Photo-crosslinking biosensor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of molecular detection, and particularly provides a photo-crosslinking biosensor as well as a preparation method and application thereof. The photo-crosslinking biosensor comprises a biosensor, the surface of the biosensor is modified with photoactive molecules, and biomolecules react with the photoactive molecules through photo-crosslinking to modify the surface of the biosensor; the photoactive molecule has at least one reaction group of amino, carboxyl, sulfydryl, maleimide group, alkynyl and azide. According to the photo-crosslinking biosensor, biomolecules can be crosslinked without marking, the universality is high, the sample detection types and detection methods of the existing BLI technology are greatly expanded, and the biomolecules are fixed on the biosensor in a random orientation manner through photo-crosslinking reaction, so that the biological activity of the biomolecules can be maintained as much as possible.

Description

Technical Field

[0001] The present invention relates to the field of molecular detection technology, and in particular to a photo-crosslinking biosensor and a preparation method and application thereof. Background Art

[0002] Biolayer-interferometry (BLI) analyzer can obtain the kinetic parameters, affinity and protein concentration information of biomolecular interactions, and is widely used in research fields such as biological structure and disease mechanism, drug development, target fishing, signal transduction, gene regulation, etc. The principle of this instrument is based on the change of membrane thickness or density caused by the binding of molecules on the surface of the biosensor, resulting in the displacement of the interference spectrum. The biosensor is immersed and read in a way to achieve real-time and rapid analysis and detection of biomolecular interactions.

[0003] When using BLI for affinity detection of proteins and small molecules, the protein must be labeled with his or biotin, and the his-tagged protein must be captured using an NTA biosensor, or the biotin-labeled protein must be fixed using an SSA biosensor, and the small molecule is used as the analyte for the experiment. However, some proteins do not have a his tag, or cannot be labeled with biotin, so it is difficult to complete the protein and small molecule affinity detection experiment using the BLI method, thus limiting the application of BLI technology in small molecule detection or small molecule drug development. In addition, the use of existing commercially available biosensors to fix biological molecules such as nucleic acids, peptides, and polysaccharides requires that biological molecules such as nucleic acids, peptides, and polysaccharides have a biotin label. If there is no biotin label, it is almost difficult to fix them on the biosensor and they can only be detected as analytes.

[0004] In addition, target fishing is also a current research hotspot. Discovering and verifying the biological targets of biomolecules helps to clarify the molecular mechanism of their function. At present, the commonly used experimental method for target fishing of biomolecules is to label biotin with biomolecules, fix them on streptavidin-modified magnetic beads or sensor chips, co-incubate cell or tissue lysate with magnetic beads as analytes, or flow through the surface of sensor chips, use eluent to elute potential target proteins, and identify potential target proteins through protein spectrum. However, biomolecules, especially small molecules, have high technical difficulties and thresholds for labeling biotin. Some biomolecules lack active groups and are difficult to label with biotin, and if the labeling site happens to be the active site of the biomolecule, it will cause the biomolecule to lose its activity. At present, there is no report on the use of BLI technology for small molecule target fishing. On the other hand, since the surface of the sensor chip is coated with gold film, the consumables cost of biomolecule target fishing experiments using surface plasmon resonance (SPR) or surface plasmon resonance imaging (SPRi) technology is high, and the instrument operation is complicated, and it often requires guidance from experienced teachers to complete the experiment.

[0005] Therefore, using BLI to detect interactions between biomolecules and achieve target fishing of biomolecules still poses great technical challenges. Summary of the invention

[0006] The present invention aims to solve one of the technical problems in the prior art at least to a certain extent. To this end, one object of the present invention is to provide a photo-crosslinking biosensor and a preparation method and application thereof.

[0007] In a first aspect of the present invention, the present invention provides a photo-crosslinking biosensor, the photo-crosslinking biosensor comprising: A biosensor, wherein the surface of the biosensor is modified with photoactive molecules, and the biomolecules are modified on the surface of the biosensor by reacting with the photoactive molecules through photocrosslinking; The photoactive molecule carries at least one reactive group selected from the group consisting of an amino group, a carboxyl group, a thiol group, a maleimide group, an alkynyl group, and an azide group.

