Surface plasmon element resonance chip for cell fixation and preparation method of surface plasmon element resonance chip
By forming a structure of hydrophobic and hydrophilic compounds on the surface of the SPR chip, the problems of non-specific adsorption and cell fixation in the prior art are solved, and better cell adhesion and survival effects are achieved.
Patent Information
- Application Number
- CN202411567830.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2025-02-07
AI Technical Summary
Existing SPR chips are difficult to take into account the needs of non-specific adsorption and cell fixation in cell detection, resulting in poor detection results.
By forming a hydrogel structure of strongly hydrophobic compounds and strongly hydrophilic compounds on the surface of the SPR chip, the compound fixation and stable cell fixation are achieved using the reaction of Au−S bonding and the reaction of active groups.
It effectively reduces the non-specific adsorption of biological molecules such as proteins, improves the adhesion, survival and proliferation of cells, and significantly improves the fixation and survival rate of chip surface cells.
Smart Images
Figure CN119394970A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of SPR technology, and in particular relates to a surface plasmon elementary resonance chip for cell fixation and a preparation method thereof. Background Art
[0002] Surface plasmon resonance (SPR) technology is one of the most commonly used label-free methods for detecting the kinetic constants and thermodynamic constants of intermolecular interactions, as well as molecular concentrations. In recent years, with the development of SPR microscopy technology developed by companies such as Biosensing, the detection range of SPR technology has gradually extended to cell-related fields, such as the quantitative analysis of the kinetic constants and thermodynamic constants of the binding of cells and molecules, cells and viruses, cells and materials, etc., which has important implications for solving mechanical quantitative technical problems in related research fields. The basis of related quantitative analysis includes the capture, fixation and detection of specific cells.
[0003] The basis of SPR technology for detection is the chip. The SPR chip is composed of a glass substrate and a metal film. The main component of the metal film modified on the surface of the common chip is gold atoms (gold chip). Other metals such as silver and copper can also be used as the metal substrate layer of the SPR chip. The gold chip has good stability and is therefore also the main component of the metal film modified on the surface of the commercial SPR chip. However, gold atoms easily bind to a variety of biological molecules (such as -NH2 in protein molecules) and produce nonspecific adsorption on the surface of the gold chip. Therefore, unmodified gold chips (bare gold chips) are usually not directly used in related detection research. The first condition for the construction of a practical commercial SPR chip is to reduce the nonspecific adsorption on the chip surface.
[0004] For cell detection, traditional chips cannot take into account the requirements of nonspecific adsorption and cell fixation. Although chips modified by methods such as poly-lysine can achieve cell fixation, nonspecific adsorption is still serious even in the process of pure protein and nucleic acid detection. In the process of complex biological system detection, nonspecific adsorption will be more serious, which directly restricts the development of SPR cell detection technology. Summary of the invention
[0005] In order to solve the above problems in the prior art, the present invention provides a surface plasmon resonance cell chip and a preparation method thereof.
[0006] The present invention first uses Au-S bonding to achieve the fixation of compounds on the surface of the SPR chip, providing conditions for subsequent modification; then a layer of strongly hydrophobic compounds is fixed on the surface of the chip to provide support for subsequent compound modification; finally, a layer of strongly hydrophilic compounds is modified on the surface of the chip to form a tightly arranged hydrogel structure. On the one hand, the surface of the chip is negatively charged by the dissociation of hydroxyl and carboxyl groups on the surface of the strongly hydrophilic compounds, and the charge repulsion reduces the adsorption of negatively charged biological molecules such as proteins on its surface; on the other hand, the hydrophilic groups also provide conditions for maintaining cell integrity. In addition, active groups such as hydroxyl and carboxyl groups also provide conditions for the binding of cell fixation sites on the surface of the chip.
[0007] A surface plasmon resonance chip for cell fixation, comprising, from bottom to top, a solid support layer, a supporting layer, a stabilizing layer and a binding layer connected in sequence; The solid support layer is an unmodified SPR gold chip; the support layer is a hydrophobic layer, one end of the support layer is fixed to the solid support layer through a thiol group, and the other end is connected to the stabilizing layer through a reaction between active groups; The stabilizing layer is composed of polysaccharide segments, and the side of the polysaccharide segments opposite to the chip surface is connected to the binding layer through the reaction between active groups; The binding layer component is selected from at least one of adhesion proteins, cell adhesion specific sites, glycoprotein recognition molecules, cell membrane surface proteins, phospholipid molecule antibodies or poly-lysine.
