阅读说明：本技术 小鼠IgG印迹聚合物的制备方法 (Preparation method of mouse IgG imprinted polymer ) 是由 孙越 王伟 赵梦元 冯雪薇 于 2019-10-21 设计创作，主要内容包括：本发明公开一种小鼠IgG印迹聚合物的制备方法,依次按照如下步骤进行：将0.01~1mol丙烯酰胺和/或N-羟基丙烯酰胺与0.002~0.02mol甲叉双丙烯酰共同溶于20 ml pH=7的PBS溶液中,超声震荡5min,得第一混合溶液；将0.001~0.01mol含溴化合物置入第一混合溶液中；在第一混合溶液中加入0.05~1ml三乙胺、0.002~0.02g小鼠IgG和0.4~4μg荧光标记的小鼠IgG,超声震荡20min,得第二混合溶液,向第二混合溶液中通入氮气10min；将通入氮气的第二混合溶液光照6小时,得到含小鼠IgG的聚合物；洗去含小鼠IgG的聚合物中的小鼠IgG模板。(The invention discloses a preparation method of a mouse IgG imprinted polymer, which comprises the following steps of dissolving 0.01 ~ 1mol of acrylamide and/or N-hydroxyacrylamide and 0.002 ~ 0.02.02 mol of methylene diacryloyl together in 20 ml of PBS solution with the pH =7, carrying out ultrasonic oscillation for 5min to obtain a first mixed solution, putting 0.001 ~ 0.01mol of a bromine-containing compound into the first mixed solution, adding 0.05 ~ 1ml of triethylamine, 0.002 ~ 0.02.02 g of mouse IgG and 0.4 ~ 4 mu g of fluorescence-labeled mouse IgG into the first mixed solution, carrying out ultrasonic oscillation for 20min to obtain a second mixed solution, introducing nitrogen into the second mixed solution for 10min, illuminating the second mixed solution introduced with nitrogen for 6 hours to obtain a polymer containing the mouse IgG, and washing a mouse IgG template in the polymer containing the mouse IgG.)

1. A preparation method of a mouse IgG imprinted polymer is characterized by sequentially comprising the following steps:

a. dissolving 0.01 ~ 1mol of acrylamide and/or N-hydroxy acrylamide and 0.002 ~ 0.02.02 mol of methylene diacryloyl together in 20 ml of PBS solution with pH =7, and ultrasonically oscillating for 5min to obtain a first mixed solution;

b. placing 0.001 ~ 0.01.01 mol of bromine-containing compound into the first mixed solution;

c. adding 0.05 ~ 1ml of triethylamine, 0.002 ~ 0.02.02 g of mouse IgG and 0.4 ~ 4 mu g of mouse IgG which is fluorescently labeled into the first mixed solution, carrying out ultrasonic oscillation for 20min to obtain a second mixed solution, and introducing nitrogen into the second mixed solution for 10 min;

d. illuminating the second mixed solution into which the nitrogen is introduced for 6 hours to obtain a polymer containing the mouse IgG;

e. placing the polymer containing mouse IgG in CH containing SDS₃In COOH solution, -0.6V

An electric field is used for 2h, the mixture is washed by PBS and dried by nitrogen to obtain a mouse IgG imprinted polymer, and the SDS and the CH₃The mass volume ratio of COOH is 10 percent, and the CH₃COOH is 10% by volume of CH₃COOH solution.

Technical Field

The invention relates to a preparation method of a mouse IgG imprinted polymer, in particular to a preparation method of a mouse IgG imprinted polymer which does not need a transition metal catalyst and carries out photoinduced ATRP (atom transfer radical polymerization) by using mouse IgG labeled by fluorescence in a water phase.

Background

When animals eat food, the immune system produces specific antibodies IgG, which form immune complexes with food and, in severe cases, can cause inflammation of body tissues. Therefore, whether the body can produce intolerance diseases to certain foods or not can be judged by detecting the IgG, and a basis is provided for diagnosis and prevention of the diseases.

The detection method of specific antibody IgG is usually enzyme-linked immunosorbent assay (ELISA) and molecular imprinting technology. The ELISA method is used as an immunoassay technology and is mainly based on the specific recognition of an antigen and an antibody, if the antigen is detected, besides the requirement that the combined antibody and the antigen have specificity, the antigen to be detected also needs to have an antigenic determinant capable of being combined with the antibody, if certain sites are not expressed or the combining sites are blocked or blocked for certain reasons due to mutation, the combination of the antigen and the antibody is influenced, a false negative result is caused, and meanwhile, the antigen and the antibody used by the method have biological activity and difficult storage conditions. The molecular imprinting technology is a novel high-efficiency separation technology, the separation effect of the molecular imprinting technology is derived from the molecular recognition function similar to natural antibodies or enzymes of Molecular Imprinted Polymers (MIPs), and the molecular imprinting technology has the advantages of strong adaptability, high chemical stability, high tolerance to pressure and temperature and the like, and meanwhile, the MIP is simple to prepare and has good repeatability.

