Protein microarray chip, preparation method and application

Abstract

The invention discloses a protein microarray chip, a preparation method and application, the chip comprises a functionalized monomolecular layer constructed on the gold surface of a substrate, the functionalized monomolecular layer comprises a compact non-specific adsorption resisting background layer formed by HS-Cx-EGy-OMe, Cx is alkyl with the carbon atom number of x, x and y are natural numbers, x is more than or equal to 5 and less than or equal to 15, and y is more than or equal to 3 and less than or equal to 6; the ATRP initiator HS-Cm-EGn-OCOCBr (CH3) 2 is dispersed in the non-specific adsorption resisting background layer, Cm is alkyl with the carbon atom number of m, m and n are natural numbers, m is larger than or equal to 5 and smaller than or equal to 15, and n is larger than or equal to 3 and smaller than or equal to 6. The protein microarray chip with the three-dimensional surface is constructed through the atom transfer radical polymerization (ATRP) technology, and the protein activity can be effectively guaranteed under the condition that the initiator is low in density. And the surface of the three-dimensional polymer has extremely high non-specific adsorption resistance, so that the authenticity of a signal subsequently used for biological detection is more reliable.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a protein microarray chip, a preparation method and application. BACKGROUND

[0002] The traditional protein microarray two-dimensional chip has the following shortcomings and deficiencies: firstly, the detection sensitivity is limited by the planar fixation mode, resulting in limited protein binding site space, which may affect the authenticity of the interaction; secondly, the problem of non-specific adsorption on the surface is prominent, and false positive signals are easy to produce; in addition, the protein activity is easy to be affected by the fixation method (such as chemical coupling) in the chip manufacturing process, which may lead to conformational changes or loss of function; at the same time, the two-dimensional environment cannot completely simulate the three-dimensional microenvironment in vivo, which may affect the physiological relevance of protein-protein, antigen-antibody and other interactions. These factors jointly limit its application in complex biological system research.

[0003] Therefore, in view of the above technical problems, it is necessary to provide a protein microarray chip, a preparation method and application. SUMMARY

[0004] The purpose of the present application is to provide a protein microarray chip, a preparation method and application.

[0005] In order to achieve the above purpose, the technical scheme provided by a specific embodiment of the present application is as follows:

[0006] The protein microarray chip comprises a functionalized monolayer constructed on the gold surface of a substrate, and the functionalized monolayer comprises:

[0007] HS-C x -EG y and a dense anti-non-specific adsorption background layer formed by -OMe, wherein C x is an alkyl group with x carbon atoms, x and y are both natural numbers and 5≤x≤15, 3≤y≤6; and

[0008] The ATRP initiator HS-C m -EG n -OCOCBr(CH3)2 is dispersed in the anti-non-specific adsorption background layer, wherein C m is an alkyl group with m carbon atoms, m and n are both natural numbers and 5≤m≤15, 3≤n≤6.

[0009] In one or more embodiments of the present application, the amount of ATRP initiator is 0.1wt%-2wt% of the amount of HS-C x -EG y -OMe.

[0010] In one or more embodiments of the present application, the functional monolayer of the chip is further formed with a three-dimensional polymer structure, which is obtained by polymerization of terminal carboxylated poly(ethylene glycol) methacrylate and hydroxyethyl methacrylate as main raw materials to form a first polymer structure, and then functional modification.

[0011] In one or more embodiments of the present application, the modification of the first polymer structure is that the terminal carboxyl groups are coupled with the amino groups of N,N-bis(carboxymethyl)-L-lysine after esterification to form a binding site that satisfies metal ion chelation and histidine tag protein capture.

[0012] In one or more embodiments of the present application, the metal ion is selected from Co 2+ , Ni 2+ , and Cu 2+ .