[0008] Preferably, the support layer is obtained by fixing a hydrophobic long-chain alkane to the solid support layer. Since the surface of the metal coating is an electron-rich structure and has a binding effect with more hydrophilic groups, the present invention selects a hydrophobic long-chain alkane that can be evenly arranged to form a dense structure on its surface, thereby preventing the binding of the hydrogel to the metal layer in subsequent modification. At the same time, since the structure is a hydrophobic structure, the interaction force between the hydrogel and the subsequent modified hydrogel is low, which also provides conditions for the uniform distribution of the hydrogel. These are all conducive to reducing nonspecific adsorption. The role of the support layer is to provide support for subsequent modification, prevent the molecules of the stabilizing layer and the binding layer from binding to the metal atoms on the chip surface, causing the hydrogel to collapse and the structure to be unstable. The hydrophobic alkane of the support layer selected by the present invention is fixed to the metal surface of the support layer through a thiol group, and the other end of the alkane chain is a highly reactive group.
[0009] Preferably, the polysaccharide is dextran or nanocellulose. Further preferably, the number of repeating units of the dextran segment or nanocellulose segment in the stabilizing layer is 10-400,000.
[0010] The function of the stabilizing layer is to reduce the non-specific adsorption on the chip surface, improve the hydrophilicity of the chip surface, and provide conditions for the stable existence of cells.
[0011] The dextran layer or nanocellulose layer is grafted onto the chip surface through a thiol reagent having a hydrophobic alkane at one end, the other end of the alkane is a highly reactive group, and the highly reactive group is a hydroxyl group, a carboxyl group, a biotin group or an aldehyde group, and the highly reactive group provides conditions for the grafting of hydroxyl groups on the surface of the dextran or nanocellulose.
[0012] The binding layer is used for cell attachment and provides conditions for cell adhesion, survival and proliferation. The binding layer is present on the other side of the glucan or nanocellulose in the stabilizing layer opposite to the chip surface.
[0013] Preferably, the adhesive protein is selected from any one of fibronectin, laminin, collagen, entactin, tenascin, thrombospondin, and heparin sulfate glycoprotein. The adhesive protein contains a specific binding site that binds to cells. The glycoprotein recognition molecule can specifically recognize cell surface glycoproteins.
[0014] like Figure 1 As shown in , the chip includes a specific adsorption component (binding layer) and an anti-nonspecific adsorption component (support layer and stabilization layer), which are distributed in layers on the chip surface and cooperate with each other to achieve the fixation of cells on the chip surface and the control of nonspecific adsorption. Considering the role of the distribution of cell surface proteins and phospholipids in cell fixation, as well as the composition structure of cell surface proteins, phospholipids and carbohydrates, the specific adsorption part is the part exposed outside the three-dimensional hydrogel on the chip surface, and needs to have the conditions to bind to the three types of substances: protein, phospholipids or carbohydrates. Considering the complexity of the sample in the actual detection process, when performing nonspecific adsorption control, it is necessary to ensure that the structure of the compound used in the anti-nonspecific adsorption component will not affect the adhesion of the cell and the integrity of the cell membrane, and cannot bind to the component to be detected. Therefore, the structure of the anti-nonspecific adsorption component should be alternating between hydrophobicity and hydrophilicity, and its distribution on the chip surface should form a structure with a constantly changing polarity distribution. At the same time, in order to coordinate the use of specific adsorption and anti-nonspecific adsorption, when designing the chip surface structure, it is also necessary to consider that the two can interact with each other to form a gel structure arrangement of a certain thickness on the chip surface, thereby blocking the nonspecific adsorption caused by the combination of one or more compounds in the component to be tested and gold.
[0015] The present invention also provides a method for preparing the surface plasmon resonance chip for cell fixation.