[0008] According to the above-mentioned photo-crosslinked biosensor of the present invention, by modifying the photoactive molecule on the surface of the biosensor, and then grafting the biomolecule on the above-mentioned photoactive molecule through photo-crosslinking, on the one hand, through the intermediate connection of the photoactive molecule, the biomolecule can avoid the labeling step, and can be grafted on the photoactive molecule through photo-crosslinking, so that the biomolecule can be fixed on the biosensor without labeling, thereby expanding the application of BLI technology in biomolecule detection or drug development. On the other hand, the photoactive molecule carries at least one reactive group of amino, carboxyl, sulfhydryl, maleimide, alkynyl, and azide, and the biosensor can carry reactive groups such as amino or NHS ester after activation. The group of the photoactive molecule and the group on the biosensor can be coupled through covalent bonds, and the photoactive molecule is modified on the biosensor. The biomolecule can be fixed on the photoactive molecule in a random orientation through the photo-crosslinking reaction, that is, the biological activity of the biomolecule is maintained to the greatest extent possible, and the application scope of BLI technology is broadened.

[0009] According to the above-mentioned photo-crosslinked biosensor of the present invention, the photoactive molecules include at least one of acetophenone compounds, benzophenone compounds, quinone anthracene compounds, aromatic azides, aromatic aziridines, and diaziridine compounds.

[0010] According to the above-mentioned photo-crosslinked biosensor of the present invention, the photoactive molecule is preferably a diaziridine compound, and the diaziridine compound is selected from at least one of the following structures: , ,

[0011] Wherein, R1 is selected from carboxyl, hydroxyl or , wherein R4 is selected from , n is a positive integer between 1 and 10; R2 is selected from , , , or , R5 is selected from an alkylene group having 1 to 20 carbon atoms; R3 is selected from an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 1 to 20 carbon atoms or an alkynyl group having 1 to 20 carbon atoms.

[0012] As an example, the diaziridine compound includes but is not limited to at least one of the following structures: , , , , , , , .

[0013] According to the above-mentioned photo-crosslinking biosensor of the present invention, the biomolecules include nucleic acids, proteins, sugars, polypeptides, viruses, small molecules or nanoparticles.

[0014] According to the above-mentioned photo-crosslinking biosensor of the present invention, the biosensor comprises an optical fiber-type transparent round rod, and the material of the biosensor comprises quartz glass or optical glass.

[0015] In a second aspect of the present invention, the present invention provides a method for preparing the above-mentioned photo-crosslinking biosensor, the method comprising: (1) activating the biosensor; (2) reacting the activated biosensor with a photoactive molecule in a solution in the dark to obtain a biosensor modified with the photoactive molecule; (3) using a blocking solution to block the biosensor modified with the photoactive molecule; (4) applying the biomolecule solution to the sealed surface of the biosensor modified with the photoactive molecule and drying it, and then causing a photo-crosslinking reaction under light to obtain a photo-crosslinked biosensor.

[0016] According to the above method for preparing a photo-crosslinked biosensor of the present invention, the biosensor is first activated, and reactive groups such as amino groups or NHS esters are modified on the surface of the biosensor through activation, and then the activated biosensor is reacted with photoactive molecules in a solution in the dark. Since the photoactive molecules carry various active groups, they can be coupled with the groups on the biosensor through covalent bonds, so that the photoactive molecules are modified on the biosensor. The biosensor modified with the photoactive molecules is then sealed with a sealing liquid, and the biomolecule solution is added to the surface of the biosensor modified with the photoactive molecules after sealing, and then dried, and then the biomolecules are photo-crosslinked with the photoactive molecules under light, thereby realizing the modification of the biomolecules on the surface of the biosensor. The method is simple to operate, easy to learn and promote, and has the advantages of non-labeling, non-destructive to biomolecules, rapid, high sensitivity, low non-specific adsorption, and good stability.

[0017] According to the above method for preparing a photo-crosslinked biosensor of the present invention, in step (1), the biosensor activation refers to modifying the biosensor with reactive groups such as NHS ester, amino, sulfhydryl, maleimide, alkynyl, azide, etc. that can undergo chemical reactions. For specific activation methods, those skilled in the art can select them according to actual conditions. For example, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are used to activate the biosensor, so that the biosensor is modified with NHS ester.