[0013] In one or more embodiments of the present application, the method for preparing a protein microarray chip comprises

[0014] preparing a substrate formed with a gold surface, HS-C x -EG y -OMe, HS-C m -EG n -OCOCBr(CH3)2;

[0015] HS-C x -EG y -OMe and HS-C m -EG n -OCOCBr(CH3)2 are uniformly dissolved in a first solvent, and the substrate is immersed in the solution and reacted at room temperature to obtain a first substrate formed with a monolayer. Preferably, the concentration of HS-C x -EG y -OMe in the solution is 0.1-10 mM. Preferably, the first solvent is selected from ethylene glycol, ethanol, dichloromethane, and dimethyl sulfoxide.

[0016] In one or more embodiments of the present application, further comprising:

[0017] A second substrate with a three-dimensional polymer on the surface is obtained by polymerization in a reaction system formed by using carboxyl-terminated poly(ethylene glycol) methacrylate and hydroxyethyl methacrylate as polymerization raw materials, CuCl2 and tris(2-dimethylaminoethyl)amine as a catalytic system, and by standing in a water / methanol mixed solvent; the first substrate is at least partially immersed in the reaction system during polymerization. Preferably, the concentration of CuCl2 in the reaction system is 0.1-10 mmol / L, and the concentration of tris(2-dimethylaminoethyl)amine is 0.1-10 mmol / L. Preferably, the concentration of carboxyl-terminated poly(ethylene glycol) methacrylate is 100-500 mM. Preferably, the proportion of hydroxyethyl methacrylate is: the concentration is 100-500 mM.

[0018] In one or more embodiments of the present application, the carboxyl-terminated poly(ethylene glycol) methacrylate is prepared by dissolving PEGMA in tetrahydrofuran, then adding succinic anhydride, DMAP and pyridine, continuously stirring the mixture at room temperature, removing the THF solvent after the reaction is completed, dissolving the residue in dichloromethane, washing and extracting with dilute hydrochloric acid, collecting the organic phase and drying with anhydrous magnesium sulfate, and filtering and removing impurities.

[0019] In one or more embodiments of the present application, the second substrate is immersed in a deionized water solution containing 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the terminal carboxyl group of the second substrate is activated to obtain an NHS ester to form a third substrate.

[0020] After the reaction is completed, the third substrate is washed and immersed in an N,N-bis(carboxymethyl)-L-lysine solution, and the amino group in the N,N-bis(carboxymethyl)-L-lysine reacts with the NHS ester group to produce a binding site to obtain a fourth substrate. Preferably, the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in the deionized water solution is 100-300 mM. Preferably, the concentration of N-hydroxysuccinimide in the deionized water solution is 50-100 mM. Preferably, the concentration of N,N-bis(carboxymethyl)-L-lysine in the N,N-bis(carboxymethyl)-L-lysine solution is 0.5-10 mM.

[0021] In one or more embodiments of the present application, the fourth substrate is further chelated with metal ions after being washed in a solution containing metal ions. Preferably, the concentration of metal ions in the metal ion solution is 10-100 mM.

[0022] In one or more embodiments of the present application, the protein microarray chip is used in protein or effector detection.

[0023] Compared with the prior art, the protein microarray chip, the preparation method and the application have the following advantages: the protein microarray chip is constructed by the technology of atom transfer radical polymerization (ATRP) to construct a three-dimensional surface, and the protein activity can be effectively guaranteed under the condition of low density of initiators; and the three-dimensional polymer surface has strong anti-non-specific adsorption capacity, and the signal reliability for subsequent biological detection is more reliable. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0025] Figure 1 The figure is a detection effect control chart of the protein microarray chip in an embodiment of the present application on specific antibodies when different concentrations of initiator INI are used;

[0026] Figure 2 The figure is an adsorption control chart of the protein microarray chip (the initiator density is 1%) in an embodiment of the present application and the chip in Comparative Example 1 on FBS;

[0027] Figure 3 The figure is a micrograph of the protein microarray chip in an embodiment of the present application;

