Polymer modified avidin as well as preparation method and application thereof

Abstract

The invention relates to polymer modified avidin as well as a preparation method and application thereof. On the premise of existence of an initiator, eosin Y is used as a catalyst, pentamethyldiethylenetriamine is used as a reducing agent to carry out polymer modification on avidin, and the polymer modified avidin is obtained. Compared with the prior art, the polymer modified on the avidin is moderate in gap, not only can allow small-molecule biotin to pass through, but also can block a biotinylated substance to be detected to pass through, so that the avidin can be combined with biotin, but also cannot be combined with the biotinylated substance to be detected, and interference of free biotin in a sample to be detected is avoided. Besides, in the polymer modification process through eosin Y and pentamethyldiethylenetriamine, the reaction temperature is moderate, and no metal is left, so that the activity of avidin is not affected in the reaction process, and effective combination of free biotin is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a polymer-modified avidin protein and a preparation method and application thereof. Background Art

[0002] Biotin-Avidin-System (BAS) is a new type of biological reaction amplification system developed in the late 1970s. With the advent of various biotin derivatives, BAS was soon widely used in various fields of medicine. A large number of studies have confirmed that the biotin-avidin system can be combined with almost all successful markers currently studied. The strong binding of biotin and avidin with high affinity and the multi-stage amplification effect make BAS immunolabeling and related tracer analysis more sensitive. It has become a new technology widely used in qualitative and quantitative detection of trace antigens and antibodies and localization observation research.

[0003] Biotin, also known as vitamin H, coenzyme R, and vitamin B7, is a water-soluble vitamin belonging to the vitamin B family. It is a nutrient necessary to maintain the natural growth, development, and normal function of the human body and is considered a key contributor to keratin. In recent years, more and more people have taken exogenous biotin to protect their skin, prevent hair loss, and lose weight. Biotin can be rapidly excreted from the body, but when high doses of biotin are supplemented, biotin cannot be excreted in time, resulting in the presence of biotin in plasma, urine, and other test samples. During the immunoassay process, the analyte in the test sample will react with the biotinylated marker to form a biotin-labeled immune complex, which will then bind to avidin. At this time, the biotin in the test sample will compete with the biotin-labeled immune complex to bind to avidin, thereby interfering with the detection of the analyte.

[0004] In order to avoid the influence of exogenous biotin on the test results, avidin is often used in the prior art. This avidin can bind to biotin but not to the biotinylated analyte. The common way to use avidin to eliminate the influence of biotin is to perform polymer modification on avidin. The polymer modified on avidin can allow small molecule biotin to pass through and then bind to avidin; while the biotinylated analyte cannot pass through the polymer and then cannot bind to avidin. Therefore, when it is necessary to use a BAS system to detect a sample and the sample contains a high dose of biotin, polymer-modified avidin can be used to bind to free biotin, thereby avoiding the binding of biotin to avidin and affecting the detection effect. Atom transfer radical polymerization is a highly efficient method for synthesizing polymer modifications of specific structures and is widely used. Photo-initiated atom transfer radicals have received great attention in recent years. Among them, metal catalysts, such as copper (Cu), iridium (Ir), and iron (Fe), are often used to assist in catalyzing photo-initiated atom transfer radical polymerization. However, this method still has problems in practical application. The use of metal catalysts will inevitably lead to metal residues and contaminate the final product, which hinders the development of this technology in the fields of biomedicine and electronic material synthesis. In addition, the degree of polymer modification also determines the efficiency of avidin binding to biotin. When the degree of polymer modification is low, not only can biotin bind to avidin through the polymer voids, but biotinylated analytes can also bind to avidin through the polymer voids, thereby affecting the detection of the analytes in the sample. Therefore, there is an urgent need for a method for modifying avidin that does not affect the activity of avidin and has a moderate degree of polymer modification. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art that there is a lack of a method for modifying avidin with a moderate degree of polymer modification without affecting the activity of avidin.

