CN104597230B - A kind of functional polymer film, preparation method and applications - Google Patents

Abstract

The invention provides a functional polymer film, a preparation method and an application thereof. The method comprises the following steps: 1) modifying the photocrosslinking agent on the surface of the substrate; 2) spin-coating the polymer solution on the surface formed in step 1); 3) irradiating the reaction with ultraviolet light, so that the polymer is covalently grafted onto Form a polymer film on the substrate; 4) further functionalize the polymer or directly perform biosensing applications. The method for preparing a three-dimensional polymer surface is simple, fast and efficient, and the density and thickness of the surface polymer can be precisely controlled by adjusting the density of the photocrosslinking agent, polymer molecular weight, spin coating parameters, etc., and the method has strong operability, Good repeatability, suitable for large-scale production. The surface of the obtained polymer film has a high immobilization amount of biomolecules, and at the same time has good anti-nonspecific adsorption ability, which can improve biosensors (such as surface plasmon resonance imaging, fluorescence, quartz crystal microbalance and electrochemical sensors, etc.) detection performance.

Description

A kind of functional polymer film, preparation method and application thereof

technical field

The invention relates to a simple, rapid and universal polymer film, a preparation method and its application in biosensing, especially in surface plasmon resonance imaging.

Background technique

Biochips mainly immobilize biological molecules such as proteins, peptides, and nucleic acids, as well as biological samples such as cells and tissues, on the surface of the chip by chemically modifying the surface of the substrate, thereby achieving accurate, rapid, and large-information detection of target biological components. Features such as high parallelism, diversity, miniaturization and automation. Reasonable surface chemical modification can make the immobilization of biological samples more efficient and stable. In order to improve the immobilization ability of biochips on biological samples, reduce non-specific adsorption, increase the signal intensity of detection, and then improve the detection sensitivity of instruments, a lot of research has been done on surface chemistry.

At present, self-assembled monolayers are widely used in the field of biosensing and chip technology. Its advantage is that it can form a stable, uniform and dense molecular layer on the surface, which can not only protect the substrate during the detection process, but also reduce non-specific adsorption. It can also have a variety of terminal functional groups for further modification; in addition, the self-assembled monolayer modification operation is extremely simple and the modification repeatability is good. However, the application of self-assembled monolayers as a two-dimensional surface is greatly limited due to the small amount of immobilized biomolecules and the weak signal when detecting biomolecular interactions. In order to further enhance the amount of immobilization of biomolecules on biochips, thereby improving the detection sensitivity of biosensors, many studies have used chip surface modification methods with three-dimensional structures. A polymer film with a three-dimensional structure and rich in various active groups is constructed on the biochip substrate. The surface prepared by this kind of modification is usually called a three-dimensional surface, because the three-dimensional space surface has a large number of biomolecules. site, greatly improving the amount of biomolecules immobilized, which can reach tens or even hundreds of times that of ordinary chips.

Existing studies on surface modification of three-dimensional structures include three-dimensional surfaces of various polymer membranes, such as dextran, polyacrylic acid, nitrocellulose, nylon, agarose, polyacrylamide, lysine-modified polyethylene glycol Hydrogels, micro/nano-scale rough surface structures, and some supramolecular self-assembled three-dimensional structures, among which dextran three-dimensional surfaces are the most widely used (Johnsson B., et al., Comparison of methods for immobilization to carboxymethyldextran sensor surfaces by analysis of the specific activity of monoclonal antibodies. Journal of Molecular Recognition, 1995.8(1-2): p.125-131.). In addition, the research on polymer brush polymers prepared by atom transfer radical polymerization (ATRP) has also received widespread attention because of its mild reaction conditions, easy operation, and strong non-specific adsorption capacity (Wang, J.- S. and K. Matyjaszewski, Controlled/"living" radical polymerization. Atom transfer radical polymerization in the presence of transition-metal complexes. Journal of the American Chemical Society, 1995.117(20):p.5614-5615.). Moreover, the material has good biocompatibility, rich terminal functional groups, and excellent properties such as anti-nonspecific adsorption, and is widely used in the surface of biosensors.

In polymer growth, there are generally two ways to connect molecular chains to the surface or interface: one is the "graft-to" method, and the other is the "graft from the surface" method (graft-to). -from). In the "grafting from the surface" method, the polymer grows directly on the surface of the substrate. Usually, the initiator of the polymer is fixed on the surface first, and then the next step of polymerization is initiated by the initiator on the surface. This method can be adjusted by adjusting the monomer The thickness of the polymer is controlled by the reaction time and the graft density of the polymer is controlled by adjusting the surface coverage of the initiator. However, the experimental steps of this method are relatively complicated, the repeatability is relatively poor when adjusting the surface polymer density, the production cost is high, and the catalyst used is usually biologically toxic and tends to remain on the surface, which is not conducive to maintaining the activity of biomolecules. The "grafting to" method is favored by people because of its simple and fast preparation and the ease of prenatal quality control of the materials used. However, the reactive functional groups of polymers are connected by chemical bonds. Different reaction systems should be used for different functional groups, and there are relatively many considerations. Moreover, for liquid-solid interface reactions, the reaction rate is usually not high, the time is long, and it is difficult for the polymer to move in the solution. Its own steric hindrance is large, resulting in limited local reactions and more prominent shortcomings.