[0028] Figure 4 The figure is a micrograph of the protein microarray chip in an embodiment of the present application;

[0029] Figure 5 The figure is a micrograph of the protein microarray chip in an embodiment of the present application;

[0030] Figure 6 The figure is a micrograph of the protein microarray chip in an embodiment of the present application;

[0031] Figure 7 The figure is a micrograph of the protein microarray chip in an embodiment of the present application;

[0032] Figure 8 The figure is a micrograph of the protein microarray chip in an embodiment of the present application;

[0033] Figure 9 The figure is a micrograph of the protein microarray chip in an embodiment of the present application. DETAILED DESCRIPTION

[0034] In order to make the technical solutions in the present disclosure better understood by the person skilled in the art, the technical solutions in the present disclosure will be clearly and completely described in the present disclosure in combination with the disclosed embodiments. Obviously, the described embodiments are only some of the embodiments of the present disclosure, but not all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by the person skilled in the art without creative labor should belong to the protection scope of the present disclosure.

[0035] The preparation of the terminal carboxylated poly(ethylene glycol) methacrylate can be, including but not limited to, as follows in the following embodiments:

[0036] Technical route:

[0037]

[0038] PEGMA360(18 g, 0.05 mol) was dissolved in tetrahydrofuran (THF), followed by the addition of succinic anhydride (6 g, 0.06 mol), DMAP (0.49 g, 0.004 mmol) and pyridine (10 mL). The mixture was continuously stirred at room temperature for 24 hours. After the completion of the reaction, the THF solvent was removed by rotary evaporation, the residue was dissolved in dichloromethane, and the extraction was washed with 0.1 M dilute hydrochloric acid three times. After the organic phase was collected, anhydrous magnesium sulfate was added and dried for 16 hours, filtered, and finally the dichloromethane was removed by rotary evaporation to obtain the carboxylated end-capped PEGMA360 polymer monomer.

[0039] C x is an alkyl group having x carbon atoms, and in the absence of an explicit indication is a straight-chain alkyl group.

[0040] Reagent source: HS-C 11 - EG3-OMe (EG3OMe), HS-C 11 - EG6-OCOCBr(CH3)2 (INI), HS-C 11 - EG3-OH (EG3OH), HS-C 11 - EG6-COOH (EG6COOH), HS-C5- EG4-OMe, HS-C5- EG5-OCOCBr(CH3)2, HS-C 15 - EG6-OMe, HS-C 15 - EG3-OCOBr(CH3)2, and the like, were purchased from Prochimia Surfaces Sp. z o.o., and all the remaining reagents were purchased from Sigma-Aldrich brand under MERCK.

[0041] Example 1

[0042] The protein microarray chip in this embodiment is fabricated as follows:

[0043] 1) 0.1mM HS-C 11 -EG3-OMe(EG3OMe); 2) HS-C 11 -EG6-OCOCBr(CH3)2(INI) was dissolved in ethylene glycol as an ATRP initiator, and then added to a gold substrate and reacted at room temperature for 16 hours to ensure the stable formation of a monolayer, thus obtaining the first substrate.

[0044] like Figure 1 As shown, samples 1-10 were prepared under the same conditions, differing only in the proportion of low-density initiator INI relative to the mass percentage of EG3OMe: 0%, 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 50%, and 100%, respectively. Verification shows that the proportion of low-density initiator INI needs to be between 0.1% and 2% of EG3OMe; higher densities can lead to poorer protein fixation or even inactivation. Figure 1 The low-density initiator INI ratio shown needs to be between 0.1% and 2% of EG3OMe to achieve good protein immobilization efficiency. For protein immobilization, specific antibodies are detected using surface plasmon resonance imaging (SPRi), with the best detection results achieved when the initiator density is between 0.1% and 2%.