[0018] According to the above method for preparing a photo-crosslinked biosensor of the present invention, in step (2), the solvent includes but is not limited to N,N-dimethylformamide (DMF) and N,N-diisopropylethylamine (DIPEA). The present invention does not specifically limit the specific light-shielding reaction conditions, and those skilled in the art can select the specific light-shielding reaction time according to actual conditions.

[0019] According to the method for preparing a photo-crosslinked biosensor of the present invention, in step (3), the blocking solution includes but is not limited to ethanolamine (DMF), and the specific blocking time can be selected by a person skilled in the art according to actual conditions. After the blocking treatment, the biosensor can be washed with DMSO and ethanol respectively to remove unreacted substances.

[0020] Preferably, in order to reduce non-specific adsorption on the biosensor, 0.1%-5% bovine serum albumin or casein may be used to block the biosensor.

[0021] According to the method for preparing a photo-crosslinked biosensor of the present invention, in step (4), the conditions for the photo-crosslinking reaction include: a light intensity of 0.1-100 mW / cm 2 , preferably 5~50 mW / cm 2 ; UV irradiation with a wavelength of 200-400 nm, preferably 300-400 nm, more preferably 365 nm; light irradiation time is 1 min~180 min, preferably 10 min~60 min.

[0022] According to the method for preparing a photo-crosslinked biosensor of the present invention, in step (4), the application method includes coating or immersion. Specifically, the biomolecule solution can be dripped or dotted onto the surface of the biosensor, or the biosensor can be immersed in the biomolecule solution for a period of time.

[0023] According to the method for preparing a photo-crosslinked biosensor of the present invention, in step (4), the drying method includes but is not limited to a high temperature oven, a freeze dryer, a vacuum dryer or direct drying.

[0024] In the third aspect of the present invention, the present invention proposes the use of the above-mentioned photo-crosslinking biosensor or the photo-crosslinking biosensor prepared by the above-mentioned method in molecular affinity detection or molecular target fishing.

[0025] According to the above-mentioned photo-crosslinked biosensor application of the present invention, the molecular affinity detection specifically includes: preparing other biomolecules into different concentration gradients, incubating with the photo-crosslinked biosensor of the present invention, detecting the binding of biomolecules in real time, and giving the affinity K D and the values ​​of kinetic parameters such as kon and koff.

[0026] According to the above-mentioned photo-crosslinking biosensor application of the present invention, molecular target fishing includes: preparing biological fluids, cells or tissue lysates as analytes, incubating them with the photo-crosslinking biosensor of the present invention, detecting the binding of biological molecules and target proteins in real time, eluting potential target proteins with eluent, and identifying them through protein spectrum.

[0027] Furthermore, the eluent includes but is not limited to PBS, HEPES, PBS containing DMSO and / or tween 20, trifluoroacetic acid, formic acid, acetic acid, glycine, hydrochloric acid, sodium hexadecyl sulfate (SDS), etc.

[0028] The present invention has at least the following technical effects: (1) The photo-crosslinking biosensor of the present invention can crosslink biological molecules without labeling, has high universality, and greatly expands the sample detection types and detection methods of the existing BLI technology. In addition, the biomolecules can be fixed on the biosensor in a random orientation through the photo-crosslinking reaction, which can maintain the biological activity of the biomolecules to the greatest extent possible.

[0029] (2) The photo-crosslinking biosensor of the present invention has the advantages of being non-labeling, non-destructive to biomolecules, rapid, highly sensitive, low in nonspecific adsorption, and good in stability, and can save time and consumables costs.