Contents of the invention

In view of the problems in the prior art, one of the objectives of the present invention is to provide a method for preparing a functional polymer film, which uses ultraviolet light cross-linking to randomly covalently fix the polymer on the surface of the substrate to obtain a polymer film , and then further functionalize it or directly capture and detect biomolecules and drug molecules.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for preparing a functional polymer film, said method comprising the steps of:

(1) Forming a terminal functionalized self-assembled monolayer on the surface of the biochip substrate;

(2) The photocrosslinking agent is grafted to the end of the self-assembled monolayer through chemical bonding;

(3) spin coating the polymer solution onto the surface formed in step (2) under light-shielding conditions, and then dry;

(4) Under the protection of an inert gas, the biochip with the polymer spin-coated on the surface is subjected to ultraviolet light, and chemically bonded under the ultraviolet light condition, so that the polymer is grafted to the surface to form a polymer film;

Optionally, proceed to step (5):

(5) Functionalize the end of the polymer to form a functional polymer film, which is used to capture biomolecules and drug molecules, etc., and perform high-throughput detection.

The invention combines the spin-coating technology and the photo-crosslinking technology to establish a simple, fast and general biochip surface chemical modification method, and prepares (functional) polymer films. The present invention mainly utilizes the method of photocrosslinking to chemically modify the surface of the biochip to form a high-density grafted three-dimensional surface, and then further functionalize it and use the terminal functional groups to realize biomolecules on the SPRi instrument. Immobilization and detection of small molecules (such as proteins, polypeptides, polysaccharides, nucleic acids) or drugs, or direct biomolecules or drug molecules.

Preferably, the biochip substrate is pretreated as follows before step (1): the biochip substrate is cleaned.

Preferably, the biochip substrate includes all materials that can be used to prepare biochip supports, such as glass, silicon wafer and quartz; polydimethylsiloxane, polystyrene, polycarbonate and polymethacrylic acid Polymer films such as methyl ester; metal and metal oxide films such as gold film, silver film and aluminum oxide film, etc.

Preferably, the cleaning method of the biochip substrate includes, but is not limited to, rinsing, shaking or ultrasonic cleaning with organic solvents (such as ethanol, methanol, and N,N-dimethylformamide, etc.) or deionized water, and also includes Use a plasma cleaner for surface cleaning, or use any combination of two or three of these methods.

Preferably, the reagents for the self-assembled monolayer in step (1) include, but are not limited to, any one of monothiol, dithiol, and silylating reagent, or a mixture of at least two of them.

Preferably, the terminal functionalized groups in step (1) include but are not limited to alkoxy, hydroxyl, carboxyl, amino, epoxy or cyano and the like.

Preferably, the photocrosslinking agent described in step (2) refers to a reagent that contains a photosensitive group and can undergo a chemical reaction under light of a specific wavelength (energy), such as aziridine, diazo, acetophenone, diphenyl Any one or a combination of at least two of ketones or anthraquinones.

Preferably, the method of grafting the photocrosslinking agent to the end of the self-assembled monolayer through chemical bonding adopts different bonding methods according to the difference of the functional group on the surface of the biochip substrate and the functional group at the end of the photocrosslinking agent , such as amidation, esterification, ring opening or nucleophilic substitution reactions, etc.

For example, the structural formula of the photocrosslinking agent that can be used in the present invention is shown in Figure 2. For the (a) structure, the esterification reaction is used to graft to the end of the self-assembled monolayer; for the (b) structure, the amidation reaction is used to graft to the Self-assembled monolayer ends. Taking the esterification method to graft the carboxyl-terminal photocrosslinking agent to the surface of hydroxythiol as an example, since the above photocrosslinking agent contains F and N elements, there are no two kinds of surface before esterification and grafting of the photocrosslinking agent. Elements exist, so it can be judged whether the grafting of the photocrosslinking agent is successful or not based on the appearance of the two element peaks of F and N. As shown in Figure 3, the XPS data of the surface after esterification showed two obvious peaks of F and N at the same time, while there were no two elements in the control, which proved that the photocrosslinker had been successfully grafted to the surface.

Preferably, the macromolecules mainly refer to functional macromolecules with biocompatibility and active end groups, including but not limited to macromolecules with good resistance to non-specific binding, such as polyethylene glycol and its derivatives, containing Fluoropolymers, polyacrylic acid, polystyrene and polylactic acid, etc.; amphoteric betaine polymers, etc.; polymers with good biocompatibility, such as nitrocellulose, chitosan, dextran and their derivatives, etc.