[0045] The second substrate was obtained by ATRP polymerization using 0.5 mmol / L CuCl2 and 0.1 mmol / L tris(2-dimethylaminoethyl)amine (CAS: 33527-91-2) as the catalytic system. The monomers and the first substrate were polymerized in a water / methanol (1:1) mixed solvent at 35°C for 3 hours to obtain the second substrate. The monomers were: terminally carboxylated poly(ethylene glycol) methacrylate (500 mM) and hydroxyethyl methacrylate (HEMA) (200 mM).

[0046] The second substrate was immersed in 50 mL of deionized water containing 200 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 50 mM N-hydroxysuccinimide (NHS) and reacted at room temperature for 30 minutes to activate the terminal carboxyl group to obtain NHS ester and thus the third substrate.

[0047] After the reaction, the third substrate was thoroughly cleaned by rinsing with pure water for 15 minutes. The chip was then immersed in a 3 mM solution of N,N-bis(carboxymethyl)-L-lysine (NH2-NTA) and reacted at room temperature for 30 minutes. The amino groups in the NH2-NTA molecule can undergo a coupling reaction with the activated NHS ester groups on the surface of the third substrate, thereby immobilizing the NTA (nitroglycerin) groups, which facilitates subsequent metal ion chelation (such as Ni...).2+ ) and His-tag protein provides the fourth substrate with binding sites.

[0048] Finally, the fourth substrate was rinsed with pure water for 15 minutes to clean the surface thoroughly, and then immersed in 100 mM NiSO4·6H2O aqueous solution, and reacted at room temperature for 30 minutes. Ni 2+ ions and NTA groups chelate, and finally successfully prepare a low-density three-dimensional polymer NTA-Ni chip. The chip can be used to efficiently immobilize His-tagged proteins.

[0049] As Figure 2 shown in the figure, the three-dimensional polymer surface has strong anti-non-specific adsorption ability. By flowing 10% fetal bovine serum (FBS), it can be observed that the adsorption value of FBS on the surface is almost zero compared to the two-dimensional surface (comparative example 1) which is only modified with carboxyl groups at the end of the self-assembled monolayer (SAM) to immobilize proteins.

[0050] As Figure 3 shown, using 1% initiator successfully constructs a low-density protein microarray three-dimensional chip, and the protein dot array is clear.

[0051] As Figure 4 shown, if 50% initiator is used, the protein immobilization efficiency is greatly reduced, and the protein dot array is blurred.

[0052] Comparative Example 1

[0053] The preparation of the protein microarray chip in this comparative example is as follows:

[0054] Dissolve 0.1 mM HS-C 11 -EG3-OH (EG3OH), 0.01 mM HS-C 11 -EG6-COOH (EG6COOH) in ethylene glycol, add gold substrate, and react at room temperature for 16 hours to ensure the stable formation of a monolayer, obtaining the first substrate.

[0055] Immerse the first substrate in a 50 mL deionized water solution containing 200 mM 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide hydrochloride (EDC·HCl) and 50 mM N-hydroxysuccinimide (NHS), and react at room temperature for 30 minutes to obtain the second substrate.

[0056] After the reaction, the second substrate was rinsed with pure water for 15 minutes to clean the surface thoroughly, and then the chip was immersed in a 3 mM N,N-bis(carboxymethyl)-L-lysine (NH2-NTA) solution for 30 minutes at room temperature. The amino group in the NH2-NTA molecule can couple with the NHS ester group on the surface of the third substrate, thereby realizing the immobilization of the NTA group, providing a binding site for subsequent metal ion chelation (such as Ni 2+ ) and capture of His-tag proteins.

[0057] Finally, the third substrate was rinsed with pure water for 15 minutes to clean the surface thoroughly, and then immersed in a 100 mM NiSO4·6H2O aqueous solution for 30 minutes at room temperature. Ni 2+ ions chelate with the NTA group, and finally a two-dimensional NTA-Ni protein chip is successfully prepared, with a clear protein dot array, Figure 9 .