[0016] When the stabilizing layer is composed of dextran, the method comprises the following steps: (1) Soaking the SPR gold chip in an ethanol solution of a thiol reagent at room temperature for 12 to 36 hours to obtain a chip modified with a thiol reagent; the thiol reagent is 6-mercapto-1-hexanol, 16-mercaptohexadecanoic acid, 11-mercaptoundecanoic acid or 6-mercaptohexylamine; (2) When the terminal of the thiol reagent is a non-carboxyl group, firstly, sodium hydroxide and bromoacetic acid or chloroacetic acid are used to convert the non-carboxyl group on the other end of the thiol reagent on the chip surface of step (1) into a carboxyl group, and then EDC / DMAP is used to fix the dextran to the SPR chip surface; when the terminal of the thiol reagent is a carboxyl group, EDC / DMAP is directly used to fix the dextran to the chip surface; (3) Soaking the chip described in step (2) in a mixed solution of sodium hydroxide and bromoacetic acid or chloroacetic acid to convert part of the hydroxyl groups on the surface of the dextran into carboxyl groups, and then cleaning the chip. The conversion ratio of the hydroxyl groups is 1-10%. (4) Use EDC / NHS to activate the carboxyl groups on the cleaned chip surface, and fix the cell-binding protein to the chip surface through amide bonds to form a binding layer, providing conditions for cell fixation.
[0017] Preferably, the concentration of the thiol reagent in ethanol in step (1) is 0.01-50 mg / mL.
[0018] When the other end of the thiol reagent in step (2) is a carboxyl group, the dextran is fixed to the chip surface through an esterification reaction catalyzed by EDC / DMAP.
[0019] Preferably, in step (2), when the end of the thiol reagent is not a carboxyl group, the chip modified with the thiol reagent in step (1) is first immersed in a mixed aqueous solution of sodium hydroxide and bromoacetic acid or chloroacetic acid to convert the non-carboxyl group at the other end of the thiol reagent on the chip surface into a carboxyl group; then the chip is immersed in a mixed aqueous solution of EDC / DMAP; and then the chip is immersed in a mixed aqueous solution of dextran and sodium hydroxide, wherein the mass fraction of dextran in the mixed aqueous solution of dextran and sodium hydroxide is 0.1-20%.
[0020] Preferably, the concentration of the chloroacetic acid or bromoacetic acid solution in step (3) is 1-3 M, the concentration of the sodium hydroxide solution is 1-5 M, and the volume ratio of the chloroacetic acid or bromoacetic acid solution to the sodium hydroxide solution is 1:1. Preferably, in step (4), a protein solution with a concentration of 0.01-0.1 mg / mL is dropped onto the surface of the chip to form a binding layer.
[0021] Or, when the stabilizing layer is composed of nanocellulose, the steps include: (1) Soak the SPR gold chip in an ethanol solution of a thiol reagent at room temperature for 12 to 36 h; (2) Using epichlorohydrin / sodium hydroxide, or acetic anhydride / sulfuric acid, or sodium hydroxide / chloroacetic acid and EDC / DMAP, the nanocellulose is fixed to the chip surface, and then chloroacetic acid / sodium hydroxide, or bromoacetic acid / sodium hydroxide, is added to react and the hydroxyl groups on the nanocellulose surface are converted into carboxyl groups; (3) Using EDC / NHS as a catalyst, the cell-binding protein was fixed to the chip surface through an amidation reaction to construct a cell-fixation chip.
[0022] Preferably, the concentration of the thiolating agent in the ethanol solution of the thiolating agent is 0.1-50 mM.
[0023] Preferably, the operation method of step (2) is as follows: when the terminal of the thiol reagent is a hydroxyl group, immersing the chip obtained in step (1) in a mixed aqueous solution of epichlorohydrin and sodium hydroxide, and leaving it to react at room temperature; after the chip is cleaned, adding it to a mixed aqueous solution of sodium hydroxide and nanocellulose, and leaving it overnight; after the chip is cleaned, immersing it in a mixed aqueous solution of sodium hydroxide and bromoacetic acid, and leaving it at room temperature overnight; Preferably, when the terminal of the thiol reagent is a carboxyl group, it is also necessary to convert the carboxyl group into a hydroxyl group by refluxing propylene glycol and sulfuric acid, and then graft the nanocellulose onto the chip surface by acetic anhydride / chloroform / pyridine; Preferably, when the terminal of the thiol reagent is an amine group, the amine group is first converted into a carboxyl group by sodium hydroxide and chloroacetic acid, and then the nanocellulose is grafted onto the chip surface under the action of EDC / DMAP.
[0024] Further preferably, in the mixed aqueous solution of epichlorohydrin and sodium hydroxide in step (2), the concentration of epichlorohydrin is 0.1 M, and the concentration of sodium hydroxide is 0.1 M.