Preferably, the inert gas in step (4) includes but not limited to nitrogen or/and argon and other inert gases.

Preferably, the wavelength of the ultraviolet light in step (4) is 365nm.

As shown in Figure 4, the surface of the chip was characterized by FT-IR (grazing angle attachment) after the polymer was cross-linked to the surface by 365nm ultraviolet light. For the surface of grafted poly(polyethylene glycol methacrylate), the OH stretching vibration peak appears at 3500cm- ¹ , the CH ₂ vibration doublet appears at 3000-3200cm ^-1 , and the obvious C2 appears around 1740cm- ¹ =O stretching vibration peak, while the low wave number region, such as around 1100cm ^-1 , has obvious peaks of CO and CC vibration. Similarly, for the surface of grafted dextran, the characteristic peak of -OH appeared at 3500cm ^-1 , the double vibration peak of CH ₂ appeared at 3000-3200cm ^-1 , and the obvious peak of COC stretching vibration appeared around 1100cm ^-1 . The above evidences all prove that the polymer has been successfully photo-crosslinked to the surface of the biochip, which also proves the feasibility of this technical solution.

Preferably, the terminal functionalized groups in step (5) include but are not limited to hydroxyl, carboxyl, aldehyde, amino, epoxy, cyano, alkynyl, azido or aziridine.

Preferably, the density and thickness of the surface polymer film can be precisely controlled by adjusting the density of the photocrosslinker (1mM-100mM), the number-average molecular weight of the polymer (100,000-2 million), and the spin-coating parameters (1000rpm-8000rpm). This method is simple, fast and efficient for preparing three-dimensional polymer surfaces.

The second object of the present invention is to provide a functional polymer film obtained by the above method.

The third object of the present invention is to provide a use of the functional polymer film as described above, which is used for immobilization and high-throughput detection of biomolecules and drug molecules.

Preferably, the biomolecules in the present invention include but are not limited to molecules such as proteins, polypeptides, nucleic acids (DNA, RNA, cDNA and peptide nucleic acids, etc.) and sugars. Drug molecules are mainly chemically synthesized pharmaceutical active compounds and active ingredients isolated and purified from natural products.

The high-throughput detection method described in the present invention mainly includes surface plasmon resonance imaging technology, fluorescent label detection technology, fluorescence intensity, fluorescence polarization, fluorescence resonance energy transfer, fluorescence quenching, quartz crystal microbalance technology and various A high-throughput electrochemical detection technology and thermal detection technology and other methods.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a new "grafting to" method, using spin-coating technology and photo-crosslinking technology to obtain a universal method for immobilizing polymers, which can non-selectively covalently immobilize polymers in one step using ultraviolet light On the surface, it is directly used to prepare a three-dimensional surface biochip with a photocrosslinked structure.

In addition, the method of the present invention uses a photocrosslinking agent to produce highly reactive carbene under ultraviolet light irradiation, which has strong reactivity and short reaction time, and can randomly perform insertion reactions with macromolecules. It is applicable to all macromolecules and has general fitness.

In addition, the method is based on the mature and reliable spin-coating technology, and the required instruments and equipment are popular and versatile. The biochip prepared by the method can significantly improve the immobilization and detection signals of various biological detection substances, has strong practicability, is suitable for large-scale popularization and commercial application, and has very broad application prospects.

In addition, the present invention preferably adopts relatively long ultraviolet light (365nm), with few side reactions and no damage to other modified molecules (such as thiol, etc.) on the surface.

Description of drawings

Figure 1 is a flowchart of three-dimensional surface modification of functional polymers based on photocrosslinking method;

Fig. 2 is the molecular structural formula of photocrosslinking agent in the present invention, wherein, (a) carboxyl terminal photocrosslinking agent; (b) amino terminal photocrosslinking agent;

Figure 3 is the X-ray Photoelectron Spectroscopy (XPS) analysis results of surface elements before and after grafting photocrosslinking agent, wherein Figures (a) and (b) are XPS figures before and after N1s element grafting, and Figure (c) and (d) are the XPS diagrams of F1s elements before and after grafting;

Figure 4 is Fourier Transform Infrared Spectroscopy (FT-IR) characterization of the surface of photocrosslinked grafted polymers, (a) poly(polyethylene glycol methacrylate); (b) dextran);

Figure 5 is a dextran three-dimensional surface biosensing chip prepared based on a photocrosslinking method, immobilizing streptavidin (SA) in a flow-through manner, and detecting 4nM biotin (Biotin);

Fig. 6 immobilizes rapamycin on the surface of poly(polyethylene glycol methacrylate), and uses SPRi to detect the interaction with 100 nM FKBP12 (PBS, pH7.4, 0.5% T20) protein, wherein, rapamycin Rapamycin-1, rapamycin-2 and rapamycin-3 represent the samples used in three repeated experiments respectively;

Figure 7 is a surface plasmon resonance imaging image of a lectin microarray;

Fig. 8 is the result of serum screening by lectin array.

detailed description

The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation methods.