[0058] Example 2

[0059] The preparation of the protein microarray chip in this example is as follows:

[0060] 1) 2 mM HS-C 11 -EG3-OMe (EG3OMe); and 2) HS-C 11 -EG6-OCOCBr(CH3)2 (INI) (INI accounts for 1% of the mass of EG3OMe) were dissolved in ethylene glycol as ATRP initiators, and a gold substrate was added for reaction at room temperature for 16 hours to ensure the stable formation of a monolayer, obtaining a first substrate.

[0061] Using ATRP, 0.1 mmol / L CuCl2 and 3 mmol / L tris(2-dimethylaminoethyl)amine (CAS: 33527-91-2) were used as the catalytic system, and the monomer was polymerized with the first substrate in a water / methanol (1:1) mixed solvent at 35°C for 3 hours to obtain a second substrate. The polymerized monomer was: carboxyl-terminated poly(ethylene glycol) methacrylate, with a concentration of 300 mM; hydroxyethyl methacrylate (HEMA), with a concentration of 100 mM.

[0062] The second substrate was immersed in a 50 mL deionized water solution containing 100 mM 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride (EDC·HCl) and 70 mM N-hydroxysuccinimide (NHS) for 30 minutes at room temperature to activate the terminal carboxyl group to obtain a NHS ester, obtaining a third substrate.

[0063] After the reaction, the third substrate was washed with pure water for 15 minutes to clean the surface thoroughly, and then the chip was immersed in a 0.5 mM N,N-bis(carboxymethyl)-L-lysine (NH2-NTA) solution for 30 minutes at room temperature. The amino group in the NH2-NTA molecule can couple with the activated NHS ester group on the surface of the third substrate, thereby realizing the immobilization of the NTA (nitrilotriacetic acid) group, providing a binding site for subsequent metal ion chelation (such as Ni 2+ ) and capture of His-tag proteins.

[0064] Finally, the fourth substrate was washed with pure water for 15 minutes to clean the surface thoroughly, and then immersed in a 100 mM NiSO4·6H2O aqueous solution for 30 minutes at room temperature. Ni 2+ ions chelate with the NTA group, and finally a low-density three-dimensional polymer NTA-Ni chip is successfully prepared. This chip can be used to efficiently immobilize proteins with His-tag (His-tag).

[0065] This embodiment successfully constructs a low-density three-dimensional protein microarray chip, and the protein dot array is clear, Figure 5 .

[0066] Example 3

[0067] The preparation of the protein microarray chip in this embodiment is as follows:

[0068] Dissolve 1) 10 mM HS-C 11 -EG3-OMe (EG3OMe) and 2) HS-C 11 -EG6-OCOCBr(CH3)2 (INI) (INI accounts for 1% of the mass of EG3OMe) as ATRP initiators in ethylene glycol, add a gold substrate, and react at room temperature for 16 hours to ensure the stable formation of a monolayer, obtaining the first substrate.

[0069] Using ATRP method, 10 mmol / L CuCl2 and 10 mmol / L tris(2-dimethylaminoethyl)amine (CAS: 33527-91-2) are used as the catalytic system, and the polymerization monomers and the first substrate are polymerized in a water / methanol (1:1) mixed solvent at 35°C for 3 hours to obtain the second substrate. The polymerization monomers are: carboxyl-terminated poly(ethylene glycol) methacrylate with a concentration of 100 mM; and hydroxyethyl methacrylate (HEMA) with a concentration of 500 mM.

[0070] The second substrate was immersed in a 50 mL deionized water solution containing 300 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) and 100 mM N-hydroxysuccinimide (NHS) for 30 minutes at room temperature to activate the terminal carboxyl group to obtain a NHS ester, thereby obtaining a third substrate.