[0025] Further preferably, in the mixed aqueous solution of sodium hydroxide and nanocellulose in step (2), the concentration of sodium hydroxide is 2M, and the mass fraction of nanocellulose is 0.1%.
[0026] Further preferably, in the mixed aqueous solution of sodium hydroxide and bromoacetic acid in step (2), the concentration of sodium hydroxide is 1-3M, and the concentration of bromoacetic acid is 1-2M.
[0027] Preferably, the operation method of step (3) is: clean the chip with water, and then immerse the chip in a mixed aqueous solution of EDC and NHS for reaction for 1 hour, wherein the concentration of EDC is 0.5M and the concentration of NHS is 0.2M.
[0028] The surface plasmon resonance chip is used for fixing SH-SY5Y cells.
[0029] Compared with the prior art, this application has the following beneficial effects: 1. Under the condition of effectively controlling non-specific adsorption on the chip surface, meet the needs of cell fixation; 2. The chip surface has good cell compatibility and can provide conditions for cell adhesion, survival, proliferation and differentiation.
[0030] 3. The fixation and survival rate of cells on the chip surface were significantly improved after modification. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 It is a schematic structural diagram of the surface plasmon resonance chip of the present invention; Figure 2 Figure 1 shows the cell fixation on the surface of the SPR gold chip (a) and the commercial dextran chip (b) in Example 1 of the present invention; Figure 3 This is a diagram showing the growth state of cells on the chip surface in Example 2 of the present invention; Figure 4 This is a diagram showing the growth state of cells on the chip surface in Example 3 of the present invention; Figure 5 is a diagram showing the growth state of cells on the chip surface in Example 4 of the present invention; Figure 6 is a diagram showing the growth state of cells on the chip surface in Example 5 of the present invention; Figure 7 This is a diagram showing the growth status of cells on the chip surface in Example 6 of the present invention. DETAILED DESCRIPTION
[0032] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.
[0033] Example 1 A method for preparing a surface plasmon resonance cell culture chip comprises the following steps: (1) Clean the SPR chip with methanol and water alternately for 3-5 times until the surface is clean, blow dry with clean air or nitrogen, and set aside.
[0034] (2) Dissolve 16-mercaptohexadecanoic acid in ethanol to prepare a 0.1 mg / mL solution, immerse the treated chip in the solution, and store it at room temperature for 96 hours.
[0035] (3) EDC and 4-dimethylaminopyridine (DMAP) were prepared into a 0.4 M:0.1 M mixed solution, and water was used as the solvent. Unless otherwise specified, all concentrations in the examples are the final concentrations in the mixed solution.
[0036] (4) Immerse the chip in the solution prepared in (3) and store it at room temperature for 30 minutes to activate the carboxyl groups on the chip surface. Then, clean it with water and methanol and set aside.
[0037] (5) Dissolve dextran in a 20 mM sodium hydroxide aqueous solution, then adjust the pH of the solution to 6.5 with high concentration of morpholineethanesulfonic acid. Place the chip in the dextran solution (0.1 g / mL), react overnight, and rinse with water.
[0038] (6) Immerse the chip in a mixture of chloroacetic acid (1.5 M) and sodium hydroxide (2.5 M), react overnight, and then rinse with water.
[0039] (7) Prepare a 0.4 M:0.1 M mixture of EDC and NHS, use water as the solvent, immerse the chip in the solution, and react at room temperature for 20 minutes.
[0040] (8) Clean the chip with water, then add laminin solution (0.01 mg / mL) and react at room temperature for 30 minutes.
[0041] (9) Clean the chip with water, sterilize it with UV light for 45 minutes, and set aside.
[0042] A culture pool was constructed on the chip surface, and the dispersed SH-SY5Y cells were cultured at a rate of 10 4 The cells were transferred to a culture tank with a volume of 100 μg / mL and cultured overnight in DMEM / F12 medium (DMEM / F12 culture medium + 10% volume of fetal bovine serum + 1% volume of penicillin / streptomycin). The original culture medium was removed and the cells were treated with polymethanol tissue fixative for 30 minutes.
[0043] Using the cell culture method in Example 1, cells were cultured on the chip obtained in this example and on the surface of a commercial dextran chip (Biosensing Instrument, CM-dextran chip). The cell culture time was 24 hours. The results were as follows: Figure 2 As shown in a and 2b, it is demonstrated that the cells can adhere and survive well.