Example 1. Dextran surface (small molecule detection: flow-through immobilized SA, detection of Biotin)

(1) Prepare a layer of chromium layer with a thickness of 3nm and a layer of gold with a thickness of 47nm on the glass substrate by thermal evaporation method, as the substrate of the biochip.

(2) Prepare an ethanol solution of hydroxyl-terminated thiol HS-(CH ₂ ) ₁₁ -EG6-OH at a concentration of 1 mM.

(3) The substrate of the biochip is cleaned with ethanol or deionized water, and then the biochip is placed in a plasma cleaner for cleaning for 3 minutes.

(4) Soak the biochip in the prepared thiol solution and incubate at 4°C for 12 hours. After the predetermined time, the biochip is taken out, washed with ethanol and deionized water alternately, and dried with nitrogen.

(5) Prepare 20 mL of the solution required for esterification and linking photocrosslinking agent, 10 mM carboxy-terminal photocrosslinking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) 10 mM, 4 - Dimethylaminopyridine (DMAP) 1 mM, solvent N,N-dimethylformamide (DMF).

(6) Immerse the biochip in the prepared esterification solution, and react in the dark at room temperature for 4 hours. After the predetermined time is reached, the biochip is washed with DMF, ethanol and deionized water in sequence, and dried with nitrogen gas for later use.

(7) Dextran with a molecular weight of 2000KDa was prepared into an aqueous solution with a mass concentration of 40%, stirred evenly, and air bubbles were removed to obtain a colorless and transparent homogeneous solution.

(8) Spread the prepared dextran solution on the surface of the biochip, spin-coat at a speed of 8000 rpm for 1 minute, and after spin-coating, place the biochip at room temperature in the dark for 1 hour to dry.

(9) Put the biochip into an ultraviolet light cross-linking apparatus, and irradiate with 365nm ultraviolet light (2.4J/cm ² ) for 15 minutes under a nitrogen atmosphere.

(10) After the predetermined time is reached, shake the biochip repeatedly with hot water at 50°C for 1 hour to remove uncovalently fixed dextran molecules on the surface, change the water 3-5 times during this period, and blow dry the biochip with nitrogen after cleaning spare.

(11) Immerse the biochip in the DMF solution of succinic anhydride (10 mg/mL) and 4-dimethylaminopyridine (15 mg/mL), and react at room temperature (25°C) for 16 hours. After the predetermined reaction time is reached, the biochip Take it out, wash it successively with DMF, ethanol, and deionized water, and dry it with nitrogen gas.

(12) Prepare 50 ug/mL streptavidin (SA) sodium acetate buffer solution (pH=4.5) for later use.

(13) Adjust the optical position of the SPRi, flow the SA solution to the surface of the biochip at a speed of 3uL/s, and continue to bind for 100s.

(14) Repeat step 12 until the amount of immobilization on the surface reaches saturation.

(15) Replace the system solution with PBS buffer (containing 1% DMSO), adjust the optical position, pass through PBS solution (1% DMSO) containing 4nM biotin, combine for 300s, dissociate for 300s, 1:100 phosphoric acid aqueous solution Regenerated, 10 mM NaOH aqueous solution regenerated.

Experimental results prove that the biochip prepared by this method has a high amount of protein immobilization, which is enough to detect small molecules bound to the protein (as shown in Figure 5).

Example 2. Poly(polyethylene glycol methacrylate) surfaces (small molecule arrays)

(1) Prepare a layer of chromium layer with a thickness of 3nm and a layer of gold with a thickness of 47nm on the glass substrate by thermal evaporation method, as the substrate of the biochip.

(2) Prepare ethanol solutions of hydroxyl-terminated and carboxyl-terminated thiols (HS-(CH ₂ ) ₁₁ -EG6-OH and HS-(CH ₂ ) ₁₁ -EG6-COOH), both at a concentration of 1 mM, and the above two The mercaptan solution was mixed according to 999:1 (v/v) and set aside.

(3) Clean the biochip with ethanol or deionized water, and then put the biochip into a plasma cleaner for cleaning for 3 minutes.

(4) Soak the biochip in the mixed thiol solution and incubate at 4°C for 12 hours. After the predetermined time, the biochip is taken out, washed with ethanol and deionized water alternately, and dried with nitrogen.

(5) Activate the carboxyl groups on the surface of the biochip, and then immerse in an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (0.4M and 0.1M), incubate at room temperature for 30 minutes until the predetermined time is reached, wash with water, and dry with nitrogen gas for later use.

(6) Immerse the biochip in N,N-dimethylformamide (DMF) solution of 10mM amino-terminal photocrosslinking agent, and react in the dark at room temperature for 4 hours. Wash with deionized water and blow dry with nitrogen gas.