The core of the molecular imprinting technology is the preparation of a Molecular Imprinted Polymer (MIP), which is generally a polymer with a rigid structure formed by a functional monomer, a cross-linking agent and a specific template molecule through hydrogen bonds, ionic bonds, van der waals forces, electrostatic forces and the like under the action of a suitable initiator, and then the template molecule is removed through a certain physical or chemical method, thereby finally preparing the MIP with selective recognition capability. There are many ways MIP can be prepared. Atom Transfer Radical Polymerization (ATRP) is the leading topic in polymer science, and is a common approach for macromolecular design, which uses halide as an initiator, low-valence transition metal (such as Cu (i), Fe (ii) and the like) complexes as catalysts, and establishes dynamic balance between Polymerization active species and dormant species through redox reaction, thereby realizing accurate control of the end group, composition, structure, molecular weight and the like of the polymer. Meanwhile, ATRP can be applied to various types of monomers, and has the advantages of simple polymerization method, various implementation means and the like, so that the ATRP is particularly suitable for preparing MIP. However, when the protein MIP is prepared by the conventional ATRP method, there are problems as follows: (1) low-valence transition metal ions are sensitive to air and the like and are not easy to store; (2) the low-valence transition metal ions have certain toxicity to biological macromolecules such as protein and the like; (3) the common post-treatment process for removing the catalyst is complex, the catalyst is always remained, and the cost is increased.

The metal-free visible light-induced atom transfer radical polymerization is a new technology, can overcome the defects of the traditional ATRP, can prepare the molecularly imprinted polymer without using a transition metal complex as a catalyst, and has the advantages of high monomer polymerization speed, high conversion rate and the like.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a preparation method of a mouse IgG imprinted polymer, which does not need a transition metal catalyst and carries out photoinduced ATRP by mouse IgG labeled by fluorescence in an aqueous phase.

The technical solution of the invention is as follows: a preparation method of a mouse IgG imprinted polymer is characterized by sequentially comprising the following steps:

a. dissolving 0.01 ~ 1mol of acrylamide and/or N-hydroxy acrylamide and 0.002 ~ 0.02.02 mol of methylene diacryloyl together in 20 ml of PBS solution with pH =7, and ultrasonically oscillating for 5min to obtain a first mixed solution;

b. placing 0.001 ~ 0.01.01 mol of bromine-containing compound into the first mixed solution;

c. adding 0.05 ~ 1ml of triethylamine, 0.002 ~ 0.02.02 g of mouse IgG and 0.4 ~ 4 mu g of mouse IgG which is fluorescently labeled into the first mixed solution, carrying out ultrasonic oscillation for 20min to obtain a second mixed solution, and introducing nitrogen into the second mixed solution for 10 min;

d. illuminating the second mixed solution into which the nitrogen is introduced for 6 hours to obtain a polymer containing the mouse IgG;

e. placing the polymer containing mouse IgG in CH containing SDS₃In COOH solution, -0.6V

An electric field is used for 2h, the mixture is washed by PBS and dried by nitrogen to obtain a mouse IgG imprinted polymer, and the SDS and the CH₃The mass volume ratio of COOH is 10 percent, and the CH₃COOH is 10% by volume of CH₃COOH solution.

The invention adopts the mouse IgG marked by fluorescence to replace the traditional photocatalyst, does not need to add a transition metal catalyst additionally, solves the problems that the low-valence metal catalyst is sensitive to air and the like and is difficult to store, the catalyst has certain toxicity to protein and other biological macromolecules, the post-treatment process for removing the catalyst is complex and the like when the existing ATRP technology is applied to the preparation of mouse IgG imprinted polymers, and also solves the problems of poor safety and high cost in the polymerization in an ethanol phase. The mouse IgG imprinted polymer prepared by the invention has better identification, and can realize better detection on the mouse IgG by an electrochemical method.

Drawings

FIG. 1 shows the results of example 1 of the present invention and various electrodes containing 0.1mol/L KCl +5 mmol/L [ Fe (CN)₆]^3-/4-(0.1 mol/L pH 7.0 PBS) electrochemical AC impedance plot in electrolyte solution.

FIG. 2 shows the results of example 1 and various electrodes of the present invention in the presence of 0.1mol/L KCl +5 mmol/L [ Fe (CN)₆]^3-/4-(0.1 mol/L pH 7.0 PBS) cyclic voltammogram in electrolyte solution.

FIG. 3 is an SEM (scanning electron microscope) knot of the surface of the mouse IgG imprinted polymer modified electrode obtained in example 1 of the invention

And (5) fruit pictures.

FIG. 4 is a schematic diagram showing the selectivity of the imprinted polymer modified electrode of example 1 of the present invention to mouse IgG, human serum albumin, hemoglobin, and myoglobin.

FIG. 5 is a DPV curve diagram obtained by detecting a series of mouse IgG with different concentrations by the imprinted polymer modified electrode in example 1 of the present invention.

Detailed Description