in the following examples, for which specific conditions are not specified, are generally determined in accordance with national standards. The experimental materials in the following examples, for which the sources are not specified, are all commercially available raw materials. The equipment used in each step in the following examples is all conventional equipment. If there are no corresponding national standards, the methods are carried out in accordance with general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise defined or indicated, all professional and scientific terms used in this application have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of this application.

[0088] Example 1: Screening of GPX4 inhibitors as an example

[0089] ① Chip preparation

[0090] a) Clean the bare gold glass chip using a plasma cleaner for 5 minutes, then rinse with isopropyl alcohol, ethanol, and water three times in sequence, and dry with nitrogen.

[0091] b) using a high-density bio-spotting instrument to inkjet print an ethylene glycol solution containing a mixture of 0.15 mol / L compound 1 (3,2-mercapto-3,6,9,12,15,18,21-heptaoxatriacontanoic acid) and 0.85 mol / L compound 2 (2-(2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethoxy)ethan-1-ol) as a substrate, and printing the solution into a matrix of 3240 dots;

[0092] c) Place the printed chip in a 4°C constant temperature humidity chamber containing saturated ethanol vapor for 12 hours, rinse three times with isopropanol, ethanol, and water, and dry with nitrogen;

[0093] d) Solution A was prepared by dissolving 312 mg of PyBOP, 100 μL of DIPEA, and 2 mL of DMF. Solution B was prepared by dissolving 216 mg of compound 3 (N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diaziridin-3-yl)benzamide) in 4 mL of DMF. Equal amounts of Solution A and Solution B were mixed and added dropwise to the cleaned and dried chip surface from step c. The mixture was incubated in the dark for 4 hours and then rinsed with ethanol.

[0094] e) Mix 30 mL of ethanolamine and 500 mL of DMF. Soak the chip cleaned in step d in the mixed solution for 1 hour. Wash it three times with DMF, ethanol, and water alternately. After drying under N2, seal it in a vacuum seal and store it in the dark.

[0095] ②Compound library printing

[0096] 3240 compounds were prepared as DMSO stock solutions with a concentration of 10 mM and placed in a 384-well plate. The ArrayJet biospotting system was used to spot the compounds according to the dot matrix format printed in step 1. After evaporating the solvent, the plate was irradiated with 365 nm UV light for 15 minutes under vacuum conditions. The plate was then washed three times with DMF, ethanol, and water, and dried with nitrogen to obtain the finished chip.

[0097] ③Protein target screening

[0098] a) The chip printing quality was checked using the SPRi device. The 3240 spotted compounds were evenly covalently fixed within the imaging field of view. Figure 5 As shown;

[0099] GPX4 protein was diluted with PB buffer to five concentrations ranging from 0.0117 mg / mL to 0.188 mg / mL and sequentially flowed through the microarray chip with an association time of 180 s, a dissociation time of 400 s, and a flow rate of 2.5 μL / s. Figure 6 shown.

[0100] ④Data processing and verification

[0101] a) The signal of each compound at the highest concentration is as follows Figure 7 As shown:

[0102] b) Based on the signal response, the following compounds were selected as potential GPX4 inhibitors:

Claims

1. A chip preparation method comprising: 1) Spotting a mixed solution containing compound 1 and compound 2 on a gold film, incubating to form a dot matrix on the gold film, washing and drying to obtain a substrate chip, wherein: The general formula of the compound 1 is: or Here, n is an integer from 1 to 15; The general formula of the compound 2 is: Here, m or n is each independently an integer from 1 to 15; And wherein the molar ratio of compound 1 to compound 2 is 1:100-100:1; 2) adding a solution containing compound 3 to the surface of the substrate chip obtained in step 1) so that the compound 3 is connected to the compound 1 on the surface of the substrate chip, washing and drying to obtain a photosensitive chip, wherein the general formula of the compound 3 is Here, m or n is an integer of 1 to 15, each independently.

2. The preparation method according to claim 1, wherein The compound 1 is 23-mercapto-3,6,9,12-tetraoxatricosanoic acid, 26-mercapto-3,6,9,12,15-pentaoxahexacosanoic acid, 32-mercapto-3,6,9,12,15,18,21-heptaoxatriacontanoic acid, 11-mercaptoundecanoic acid, 12-mercaptododecanoic acid or 16-mercaptohexadecanoic acid; the compound 2 is 2-((11-mercaptoundecyl)oxy) ) ethane-1-ol, 2-(2-((6-mercaptohexyl)oxy)ethoxy)ethane-1-ol, 2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethane-1-ol, 2-(2-(2-((6-mercaptohexyl)oxy)ethoxy)ethoxy)ethane-1-ol, 2-(2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethane-1-ol, 18-mercaptohexyl 2-Mercapto-3,6,9,12-tetraoxaoctadecane-1-ol, 23-Mercapto-3,6,9,12-tetraoxatricosan-1-ol, 24-Mercapto-3,6,9,12,15,18-hexaoxatetracosan-1-ol, 26-Mercapto-3,6,9,12,15-pentaoxahexacosacan-1-ol, 29-Mercapto-3,6,9,12,15,18-hexaoxanonacosacan-1-ol , 2,5,8,11-tetraoxadocosapentadecane-22-thiol, 2,5,8,11,14,17,20-heptaoxatriacontane-31-thiol, 11-mercaptoundecan-1-ol or 16-mercaptohexadecane-1-ol; the compound 3 is N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diazinc-3-yl)benzamide.