[0030] (3) The method for preparing the photo-crosslinking biosensor of the present invention has the advantages of simple operation steps and is easy to learn and promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 is a diagram of the chemical synthesis reaction process of the photoactive molecule of Example 1 of the present invention; Figure 2 is the nuclear magnetic resonance spectrum of the photoactive molecule of Example 1 of the present invention; Figure 3 is the mass spectrum of the photoactive molecule of Example 1 of the present invention; Figure 4 is the binding and dissociation curves of carbonic anhydrase II and furosemide of Example 1 of the present invention; Figure 5 is the affinity and other values ​​of carbonic anhydrase II and furanilic acid in Example 1 of the present invention; Figure 6is the binding and dissociation curves of carbonic anhydrase II and furosemide after the photo-crosslinked biosensor of Example 1 of the present invention was placed for 2 weeks; Figure 7 is the affinity and other values ​​of carbonic anhydrase II and furosemide of the photo-crosslinked biosensor of Example 1 of the present invention after being placed for 2 weeks; Figure 8 is the binding and dissociation curves of semaglutide and GLP1R protein in Example 1 of the present invention; Fig. 9 is the affinity and other values ​​of semaglutide and GLP1R protein of Example 1 of the present invention; Fig.10 is the binding and dissociation curves of the RBD protein and the neutralizing antibody of Example 1; Fig.11 is the affinity and other values ​​of the RBD protein and the neutralizing antibody in Example 1; Fig.12 is the binding and dissociation curves of the lipid nanoparticles and the protein in Example 1; Fig.13 is the affinity and other values ​​of the lipid nanoparticles and proteins of Example 1; Fig.14 This is a target fishing experiment diagram of the photo-crosslinking biosensor according to Example 1 of the present invention. DETAILED DESCRIPTION

[0033] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0034] Example 1

[0035] (1) Preparation of photoactive molecules

[0036] 1,2-bis(2-aminoethoxy)ethane (1.96 g) was added to the flask and dissolved in dichloromethane (CH2Cl2). The reaction system was cooled to 0°C in an ice bath, and then a CH2Cl2 solution of di-tert-butyl dicarbonate (0.30 g) was added dropwise to the flask under nitrogen protection. The addition process lasted for 3 hours, after which the reaction solution was stirred at room temperature overnight. The reacted solution was extracted with a saturated NaCl solution and CH2Cl2, and the organic phase was dried with Na2SO4, filtered and concentrated to obtain 0.59 g of a colorless transparent liquid, namely compound 1.

[0037] 1,1'-Carbonyldiimidazole (CDI, 0.40 g) was added to a CH2Cl2 solution of trifluoromethylphenylbis(aziridine) (TAD, 0.25 g) at 0°C and stirred for 1 hour. The carboxyl activation process was monitored by TLC. After the carboxyl activation, compound 1 dissolved in CH2Cl2 was added dropwise. The reaction solution was stirred at room temperature overnight. The mixture was extracted with saturated NaCl solution, NaHCO3 solution and CH2Cl2, and the organic phase was dried with Na2SO4, filtered and concentrated. The crude product was purified by column chromatography to obtain 0.21 g of yellow oily solid, compound 2.

[0038] 4 M HCl was added to a MeOH solution of compound 2 (0.21 g) and stirred at room temperature overnight. After the reaction was completed as monitored by TLC, it was extracted with saturated NaCl solution, NaHCO3 solution and CH2Cl2, and then the organic phase was dried with Na2SO4, filtered and concentrated to obtain 0.16 g of a light yellow viscous liquid, namely compound 3 ( ). The specific reaction process can be referred to Figure 1 The NMR spectrum of compound 3 is shown in Figure 2 As shown, the mass spectrum of compound 3 is Figure 3 shown.

[0039] (2) Preparation of photocrosslinking biosensors

[0040] The carboxyl-modified biosensor was activated with EDC and NHS for 0.5 h. 50 mM N,N-diisopropylethylamine (DIPEA) and 10 mM of the above compound 3 were added to the DMF solution, and the biosensor was reacted at 500 rpm in the dark for 4 h. The biosensor was then blocked with 1 M ethanolamine DMF solution for 1 h. The biosensor was washed with DMSO and ethanol to remove unreacted products.

[0041] In order to illustrate the performance of the photo-crosslinked biosensor of the present application, the photo-crosslinked biosensor prepared in Example 1 was tested in terms of molecular affinity detection or molecular target fishing, as follows: (1) BLI technology detects the affinity of proteins and small molecules

[0042] A 20 mM solution of small molecule furanoselic acid was prepared with DMSO, and the photocrosslinked biosensor was immersed in the furanoselic acid solution for 5 minutes, then placed in a 40°C oven for 2 hours, irradiated with a 365 nm ultraviolet lamp for 30 minutes, and washed with DMSO and ethanol respectively to remove unreacted furanoselic acid.