[0006] In order to solve the above technical problems, the present invention provides a polymer-modified avidin protein and its preparation method and application. When the analyte is detected, the analyte in the sample is combined with the biotin labeling reagent and the luminescent substance labeling reagent in the kit to form a biotin-analyte-luminescent substance label, and the biotin-analyte-luminescent substance label will be captured by the avidin magnetic particles, so as to reflect the content of the analyte by detecting the luminescent signal of the avidin magnetic particles. Taking the sandwich method as an example, when biotin is present in the sample, the biotin in the sample will compete with the biotin-analyte-luminescent substance label to bind to the avidin magnetic particles, resulting in insufficient capture of the biotin-analyte-luminescent substance label by the avidin magnetic particles. When the light signal is detected, the biotin in the sample is captured by the avidin magnetic beads but cannot emit light because it does not carry the luminescent substance, and part of the biotin-analyte-luminescent substance label is not captured by the avidin magnetic beads and is washed away during washing, thereby causing the measurable light signal to weaken, thereby affecting the detection of the analyte. Therefore, the present invention utilizes the maleimide-protected halogenated hydrocarbon in the initiator to react with the amine group on the surface of avidin to form a covalent bond, thereby introducing a halogenated hydrocarbon group on the surface of avidin. At the same time, using Eosin Y as a catalyst and PMDETA as a reducing agent, Eosin Y reaches an excited state under illumination, and is then reduced by PMDETA to generate a relatively stable free radical anion. The free radical anion further reduces the halogenated hydrocarbon to form an initial initiating free radical, and then the vinyl monomer is polymerized by activation in the atom transfer free radical process, and finally a polymer with a controllable molecular weight is generated on the surface of avidin. Subsequently, the interference of biotin in the sample to be tested is eliminated or reduced by utilizing the small size of biotin to pass through the polymer gap and bind to avidin, while the biotin-test object-luminescent substance marker cannot pass through the polymer gap and bind to avidin.

[0007] The first object of the present invention is to provide a method for preparing a polymer-modified avidin, comprising the following steps:

[0008] S1. Mixing avidin and an initiator in the presence of an organic solvent to react to obtain an initiator-modified avidin;

[0009] S2, mixing the initiator-modified avidin with a monomer in the presence of a photocatalyst and a reducing agent, and reacting them under light conditions to obtain the polymer-modified avidin;

[0010] Wherein, the structural formula of the initiator is as shown in Formula I or Formula II:

[0011]

[0012]

[0013] The structural formula of the monomer is shown in Formula III or Formula IV:

[0014]

[0015] Wherein, n is an integer of 10-200, R is a C1-C6 alkylene group, the photocatalyst is eosin Y, and the reducing agent is pentamethyldiethylenetriamine.

[0016] Furthermore, the avidin obtained by polymer modification using eosin Y and pentamethyldiethylenetriamine in the present invention can avoid binding with biotinylated analytes while binding with biotin. In the process of immunoassay using the BAS system, the biotinylated analyte will bind with avidin and a luminescent substance, thereby detecting the concentration of the analyte by detecting the luminescent signal. In this process, the biotin in the sample will competitively bind with the biotinylated analyte to avidin, thereby affecting the detection effect. When the gap of the polymer modified on the added avidin is too large, the biotinylated analyte can bind with the avidin through the gap of the polymer, thereby failing to bind with avidin and a luminescent substance, thereby affecting the detection effect. When the gap of the polymer modified on the avidin is too small, neither biotin nor the biotinylated analyte can bind with the avidin through the gap of the polymer, and biotin still exists in the sample in a free state, thereby interfering with the detection of the biotinylated analyte. The present invention selects eosin Y and pentamethyldiethylenetriamine to perform polymer modification on avidin, and the polymer gap on the obtained avidin is moderate, which can prevent the combination of biotinylated test objects and avidin while allowing biotin to pass through.

[0017] Furthermore, the organic solvent in step S1 is 2-(4-hydroxybenzoxazole)benzoic acid.

[0018] Furthermore, the reaction temperature in step S1 is 0-10°C.