(7) A methanol/water solution of poly(polyethylene glycol methacrylate) high molecular polymer was synthesized by atom radical transfer polymerization (ATRP). Mix the copper chloride (CuCl ₂ ) solution with its ligand bipyridine (Bpy) and stir for 15 min. First, monomer OEGMA and monomer HEMA were added into a mixed solution of methanol and water (1:1, volume ratio) at a ratio of 1:1 (molar ratio), ultrasonicated for 15 minutes, and nitrogen gas was continuously introduced for 30 minutes. Then add 0.04M newly configured ascorbic acid aqueous solution, stir for 10min and mix well, then add 1mM ethanol solution of dithiol initiator, wherein the molar ratio of initiator to monomer is 1:25000, continue to feed nitrogen, and react for 16 hours .

(8) The ATRP polymer stock solution obtained in step 7 was rotary-evaporated (35° C.) to remove methanol, and the remaining liquid was extracted with dichloromethane solvent and centrifuged. The lower dichloromethane solution part was taken out and spin-dried.

(9) Dissolving the polymer purified in step (8) with ethanol to obtain a 2 μM ethanol solution of poly(polyethylene glycol methacrylate) polymer, which is set aside.

(10) Spread the configured polymer solution on the surface of the biochip, and spin-coat it at a speed of 8000rpm for 1 minute. After spin-coating, place the biochip at room temperature in the dark for 1 hour to dry. After reaching the predetermined time , washed with ethanol and water alternately, and blown dry with nitrogen.

(11) Put the biochip into an ultraviolet light cross-linking apparatus, and irradiate with 365nm ultraviolet light (2.4J/cm ² ) for 15 minutes under nitrogen atmosphere.

(12) After the predetermined time is reached, the biochip is ultrasonically cleaned with ethanol and water, and the spare core is blown dry with nitrogen gas.

(13) Immerse the biochip in the DMF solution of succinic anhydride (10mg/mL) and 4-dimethylaminopyridine (15mg/mL), react at room temperature for 12 hours, after reaching the predetermined reaction time, take out the biochip, and use Wash with DMF, ethanol and deionized water, and blow dry with nitrogen.

(14) Cover the surface of the biochip with 1 mM ethanol solution of EG3, seal for 30 min, then wash with ethanol and water alternately, and blow dry with nitrogen. So far, the poly(polyethylene glycol methacrylate) surface chip prepared by the photocrosslinking method has been completed, and the small molecules are immobilized by the photocrosslinking method and the related proteins are detected by SPRi, as follows:

(15) The surface of the biochip is reactivated and immersed in an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) ( 0.4M and 0.1M), incubate at room temperature for 30 minutes until the predetermined time is reached, wash with water, and dry with nitrogen gas for later use.

(16) Immerse the biochip again in the N,N-dimethylformamide (DMF) solution of 10mM photocrosslinking agent, and react in the dark at room temperature for 4 hours. Wash with deionized water and dry with nitrogen gas.

(17) Prepare 100 nM rapamycin small molecule solution and 100 ug/mL FKBP12 protein PBST buffer solution (Tween 0.05%) for later use.

(18) Spot the small molecule solution on the surface prepared in step (16) through a spotting instrument, and dry it in vacuum.

(19) Put the chip into the ultraviolet light cross-linking apparatus again, and irradiate with 365nm ultraviolet light (2.4J/cm ² ) for 15 minutes under nitrogen environment to carry out photo-crosslinking.

(20) Adjust the optical position of SPRi, flow the FKBP12 protein solution to the chip surface at a speed of 3uL/s, continue to bind for 300s, dissociate for 300s, and regenerate with glycine-hydrochloric acid.

Experimental results prove that the biochip prepared by this method has a high amount of immobilized small molecules, which is sufficient to detect the interaction between small molecules and their associated proteins (as shown in Figure 6).

Embodiment 3. Dextran surface (antigen antibody fluorescent detection)

(1) The glass substrate is cleaned with ethanol or deionized water, and the biochip is cleaned in a plasma cleaner for 3 minutes to be used as the substrate of the biochip.

(2) Prepare 3-aminopropyltriethoxysilane (APES) diluted with acetone at 1:50, take it out after 20-30s, and then use pure acetone solution to remove unbound APES, so that APES can be combined with glass, A monolayer is formed on the surface.

(3) Soak in 25% glutaraldehyde for 30 minutes, wash with acetone, immerse in 1 mM aqueous solution of PEG with one end being an amino group and one end being a hydroxyl group, and incubate at room temperature for 1 h. After the predetermined time is reached, the biochip is taken out and used Rinse with deionized water and dry with nitrogen gas.

(4) Prepare 20 mL of the solution required for esterification and linking photocrosslinking agent, 10 mM carboxy-terminal photocrosslinking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) 10 mM, 4 - Dimethylaminopyridine (DMAP) 1 mM, solvent N,N-dimethylformamide (DMF).

(5) The biochip was immersed in the prepared esterification solution, and reacted at room temperature in the dark for 4 hours. After the predetermined time was reached, the biochip was washed with DMF, ethanol and deionized water in sequence, and dried with nitrogen gas for later use.