[0071] After the reaction was completed, the third substrate was shaken with pure water for 15 minutes to thoroughly clean the surface, and then the chip was immersed in a 10 mM N,N-bis(carboxymethyl)-L-lysine (NH2-NTA) solution for 30 minutes at room temperature. The amino group in the NH2-NTA molecule can couple with the activated NHS ester group on the surface of the third substrate, thereby realizing the immobilization of the NTA (nitrilotriacetic acid) group, providing a binding site for subsequent metal ion chelation (such as Ni 2+ ) and capture of His-tag proteins, thereby obtaining a fourth substrate.

[0072] Finally, the fourth substrate was shaken with pure water for 15 minutes to thoroughly clean the surface, and then immersed in a 100 mM NiSO4·6H2O aqueous solution for 30 minutes at room temperature. Ni 2+ ions chelate with the NTA group, and finally a low-density three-dimensional polymer NTA-Ni chip is successfully prepared. The chip can be used for efficient immobilization of proteins with His-tag (His-tag).

[0073] In this embodiment, a low-density three-dimensional protein microarray chip is successfully constructed, and the protein dot array is clear, Figure 6 .

[0074] Example 4

[0075] The difference between this embodiment and Example 1 is that the raw material of the background layer is 1) HS-C5-EG4-OMe; 2) HS-C5-EG5-OCOCBr(CH3)2 (INI) (the mass ratio of INI to EG6OMe is 1%).

[0076] In this embodiment, a low-density three-dimensional protein microarray chip is successfully constructed, and the protein dot array is clear, Figure 7 .

[0077] Example 5

[0078] The difference between this embodiment and Example 1 is that the raw material of the background layer is 1) HS-C 15 -EG6-OMe; 2) HS-C 15 -EG3-OCOBr(CH3)2 (INI) (the mass ratio of INI to EG6OMe is 1%).

[0079] The low-density three-dimensional protein microarray chip is successfully constructed, and the protein dot array is clear, Figure 8 .

[0080] Example 6

[0081] The difference between this example and example 1 is that the mass ratio of INI to EG4OMe is 1%, the fourth substrate is washed with pure water for 15 minutes to clean the surface, and then soaked in 100mM CuSO4·6H2O aqueous solution, and reacted at room temperature for 30 minutes. Cu 2+ The ions are chelated with the NTA groups, and finally a low-density three-dimensional polymer NTA-Cu chip is successfully prepared. The chip can be used for efficient fixation of proteins with His-tag (His-tag).

[0082] The three-dimensional polymer surface has anti-non-specific adsorption ability, and 10% fetal bovine serum FBS is flowed through. The adsorption value of FBS on the surface is observed, and the three-dimensional surface has almost no adsorption compared with the two-dimensional surface (comparative example 1) which only fixes proteins on the terminal carboxyl group modified self-assembled monolayer (SAM). The protein dot array of the constructed low-density protein microarray three-dimensional chip is clear.

[0083] Example 7

[0084] The difference between this example and example 1 is that the mass ratio of INI to EG4OMe is 1%, the fourth substrate is washed with pure water for 15 minutes to clean the surface, and then soaked in 100mM CoSO4·6H2O aqueous solution, and reacted at room temperature for 30 minutes. Co 2+ The ions are chelated with the NTA groups, and finally a low-density three-dimensional polymer NTA-Co chip is successfully prepared. The chip can be used for efficient fixation of proteins with His-tag (His-tag).

[0085] The three-dimensional polymer surface has anti-non-specific adsorption ability, and 10% fetal bovine serum FBS is flowed through. The adsorption value of FBS on the surface is observed, and the three-dimensional surface has almost no adsorption compared with the two-dimensional surface (comparative example 1) which only fixes proteins on the terminal carboxyl group modified self-assembled monolayer (SAM). The protein dot array of the constructed low-density protein microarray three-dimensional chip is clear.

[0086] It will be apparent to those skilled in the art that the disclosure is not limited to the details of the above-exemplified embodiments and that the disclosure can be implemented in other particular forms without departing from the spirit or essential characteristics of the disclosure. The presently disclosed embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the disclosure being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. No feature of the claims is to be construed as limiting the claims to the exact nature of the features described therein.