[0019] Furthermore, in step S2, a monomer is added to the initiator-modified avidin in the presence of a photocatalyst, a reducing agent and a developer, and the reaction is carried out under light conditions to obtain a polymer-modified avidin; the developer is 4-hydroxyazobenzene-2-carboxylic acid. 4-Hydroxyazobenzene-2-carboxylic acid (HABA) can protect the binding site of avidin and biotin from being modified by the initiator and the polymer, thereby ensuring that the binding ability of avidin and biotin remains constant.

[0020] Furthermore, the molar ratio of the monomer, the initiator, eosin Y and pentamethyldiethylenetriamine is (100-500):1:1:10.

[0021] Furthermore, the illumination wavelength of the illumination condition in step S2 is 500-540nm and the light intensity is 20-30mw / cm -2 Light and light intensity will also affect the activity of avidin and the polymer modification of avidin. On the one hand, the wavelength has an effect on the efficiency of polymerization grafting. The penetration depth of 500-540nm in the solution is higher than that of common ultraviolet light, which can improve the efficiency of grafting polymerization. On the other hand, light irradiation at this wavelength will not affect the activity of avidin.

[0022] Furthermore, the reaction in step S2 is carried out under an inert atmosphere, and the reaction temperature is 20-28°C.

[0023] Furthermore, the inert atmosphere includes nitrogen.

[0024] The second object of the present invention is to provide a polymer-modified avidin obtained by the above preparation method.

[0025] The third object of the present invention is to provide a method for capturing biotin from a system containing biotin and a biotinylated analyte, wherein the method comprises adding the above-mentioned polymer-modified avidin to a system containing biotin and a biotinylated analyte for reaction. In a system containing biotin and a biotinylated analyte, the polymer-modified avidin of the present invention can bind to biotin but cannot bind to the biotinylated analyte, thereby avoiding interference from free biotin in the system.

[0026] Furthermore, the biotinylated analyte includes a protein or a nucleic acid covalently linked to biotin.

[0027] Beneficial effects of the present invention:

[0028] (1) The present invention provides a polymer-modified avidin and a preparation method and application thereof. The present invention uses light-induced atom transfer radical polymerization technology to modify a polymer on the surface of avidin to form a polymer-modified avidin. The surface of avidin is modified with a polymer. When biotin and a biotinylated analyte are present in a sample to be tested, biotin can pass through the gaps in the polymer and specifically bind to the avidin, while the biotinylated analyte cannot pass through the gaps in the polymer due to its large molecular weight and thus cannot bind to the avidin, thereby achieving the capture of free biotin in the sample to be tested.

[0029] (2) The present invention achieves metal-free atom transfer radical polymerization by replacing the metal catalyst with eosin Y and pentamethyldiethylenetriamine (PMDETA), thereby avoiding metal residues in the final product. Furthermore, when the photocatalytic reaction is carried out with eosin Y and pentamethyldiethylenetriamine (PMDETA), the reaction conditions will not affect the activity of avidin, and the polymer gap modified on the surface of avidin is moderate, which can allow small molecule biotin to pass through and block the passage of biotinylated analytes. Therefore, the polymer-modified avidin of the present invention can bind to biotin while avoiding binding to the biotinylated analyte, thereby providing an effective method for removing the interference of free biotin in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0031] Figure 1 It is a reaction principle diagram of the present invention;

[0032] Figure 2 is the hydrogen spectrum nuclear magnetic spectrum of the single-head initiator of the present invention;

[0033] Figure 3 is the hydrogen spectrum nuclear magnetic spectrum of the double-head initiator of the present invention;

[0034] Figure 4 This is the effect test result of the polymer-modified avidin protein prepared in Example 5 of the present invention;