[0044] Example 2 A method for preparing a surface plasmon resonance cell culture chip comprises the following steps: (1) Clean the SPR chip with methanol and water alternately for 3-5 times until the surface is clean, blow dry with clean air or nitrogen, and set aside.
[0045] (2) Dissolve 6-mercapto-1-hexanol in ethanol to a concentration of 20 mM, immerse the treated chip in the solution, and react overnight.
[0046] (3) After cleaning the chip with water and methanol, immerse the chip in a mixed aqueous solution of epichlorohydrin (0.1 M) and sodium hydroxide (0.1 M) at room temperature for 3 hours.
[0047] (4) After cleaning the chip with water and methanol, add a mixed solution of sodium hydroxide (2 M) and nanocellulose (0.1% by mass in water) and leave it overnight. Through the action of epichlorohydrin, the nanocellulose is combined with the hydroxyl groups on the chip surface.
[0048] (5) Clean the chip with water, then immerse the chip in a mixed aqueous solution of sodium hydroxide (3M) and bromoacetic acid (2M) and leave it at room temperature overnight.
[0049] (6) Clean the chip with water, and then immerse the chip in a mixture of EDC and NHS (0.5M:0.2M) for 1 hour.
[0050] (7) Clean the chip with water, then add nestin (0.02 mg / mL) dropwise onto the chip surface and leave at room temperature for 1 hour.
[0051] (8) Rinse the chip with water and sterilize it under UV for 45 minutes.
[0052] The cell culture method in Example 1 was used to obtain the following experimental results: Figure 3 As shown. This indicates that cells can adhere to and survive on the chip surface.
[0053] Example 3 A method for preparing a surface plasmon resonance cell culture chip comprises the following steps: (1) Clean the SPR chip with methanol and water alternately for 3-5 times until the surface is clean, blow dry with clean air or nitrogen, and set aside.
[0054] (2) Dissolve 11-mercaptoundecanoic acid in ethanol solution to prepare a 10 mM solution, immerse the chip in the solution, store it at room temperature overnight, and rinse the chip with water and methanol.
[0055] (3) Prepare a mixture of propylene glycol and sulfuric acid (1 M) (the concentration of concentrated sulfuric acid in propanol), then immerse the chip in the solution and reflux at 80°C for 48 hours to graft hydroxyl groups onto the chip surface, and then rinse with water and methanol.
[0056] (4) Dissolve acetic anhydride in chloroform, add pyridine to the chloroform solution of acetic anhydride so that the mass fraction of pyridine in the above solution is 5%, add nanocellulose, react at 30°C overnight, and rinse the chip with water.
[0057] (5) Soak the chip in bromoacetic acid and sodium hydroxide solution (0.5M / 3M), react overnight, rinse with water, and then prepare a mixture of EDC and NHS (0.25M:0.1M), add thrombospondin (0.05mg / mL), and then drop the mixture onto the chip surface to react for 1 hour, and rinse with water.
[0058] (6) Rinse the chip with water and sterilize it under UV for 45 minutes.
[0059] The cell culture method in Example 1 was used to obtain the following results: Figure 4 As shown in , it is demonstrated that cells can achieve survival and adhesion.
[0060] Example 4 A method for preparing a surface plasmon resonance cell culture chip comprises the following steps: (1) Clean the SPR chip with methanol and water alternately for 3-5 times until the surface is clean, blow dry with clean air or nitrogen, and set aside.
[0061] (2) Dissolve 6-mercaptohexylamine (5 mM) in ethanol, place the treated chip in the above solution, react at 45°C for 4 hours, and rinse with water and methanol.
[0062] (3) Prepare a mixture of sodium hydroxide (1 M) and chloroacetic acid (0.5 M), immerse the chip in the solution, react overnight, and rinse with water and methanol.
[0063] (4) Prepare a mixture of EDC and DMAP (0.5M:0.05M), immerse the chip in the solution, react for 30 minutes, and rinse with water.
[0064] (5) The chip was immersed in a nanocellulose sodium hydroxide solution (mass fraction 0.1%, sodium hydroxide concentration 50 mM) for 2 hours. After rinsing with water, the chip was immersed in a mixed solution of chloroacetic acid (1 M) and sodium hydroxide (1 M), allowed to react overnight, and then rinsed with water.