[0087] Furthermore, it should be understood that although the description is made on the basis of the embodiments, not every embodiment contains only one independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A protein microarray chip comprising a functional monolayer constructed on a gold surface of a substrate, the functional monolayer comprising: HS-C x -EG y -OMe forms a dense anti-non-specific adsorption background layer, wherein C x is an alkyl group having x carbon atoms, x, y are natural numbers and 5≤x≤15, 3≤y≤6; and ATRP initiator HS-C dispersed in an anti-non-specific adsorption background layer m -EG n -OCOCBr(CH3)2, wherein C m is an alkyl group having m carbon atoms, m, n are natural numbers and 5 ≤ m ≤ 15, 3 ≤ n ≤ 6.

2. The protein microarray chip according to claim 1, wherein The amount of said ATRP initiator is 0.1 wt% - 2 wt% of the amount of HS-C x -EG y -OMe 3. The protein microarray chip according to any one of claims 1-2, wherein The functional monolayer of the chip is further formed with a three-dimensional polymer structure, which is obtained by polymerization of terminal carboxylated poly(ethylene glycol) methacrylate and hydroxyethyl methacrylate as main raw materials to form a first polymer structure, and then functional modification.

4. The protein microarray chip according to claim 3, wherein The modification of the first polymer structure is that the terminal carboxyl groups are coupled with the amino groups of N,N-bis(carboxymethyl)-L-lysine after esterification to form binding sites that satisfy metal ion chelation and histidine tag protein capture.

5. The protein microarray chip according to claim 4, wherein The metal ion is selected from the group consisting of: Co 2+ , Ni 2 + , Cu 2+ .

6. The method for fabricating a protein microarray chip according to any one of claims 1-5, characterized in that, comprising substrate to be formed with a gold surface, HS-C x -EG y -OMe, HS-C m -EG n -OCOCBr(CH3)2; HS-C x -EG y -OMe and HS-C m -EG n -OCOCBr(CH3)2are uniformly dissolved in the first solvent, and the substrate is immersed in the solution, and a first substrate with a monomolecular layer formed is obtained by reacting at room temperature.

7. The method for fabricating a protein microarray chip according to claim 6, characterized in that, further comprising: a second substrate with a three-dimensional polymer on the surface is obtained by static polymerization in a reaction system of CuCl2 and tris(2-dimethylaminoethyl)amine as a catalytic system in a water / methanol mixed solvent, with terminal carboxylated poly(ethylene glycol) methacrylate and hydroxyethyl methacrylate as polymerization raw materials; the first substrate is at least partially immersed in the reaction system during polymerization.

8. The method for fabricating a protein microarray chip according to claim 7, characterized in that, The terminal carboxylated poly(ethylene glycol) methacrylate is prepared by dissolving PEGMA in tetrahydrofuran, then adding succinic anhydride, DMAP and pyridine, continuously stirring the mixture at room temperature, removing the THF solvent after the reaction is completed, dissolving the residue in dichloromethane, washing and extracting with dilute hydrochloric acid, collecting the organic phase and drying with anhydrous magnesium sulfate, and filtering and removing impurities.

9. The method for fabricating a protein microarray chip according to claim 7, characterized in that, further comprising: immersing the second substrate in a deionized water solution containing 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride and N-hydroxysuccinimide, and reacting at room temperature to activate the terminal carboxyl groups of the second substrate to obtain NHS ester as a third substrate; After the reaction is completed, the third substrate is washed and immersed in an N,N-bis(carboxymethyl)-L-lysine solution, and reacted at room temperature, and the amino groups in the N,N-bis(carboxymethyl)-L-lysine are coupled with the NHS ester groups to produce binding sites.

10. Use of the protein microarray chip according to any one of claims 1-5 in protein or effector detection.

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