(6) Dextran with a molecular weight of 2000KDa was formulated into an aqueous solution with a mass concentration of 40%, stirred evenly, and air bubbles were removed to obtain a colorless and transparent homogeneous solution.

(7) Spread the prepared dextran solution on the surface of the biochip, spin-coat at a speed of 8000 rpm for 1 minute, and after spin-coating, place the chip at room temperature in the dark for 1 hour to dry.

(8) Put the biochip into an ultraviolet light cross-linking apparatus, and irradiate with 365nm ultraviolet light (2.4J/cm ² ) for 15 minutes under a nitrogen environment.

(9) After the predetermined time is reached, shake the biochip repeatedly with hot water at 50°C for 1 hour to remove uncovalently immobilized dextran molecules on the surface, change the water 3-5 times during this period, and blow dry the biochip with nitrogen after cleaning spare.

(10) Immerse the biochip in the DMF solution of succinic anhydride (10mg/mL) and 4-dimethylaminopyridine (15mg/mL), and react at room temperature (25°C) for 16 hours. After the predetermined reaction time is reached, the biochip Take it out, wash it successively with DMF, ethanol, and deionized water, blow dry with nitrogen, and set aside.

(11) Use a pipette gun to take 3 μl of 1 mg/ml human IgG (the solvent is a sodium acetate solution with a pH value of 4.5) and drop it on the table obtained in step (10), supplemented with 1 mg/ml of BSA as a negative control , incubated at room temperature for 1 hour, washed the surface twice (5 minutes each time) with phosphate buffer solution containing 0.0005 g/ml Tween-20, washed with deionized water, and dried with nitrogen gas.

(12) Use 0.05g/ml skim milk solution (Skim milk) (prepared by weighing 5g skim milk powder and dissolving 100ml of 0.1M phosphate buffer) to seal the surface at room temperature for 1 hour, then use 0.0005g/ml Wash the surface twice (5 minutes each time) with phosphate buffer solution of Tween-20 in ml, wash with deionized water, and blow dry with nitrogen gas.

(13) Dilute goat anti-human IgG labeled with fluorescein isothiocyanate (FITC) in 0.005 mg/ml skim milk aqueous solution at a dilution factor of 1:50, and place the chip after processing in step 12 Immerse in this skimmed milk, place at room temperature for 1 hour, carry out antigen-antibody reaction, wash the surface twice (5 minutes each time) with the phosphate buffer solution of 0.0005g/ml Tween-20, and after cleaning with deionized water, blow with nitrogen Dry.

(14) Perform fluorescence imaging on the chips processed in step 13 with a Leica M-6000 fluorescence microscope under a 480 nm halogen light lamp with an exposure time of 300 ms and a light intensity of 5 with a 20-fold objective lens.

Example 4. Poly(polyethylene glycol methacrylate) surface (serum detected by SPRi glycoprotein array)

(1) Prepare a layer of chromium layer with a thickness of 3nm and a layer of gold with a thickness of 47nm on the glass substrate by thermal evaporation method, as the substrate of the biochip.

(2) Prepare ethanol solutions of hydroxyl-terminated and carboxyl-terminated thiols (HS-(CH ₂ ) ₁₁ -EG6-OH and HS-(CH ₂ ) ₁₁ -EG6-COOH), both at a concentration of 1 mM, and the above two The mercaptan solution was mixed according to 999:1 (v/v) and set aside.

(3) Clean the biochip with ethanol or deionized water, and then put the biochip into a plasma cleaner for cleaning for 3 minutes.

(4) Soak the biochip in the mixed thiol solution and incubate at 4°C for 12 hours. After the predetermined time, the biochip is taken out, washed with ethanol and deionized water alternately, and dried with nitrogen.

(5) Activate the carboxyl groups on the surface of the biochip, and immerse the chip in an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) Medium (0.4M and 0.1M), incubate at room temperature for 30 minutes until the predetermined time is reached, wash with water, and dry with nitrogen gas for later use.

(6) Immerse the biochip in N, N-dimethylformamide (DMF) solution of 10 mM photocrosslinking agent, and react in the dark at room temperature for 4 hours. Deionized water was used for cleaning, and nitrogen gas was blown dry for later use.

(7) A methanol/water solution of poly(polyethylene glycol methacrylate) high molecular polymer was synthesized by atom radical transfer polymerization (ATRP). Mix copper chloride (CuCl ₂ ) solution with its ligand bipyridine, and stir for 15 min. First, monomer OEGMA and monomer HEMA were added into a mixed solution of methanol and water (1:1, volume ratio) at a ratio of 1:1 (molar ratio), ultrasonicated for 15 minutes, and nitrogen gas was continuously introduced for 30 minutes. Then add 0.04M freshly prepared aqueous solution of ascorbic acid, and stir for 10 minutes to mix well. Then add 1 mM ethanol solution of dithiol initiator, wherein the molar ratio of initiator to monomer is 1:25000, continuously feed nitrogen, and react for 16 hours.