[0043] Taking the Sartorius biofilm interferometry Octet instrument as an example, two of the above-mentioned photo-crosslinked biosensors immobilized with furanilic acid were first used to bind and dissociate with carbonic anhydrase II and buffer at different concentrations, and then two of the above-mentioned photo-crosslinked biosensors immobilized with only compound 3 but not furanilic acid were used as blank controls to bind and dissociate with carbonic anhydrase II and buffer at different concentrations, and the double subtraction method was used for data analysis. Figure 4 The binding of carbonic anhydrase II and furanoseline has obvious kinetic characteristics, with an affinity of 3.9×10 -6 M, R 2 It is 0.9742. Figure 5 The concentration, signal response value, kon, koff, Rmax, error bar, etc. of carbonic anhydrase II and furosemide are displayed.

[0044] In order to investigate the stability of the photo-crosslinked biosensor, the photo-crosslinked biosensor prepared by the present invention was placed at room temperature for 2 weeks, and then the affinity detection experiment of carbonic anhydrase II and furanilic acid was performed. Figure 6 Using this method and process, carbonic anhydrase II and furanilamide still have good binding, with an affinity of 4.58×10 -6 M, R 2 It is 0.9756. Figure 7 The concentrations of carbonic anhydrase II and furosemide, signal response values, kon, koff, Rmax, error bar, etc. are displayed. Therefore, it is shown that the properties of the photo-crosslinked biosensor have not changed significantly after being placed at room temperature for 2 weeks, and it has good stability.

[0045] (2) BLI technology detects the affinity of proteins and peptides

[0046] A 1 mg / mL semaglutide solution was prepared with water, and the photocrosslinked biosensor was immersed in the semaglutide solution for 5 minutes, then placed in a 37°C oven for 20 minutes, irradiated with a 365 nm UV lamp for 30 minutes, and washed with PBS to remove unreacted semaglutide.

[0047] Taking the Sartorius biofilm interferometry Octet instrument as an example, two of the above-mentioned photo-crosslinking biosensors with fixed semaglutide were incubated with different concentrations of GLP1R protein and buffer respectively to detect the binding and dissociation process of protein and peptide in real time. Figure 8 The binding of semaglutide to GLP1R protein has obvious kinetic characteristics, with an affinity of 1.1×10 -7 M, R 2 It is 0.9879. Fig. 9The concentration, signal response value, kon, koff, Rmax, error bar, etc. of semaglutide and GLP1R protein are displayed.

[0048] (3) BLI technology detects protein-protein affinity

[0049] Prepare a 1 mg / mL SARS-CoV-2 RBD protein solution with water, soak the photocross-linked biosensor in the SARS-CoV-2 RBD protein solution for 5 minutes, then place it in a 37°C oven for 20 minutes, irradiate it with a 365 nm ultraviolet lamp for 30 minutes, and wash the biosensor with PBS to remove unreacted SARS-CoV-2 RBD protein.

[0050] Taking the Sartorius Biofilm Interferometry Octet instrument as an example, two of the above-mentioned photo-crosslinked biosensors that fixed the SARS-CoV-2 RBD protein were incubated with neutralizing antibodies and buffers of different concentrations to detect the binding and dissociation process of proteins in real time. Fig.10 The binding of SARS-CoV-2 RBD protein and neutralizing antibodies has obvious kinetic characteristics, with an affinity of 6.5×10 -9 M, R 2 It is 0.9854. Fig.11 The concentration, signal response value, kon, koff, Rmax, error bar, etc. of the SARS-CoV-2 RBD protein and neutralizing antibodies are displayed.

[0051] (4) BLI technology detects the affinity between nanoparticles and proteins

[0052] A 1 mg / mL lipid nanoparticle solution was prepared with water, and the photocrosslinked biosensor was immersed in the lipid nanoparticle solution for 5 minutes, then placed in a 37°C oven for 20 minutes, irradiated with a 365 nm UV lamp for 30 minutes, and washed with PBS to remove unreacted lipid nanoparticles.