3. The preparation method according to claim 2, wherein The compound 1 is 32-mercapto-3,6,9,12,15,18,21-heptaoxatriacontanoic acid; the compound 2 is 2-(2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethoxy)ethan-1-ol; and the compound 3 is N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diazinc heterocyclopropene-3-yl)benzamide.

4. The preparation method according to claim 1, wherein The method further comprises: 3) Spotting the analyte onto the same dot array on the photosensitive chip as in step 1) and irradiating the chip with light to cause a carbene radical reaction between the analyte and the compound 3, thereby connecting the analyte to the compound 3 to obtain a finished chip.

5. The preparation method according to any one of claims 1 to 4, wherein The method comprises: 1) Use a plasma cleaner to clean the gold film chip, or use piranha solution to clean the gold film chip, and then use isopropyl alcohol, ethanol, and water to clean it alternately, and dry it; 2) using a high-density biospotter, spotting an ethylene glycol solution containing a mixture of 3,2-mercapto-3,6,9,12,15,18,21-heptaoxatriacontanoic acid and 2-(2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethoxy)ethane-1-ol at a molar ratio of 1:10-1:3 on a gold film, incubating the solution in a constant temperature humidity chamber containing saturated ethanol vapor to form a dot matrix on the gold film, and washing the solution alternately with isopropyl alcohol, ethanol, and water, and drying the solution to obtain the substrate chip; 3) PyBOP, DIPEA, and DMF were dissolved and mixed to prepare solution A, N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diaziridin-3-yl)benzamide was dissolved in DMF to prepare solution B, solution A and solution B were mixed and then dropwise added to the surface of the substrate chip, incubated in the dark, and then rinsed with ethanol. The washed chip was immersed in a mixed solution of ethanolamine and DMF for 1 hour, and then washed alternately with DMF, ethanol, and water, and dried to obtain the photosensitive chip; 4) Spotting the analyte onto the photosensitive chip in the same position as in step 2) using a biological spotter, evaporating the solvent, and then irradiating with ultraviolet light under vacuum conditions. The chip is then washed alternately with DMF, ethanol, and water, and dried to obtain the finished chip.

6. The preparation method according to claim 5, wherein The method comprises: 1) Clean the gold film chip using a plasma cleaner for 5 minutes, or clean it with piranha solution for 2 minutes, then rinse it three times with isopropyl alcohol, ethanol, and water, and dry it with N2; 2) using a high-density bio-spotting instrument to inkjet print a solution of ethylene glycol containing a mixture of 0.15 mol / L 3,2-mercapto-3,6,9,12,15,18,21-heptaoxatriacontanoic acid and 0.85 mol / L 2-(2-(2-((11-mercaptoundecyl)oxy)ethoxy)ethoxy)ethane-1-ol onto a gold film, incubating the solution in a 4°C constant temperature humidity chamber containing saturated ethanol vapor for 12 hours to form a dot matrix on the gold film, and then washing the solution three times with isopropyl alcohol, ethanol, and water, followed by drying with nitrogen to obtain the substrate chip; 3) 312 mg of PyBOP, 100 μL of DIPEA, and 2 mL of DMF were dissolved and mixed to prepare solution A. 216 mg of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diaziridin-3-yl)benzamide was dissolved in 4 mL of DMF to prepare solution B. Equal amounts of solution A and solution B were mixed and then dropwise added to the surface of the substrate chip. The chip was incubated in the dark for 4 hours and then rinsed with ethanol. The washed chip was immersed in a mixed solution containing 30 mL of ethanolamine and 500 mL of DMF for 1 hour. The chip was then rinsed three times alternately with DMF, ethanol, and water, and dried with nitrogen to obtain the photosensitive chip, which was sealed and stored in a vacuum chamber in the dark. 4) The analyte is spotted onto the photosensitive chip using a bio-spotting instrument in the same dot array as in step 2). After evaporating the solvent, the chip is irradiated with 365 nm UV light for 15 minutes under vacuum conditions. The chip is then washed three times alternately with DMF, ethanol, and water, and dried with nitrogen to obtain the finished chip containing the analyte.

7. A photosensitive chip prepared by the preparation method according to any one of claims 1 to 3.

8. A finished chip produced by the method according to any one of claims 4 to 6.

9. Use of the photosensitive chip according to claim 7 or the finished chip according to claim 8 in SPRi detection.

10. Use of the photosensitive chip according to claim 7 or the finished chip according to claim 8 in drug screening.

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