(8) The ATRP polymer stock solution obtained in step (7) was rotary evaporated (35 degrees), methanol was removed, and the remaining liquid was extracted with dichloromethane solvent, centrifuged, and the lower layer of dichloromethane solution was taken out, and spin-dried.

(9) Dissolving the purified macromolecule in step (8) with ethanol to obtain an ethanol solution of the purified poly(polyethylene glycol methacrylate) macromolecular polymer, which is set aside.

(10) Spread the prepared polymer solution on the surface of the biochip, spin-coat at a speed of 8000 rpm for 1 minute, and after spin-coating, place the biochip at room temperature in the dark for 1 hour to dry.

(11) Put the biochip into an ultraviolet light cross-linking apparatus, and irradiate with 365nm ultraviolet light (2.4J/cm ² ) for 15 minutes in a nitrogen environment.

(12) After the predetermined time is reached, the biochip is ultrasonically cleaned with ethanol and water, and dried with nitrogen gas for later use.

(13) Immerse the biochip in the DMF solution of succinic anhydride (10mg/mL) and 4-dimethylaminopyridine (15mg/mL), react at room temperature for 16 hours, after reaching the predetermined reaction time, take out the biochip, and use Wash with DMF, ethanol, and deionized water, and blow dry with nitrogen. So far, the poly(polyethylene glycol methacrylate) surface chip prepared by the photocrosslinking method has been completed, and the immobilization of lectin (lectin) by the spotting instrument and the detection of type 1 diabetes (T1) and type 2 diabetes by SPRi have been completed. (T2) and related detection of type 1.5 diabetes mellitus (LADA) human serum, and normal human serum was used as control (C).

(14) The surface of the biochip is activated and immersed in an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (0.4 M and 0.1M), incubate at room temperature for 30 minutes until the predetermined time, wash with water, and dry with nitrogen gas for later use.

(15) Spotting 49 different lectins on the surface of the prepared chip with a spotting instrument, dried in vacuum, and set aside.

(16) Block the chip in step (15) with 2% skimmed milk (PBS solution), overnight at 4°C, wash with 10*PBST, 1*PBST, 0.1*PBST and water for 10 minutes, blow with nitrogen Dry and set aside.

(17) Dilute 4 different serum samples at 1:4000 with HEPES buffer, and set aside.

(18) Adjust the optical position of the SPRi, first pass through the HEPES buffer at a flow rate of 4ul/s for 10min, then use 1:200 hydrochloric acid (adding 0.05% Tween) to regenerate the surface 3 times, and the serum sample solution was injected at a flow rate of 3uL/s Flow to the surface of the chip in random order, continue to bind for 300s, dissociate for 300s, and regenerate with 1:200 hydrochloric acid (adding 0.05% Tween).

Experimental results prove that the chip prepared by this method has a high amount of lectin immobilization, and can detect different types of diabetic serum well (as shown in Fig. 7 and Fig. 8).

Embodiment 5. Polyacrylic acid surface (PAA) prepares protein microarray

(1) Prepare a layer of chromium layer with a thickness of 3nm and a layer of gold with a thickness of 47nm on the glass substrate by thermal evaporation method, as the substrate of the biochip.

(2) Ethanol solutions (HS-(CH ₂ ) ₁₁ -EG6-OH and HS-(CH ₂ ) ₁₁ -EG6-COOH) of hydroxyl-terminated and carboxyl-terminated thiols were prepared, both at a concentration of 1 mM. Mix the two mercaptan solutions (EG6-OH:EG6-COOH) according to 999:1 and set aside.

(3) Clean the biochip with ethanol or deionized water, and then put the biochip into a plasma cleaner for cleaning for 3 minutes.

(4) Soak the biochip in the mixed thiol solution and incubate at 4°C for 12 hours. After the predetermined time, the biochip is taken out, washed with ethanol and deionized water alternately, and dried with nitrogen.

(5) The carboxyl group on the surface of the biochip is activated, and the biochip is immersed in 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) In an aqueous solution (0.4M and 0.1M), incubate at room temperature for 30 minutes until the predetermined time is reached, wash with water, and dry with nitrogen gas for later use.

(6) Immerse the biochip in the N, N-dimethylformamide (DMF) solution of 10mM carboxyl-terminal photocrosslinking agent, and react in the dark at room temperature for 4 hours. Wash with deionized water and blow dry with nitrogen gas.

(7) Polyacrylic acid polymer (PAA) with a molecular weight of 140,000 was formulated into an aqueous solution with a volume concentration of 1%, stirred evenly, and air bubbles were removed to obtain a colorless and transparent homogeneous solution.

(8) The prepared PAA solution was spread on the surface of the biochip, and spin-coated at a speed of 8000 rpm for 1 minute. After spin-coating, the biochip was left at room temperature in the dark for 1 hour to dry.

(9) Put the biochip into an ultraviolet light cross-linking apparatus, and irradiate with 365nm ultraviolet light (2.4J/cm ² ) for 15 minutes under a nitrogen environment.