[0053] Taking the Sartorius Biomembrane Interferometry Octet instrument as an example, two optical cross-linking biosensors with fixed lipid nanoparticles were incubated with different concentrations of immunoglobulins and buffers to detect the binding and dissociation process of lipid nanoparticles and proteins in real time. Fig.12 The binding of lipid nanoparticles and immunoglobulins has obvious kinetic characteristics, with an affinity of 2.1×10 -8 M. Fig.13 The concentration, signal response value, kon, koff, Rmax, error bar, etc. of lipid nanoparticles and immunoglobulins are displayed.

[0054] (5) Using BLI technology for target fishing of small molecule drugs

[0055] Taking the Sartorius biofilm interferometry Octet instrument as an example, the above-mentioned furanilamide fixed photocrosslinking biosensor and tissue lysate are first incubated, and then the non-specifically bound proteins are washed away in PBS-T solution, and then the target protein bound to furanilamide is eluted and collected in 0.5% trifluoroacetic acid eluent, and identified by protein spectrum, such as Fig.14 shown.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A photo-crosslinking biosensor, characterized in that: include: A biosensor, wherein the surface of the biosensor is modified with photoactive molecules, and the biomolecules are modified on the surface of the biosensor by reacting with the photoactive molecules through photocrosslinking; The photoactive molecule carries at least one reactive group selected from the group consisting of an amino group, a carboxyl group, a thiol group, a maleimide group, an alkynyl group, and an azide group.

2. The photo-crosslinking biosensor according to claim 1, characterized in that: The photoactive molecules include at least one of acetophenone compounds, benzophenone compounds, quinone anthracene compounds, aromatic azides, aromatic aziridines, and diaziridine compounds.

3. The photo-crosslinking biosensor according to claim 2, characterized in that: The photoactive molecule is preferably a diaziridine compound, and the diaziridine compound is selected from at least one of the following structures: ， ， Wherein, R1 is selected from carboxyl, hydroxyl or , wherein R4 is selected from , n is a positive integer between 1 and 10; R2 is selected from , , , or , R5 is selected from an alkylene group having 1 to 20 carbon atoms; R3 is selected from an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 1 to 20 carbon atoms or an alkynyl group having 1 to 20 carbon atoms.

4. The photo-crosslinking biosensor according to claim 3, characterized in that: The diaziridine compound includes at least one of the following compounds: 、 、 、 、 、 、 、 。 5. The photo-crosslinking biosensor according to claim 1 or 2, characterized in that: The biomolecules include nucleic acids, proteins, sugars, polypeptides, viruses, small molecules or nanoparticles.

6. The photo-crosslinking biosensor according to claim 1 or 2, characterized in that: The biosensor comprises a transparent round rod of optical fiber type; The material of the biosensor includes quartz glass or optical glass.

7. The method for preparing the photocrosslinking biosensor according to any one of claims 1 to 6, characterized in that: include: (1) activating the biosensor; (2) reacting the activated biosensor with a photoactive molecule in a solution in the dark to obtain a biosensor modified with the photoactive molecule; (3) using a blocking solution to block the biosensor modified with the photoactive molecule; (4) applying the biomolecule solution to the sealed surface of the biosensor modified with the photoactive molecule and drying it, and then causing a photo-crosslinking reaction under light to obtain a photo-crosslinked biosensor.

8. The method according to claim 7, characterized in that: In step (1), the biosensor activation refers to modifying the biosensor with NHS ester, amino, sulfhydryl, maleimide, alkyne, or azide groups that can undergo chemical reactions.

9. The method according to claim 7 or 8, characterized in that: In step (4), the conditions for the photocrosslinking reaction include: light intensity of 0.1-100 mW / cm 2 , preferably 5~50 mW / cm 2 ; UV irradiation with a wavelength of 200-400 nm, preferably 300-400 nm, more preferably 365 nm; light irradiation time is 1 min~180 min, preferably 10 min~60 min.

10. Use of the photo-crosslinking biosensor according to any one of claims 1 to 6 or the photo-crosslinking biosensor prepared by the method according to any one of claims 7 to 9 in molecular affinity detection or molecular target fishing.

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