[0065] (6) Prepare a mixture of EDC and NHS (0.1M:0.2M), immerse the chip in it, react for 1 hour, and rinse with water.
[0066] (7) Add poly-lysine (0.05 mg / mL) onto the chip surface and allow to react for 1 hour. Then rinse with water and sterilize under UV for 45 minutes.
[0067] The cell culture method in Example 1 was used to obtain the following results: Figure 5 As shown, it proves that cells can survive and adhere to the chip surface.
[0068] Example 5 A method for preparing a surface plasmon resonance cell culture chip comprises the following steps: (1) The method is the same as in Example 1. After EDC and NHS treatment, membrane protein antibodies (0.01 mg / mL) are fixed on the chip surface. After reacting for 1 hour, the chip is rinsed with water and sterilized with ultraviolet light for 45 minutes. The cell culture method in Example 1 is used, and the results are as follows: Figure 6 As shown, it proves that cells can survive and adhere well on the chip surface.
[0069] Example 6 A method for preparing a surface plasmon resonance cell culture chip comprises the following steps: (1) The method is the same as in Example 7. After EDC and DMAP treatment, Her 2 protein antibody (0.05 mg / mL) is fixed on the chip surface. After reacting for 1 hour, the chip is rinsed with water and sterilized with ultraviolet light for 45 minutes. The cell culture method in Example 1 is used, and the results are as follows: Figure 7 As shown in , it is proved that the above chip can achieve cell adhesion.
Claims
1. A surface plasmon resonance chip for cell fixation, characterized in that: From bottom to top, it includes a solid support layer, a supporting layer, a stabilizing layer and a bonding layer connected in sequence; The solid support layer is an unmodified SPR gold chip; the support layer is a hydrophobic layer, one end of the support layer is fixed to the solid support layer through a thiol group, and the other end is connected to the stabilizing layer through a reaction between active groups; The stabilizing layer is composed of polysaccharide segments, and the side of the polysaccharide segments opposite to the chip surface is connected to the binding layer through the reaction between active groups; The binding layer component is selected from at least one of adhesion proteins, cell adhesion specific sites, glycoprotein recognition molecules, cell membrane surface proteins or phospholipid molecule antibodies.
2. The surface plasmon resonance chip for cell fixation according to claim 1, characterized in that: The support layer is obtained by fixing hydrophobic long-chain alkanes on the solid support layer; preferably, the polysaccharide is dextran or nanocellulose.
3. The surface plasmon resonance chip for cell fixation according to claim 1, characterized in that: The number of repeating units of the polysaccharide chain segment in the stabilizing layer is 10-400000; preferably, the binding layer protein is selected from any one of fibronectin, laminin, collagen, nidogen antibody, tenascin, thrombospondin antibody, and heparin sulfate glycoprotein antibody.
4. The method for preparing a surface plasmon resonance chip for cell fixation according to claim 2 or 3, characterized in that: When the stabilizing layer is composed of dextran, the method comprises the following steps: (1) Soaking the SPR gold chip in an ethanol solution of a thiol reagent at room temperature for 12 to 36 hours to obtain a chip modified with a thiol reagent; the thiol reagent is 6-mercapto-1-hexanol, 16-mercaptohexadecanoic acid, 11-mercaptoundecanoic acid or 6-mercaptohexylamine; (2) When the terminal of the thiol reagent is a non-carboxyl group, firstly, sodium hydroxide and bromoacetic acid or chloroacetic acid are used to convert the non-carboxyl group on the other end of the thiol reagent on the chip surface of step (1) into a carboxyl group, and then EDC / DMAP is used to fix the dextran to the SPR chip surface; when the terminal of the thiol reagent is a carboxyl group, EDC / DMAP is directly used to fix the dextran to the chip surface; (3) Soaking the chip described in step (2) in a mixed solution of sodium hydroxide and bromoacetic acid or chloroacetic acid to convert part of the hydroxyl groups on the surface of the dextran into carboxyl groups, and then cleaning the chip. The conversion ratio of the hydroxyl groups is 1-10%. (4) Use EDC / NHS to activate the carboxyl groups on the cleaned chip surface, and fix the cell-binding protein to the chip surface through amide bonds to form a binding layer, providing conditions for cell fixation.