(10) After the predetermined time is reached, the biochip is washed with water and dried with nitrogen gas for later use.

(11) The surface of the biochip is activated and immersed in an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (0.4 M and 0.1M), incubate at room temperature for 30 minutes. When the predetermined time is reached, wash it with water and dry it with nitrogen gas for later use. So far, the polyacrylic acid surface chip prepared by the photocrosslinking method has been completed, and SPRi is used for antigen immobilization and antibody detection.

(12) Prepare 1mg/ml H-IgG PBS solution and 100ug/ml Goat-anti-H-IgG PBS solution for later use.

(13) Spot the prepared H-IgG solution on the surface of the chip prepared in step (11), dry it, and set it aside.

(14) The biochip was blocked with 1 mg/ml BSA solution at room temperature for 10 min, washed with PBS solution and water, dried with nitrogen gas, and set aside.

(15) Adjust the optical position of SPRi, flow the PBS solution of Goat-anti-H-IgG to the surface of the chip at a speed of 3uL/s, continue to bind for 300s, dissociate for 300s, and regenerate with NaOH solution.

Experimental results prove that the biochip prepared by this method has a high immobilized amount of antigen, and can detect the interaction between antigen and its antibody well.

The applicant declares that the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (14)

1. a kind of preparation method of functional polymer film, it is characterised in that the method comprising the steps of：

(1) self assembled monolayer of end-functionalization is formed in biochip substrate surface；

(2) photocrosslinking agent is grafted to into self assembled monolayer end by chemical bonding；

(3) Polymer Solution is spun to into the surface that step (2) is formed under the conditions of lucifuge, is then dried；

(4) there is high molecular biochip that ultraviolet lighting is carried out under inert gas shielding surface spin coating, in ultraviolet light conditions Under be chemically bonded, make macromolecular grafted to surface, form macromolecule membrane；

Optionally, carry out step (5)：

(5) macromolecule is carried out into end-functionalization, forms functional polymer film；

Photocrosslinking agent density is 1mM～100mM, and macromolecule number-average molecular weight is 100,000～2,000,000, and spin coating rotating speed is 1000rpm ～8000rpm；In step (1), the reagent of self assembled monolayer is any one in single mercaptan, dithiol and silylating reagent Kind or at least two mix reagent, in step (5) group of end-functionalization be hydroxyl, carboxyl, aldehyde radical, amino, epoxy Base, cyano group, alkynyl, azido or azirine.

2. the method for claim 1, it is characterised in that biological chip base is pre-processed as follows before step (1)： Biochip substrate is cleaned up.

3. the method for claim 1, it is characterised in that the biochip substrate be glass, silicon chip, quartz, poly- two Methylsiloxane, polystyrene, Merlon, polymethyl methacrylate, golden film, silverskin or di-aluminium trioxide film.

4. the method for claim 1, it is characterised in that the cleaning way of the biochip substrate is to use organic solvent Or deionized water is rinsed, shakes and wash or be cleaned by ultrasonic, or surface clean, or its combination are carried out using plasma cleaning instrument.

5. the method for claim 1, it is characterised in that the group of end-functionalization is alkoxyl, hydroxyl in step (1) Base, carboxyl, amino, epoxy radicals or cyano group.

6. the method for claim 1, it is characterised in that photocrosslinking agent described in step (2) is azirine, diazonium, second In acyl benzene, benzophenone or Anthraquinones any one or at least two combination.

7. the method for claim 1, it is characterised in that make photocrosslinking agent by chemical bonding, be grafted to self assembly list The method of molecular layer end is amidatioon, esterification, open loop or nucleophilic substitution.

8. the method for claim 1, it is characterised in that the macromolecule is polyethylene glycol and its derivative, fluorine-containing poly- Compound, polyacrylic acid, polystyrene, PLA, both sexes betaines polymer, nitrocellulose, shitosan or glucan and its Derivative.

9. the method for claim 1, it is characterised in that step (4) inert gas is nitrogen or/and argon gas.

10. the method for claim 1, it is characterised in that a length of 365nm of step (4) ultraviolet light wave.

A kind of 11. functional polymer films obtained by one of claim 1-10 methods described.

A kind of 12. purposes of functional polymer film as claimed in claim 11, which is used for biomolecule and drug molecule Fixed and high flux is detected.

13. purposes as claimed in claim 12, it is characterised in that the biomolecule is albumen, polypeptide, nucleic acid and sugar； The drug molecule purifies the active component for obtaining for the pharmaceutical active compounds and Separation of Natural Products of chemical synthesis.

14. purposes as claimed in claim 12, it is characterised in that high flux detection include surface plasmon resonance into As technology, fluorescence labeling detection technique, fluorescence intensity, fluorescence polarization, FRET, fluorescent quenching, quartz crystal Micro- balance technology, high-throughout electrochemical measuring technique and high-throughout hot detection technique.