5. The method for preparing a surface plasmon resonance chip for cell fixation according to claim 4, characterized in that: In step (1), the concentration of the thiol reagent in ethanol is 0.01-50 mg / mL; Preferably, in step (2), when the end of the thiol reagent is not a carboxyl group, firstly, the chip modified with the thiol reagent in step (1) is immersed in a mixed aqueous solution of sodium hydroxide and bromoacetic acid or chloroacetic acid to convert the non-carboxyl group at the other end of the thiol reagent on the surface of the chip into a carboxyl group; then, the chip is immersed in a mixed aqueous solution of EDC / DMAP; and then, the chip is immersed in a mixed aqueous solution of dextran and sodium hydroxide, wherein the mass fraction of dextran in the mixed aqueous solution of dextran and sodium hydroxide is 0.1-20%; Preferably, in the mixed solution of step (3), the concentration of the chloroacetic acid or bromoacetic acid solution is 1-3M, the concentration of the sodium hydroxide solution is 1-5M, and the volume ratio of the chloroacetic acid or bromoacetic acid solution to the sodium hydroxide solution is 1:1; Preferably, in step (4), a protein solution with a concentration of 0.01-0.1 mg / mL is dropped onto the surface of the chip to form a binding layer.
6. The method for preparing a surface plasmon resonance chip for cell fixation according to claim 4, characterized in that: When the stabilizing layer is composed of nanocellulose, the method comprises the following steps: (1) Soak the SPR gold chip in an ethanol solution of a thiol reagent at room temperature for 12 to 36 h; (2) Using epichlorohydrin / sodium hydroxide, or acetic anhydride / sulfuric acid, or sodium hydroxide / chloroacetic acid and EDC / DMAP, the nanocellulose is fixed to the chip surface, and then chloroacetic acid / sodium hydroxide, or bromoacetic acid / sodium hydroxide, is added to react and the hydroxyl groups on the nanocellulose surface are converted into carboxyl groups; (3) Using EDC / NHS as a catalyst, the cell-binding protein was fixed to the chip surface through an amidation reaction to construct a cell-fixation chip.
7. The method for preparing a surface plasmon resonance chip for cell fixation according to claim 6, characterized in that: The concentration of the thiolating agent in the ethanol solution of the thiolating agent in step (1) is 0.1-50 mM; Preferably, the operation method of step (2) is as follows: when the terminal of the thiol reagent is a hydroxyl group, immersing the chip obtained in step (1) in a mixed aqueous solution of epichlorohydrin and sodium hydroxide, and leaving it to react at room temperature; after the chip is cleaned, adding it to a mixed aqueous solution of sodium hydroxide and nanocellulose, and leaving it overnight; after the chip is cleaned, immersing it in a mixed aqueous solution of sodium hydroxide and bromoacetic acid, and leaving it at room temperature overnight; Preferably, when the terminal of the thiol reagent is a carboxyl group, it is also necessary to convert the carboxyl group into a hydroxyl group by refluxing propylene glycol and sulfuric acid, and then graft the nanocellulose onto the chip surface by acetic anhydride / chloroform / pyridine; Preferably, when the terminal of the thiol reagent is an amine group, the amine group is first converted into a carboxyl group by sodium hydroxide and chloroacetic acid, and then the nanocellulose is grafted onto the chip surface under the action of EDC / DMAP.
8. The method for preparing a surface plasmon resonance chip for cell fixation according to claim 7, characterized in that: In the mixed aqueous solution of epichlorohydrin and sodium hydroxide in step (2), the concentration of epichlorohydrin is 0.1 M, and the concentration of sodium hydroxide is 0.1 M; Preferably, in the mixed aqueous solution of sodium hydroxide and nanocellulose in step (2), the concentration of sodium hydroxide is 2M, and the mass fraction of nanocellulose is 0.1%; Preferably, in the mixed aqueous solution of sodium hydroxide and bromoacetic acid in step (2), the concentration of sodium hydroxide is 1-3M, and the concentration of bromoacetic acid is 1-2M.
9. The method for preparing a surface plasmon resonance chip for cell fixation according to claim 6, characterized in that: The operation method of step (3) is as follows: the chip is cleaned with water, and then the chip is immersed in a mixed aqueous solution of EDC and NHS for reaction for 1 hour, wherein the concentration of EDC is 0.5M and the concentration of NHS is 0.2M.
10. The use of the surface plasmon resonance chip for cell fixation according to claim 2 or 3, characterized in that: Used for SH-SY5Y cell fixation.