[0035] Figure 5 This is the effect test result of the polymer-modified avidin protein prepared in Example 6 of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0037] In all aspects and embodiments mentioned herein, the term "avidin" and avidin-type protein can be used interchangeably. Avidin-type protein is generally understood to be a protein having at least one binding pocket that can specifically bind to a heterocyclic structure of biotin, represented by a urea ring fused to a tetrahydrothiophene ring. By virtue of this property, avidin-type protein can bind to a biotinylated target molecule, wherein biotin is covalently bound to the molecule through the carbon atom of the carboxyl functional of the pentanoic acid side chain of biotin. Several embodiments of avidin-type protein are known in the art. More specifically, avidin-type protein can be selected from avidin, neutravidin, streptavidin, bradavidin, traptavidin, its biotin binding variants, its mixtures, its monomers, dimers, trimers, tetramers or polymers, its conjugated forms and antibodies bound to conventional biotinylated molecules of interest. It is known that in their naturally occurring forms, many avidin-type proteins (especially those that are not antibodies), in particular avidin and streptavidin, are homotetramers; that is, they are composed of four identical subunits. In embodiments of variants of monomeric avidin-type proteins, the naturally occurring form may be a di-tri- or tetra-oligomer, in which each monomer has a biotin binding pocket. In embodiments, the avidin-type protein is selected from a monomer, a homodimer, a homotrimer and a homotetramer.

[0038] Also more specifically, the avidin-type protein may be an antibody having an antigen binding pocket capable of specifically binding to the heterocyclic structure of biotin represented by a urea ring fused to a tetrahydrothiophene ring.

[0039] For purposes of this disclosure, it is understood that the terms "biotin" or "free biotin" are used interchangeably and refer to the naturally occurring compound, ie, D(+)-biotin.

[0040] Biotin (D(+)-biotin; C 10 H 16 N2O3S; MW = 244.31 g / mol; IUPAC name: 5- [(3aS, 4S, 6aR) -2-oxo-1, 3, 3a, 4, 6, 6a- hexahydrothieno [3, 4-d] imidazol-4-yl] pentanoic acid), CAS registry number 58-85-5, comprising a urea ring fused to a tetrahydrothiophene ring and a pentanoic acid substituent attached to one of the carbon atoms of the tetrahydrothiophene ring.

[0041] The appropriate chemical group that attaches biotin to a molecule of interest via the carbon atom of the carboxyl function of the pentanoic acid side chain is referred to as "biotinylation" or "conventional biotinylation". Thus, the biotin residue of the "biotinylated" molecule of interest has the ring structure (i.e., the urea ring fused to the tetrahydrothiophene ring) facing outward, while the linear portion of the biotin residue faces inward, toward the surface of the biotinylated molecule. The ring structure facing outward can be bound by avidin-type proteins. Therefore, it is important that the heterocyclic structure of biotin needs to be exposed for specific binding by avidin-type proteins.

[0042] The term "avidin / biotin binding pair" is well known to the person skilled in the art. It refers to the fact that biotin (including the biotin moiety of the biotinylated molecule) on the one hand and avidin on the other hand represent the two members of this binding pair. As mentioned above, this binding pair is notable for having one of the highest binding affinities known for non-covalent interactions.

[0043] The term "sandwich method" is a type of assay that is one of the most useful and commonly used assays. In a typical forward assay, an unlabeled antibody is immobilized on a "solid phase" and the sample to be tested is contacted with the bound molecule. After incubation for a certain period of time to form an antibody-antigen complex, a second antibody labeled with a reporter molecule that binds to the antigen and is capable of producing a detectable signal is added and incubated to form a sandwich complex of antibody-antigen-labeled second antibody antibody. Any unreacted material is washed away, and the presence of the analyte is determined by observing the signal produced by the reporter molecule. The results can be qualitative, by simply observing the visible signal, or can be quantitative by comparison with a control sample containing a known amount of analyte. There are two types of sandwich methods: double antibody sandwich method for detecting antigens and double antigen sandwich method for detecting antibodies.

[0044] The term "competitive method" is a type of assay in which an unlabeled antibody is fixed to a "solid phase" and the sample to be tested is contacted with the bound molecule. After an appropriate incubation period sufficient to allow the formation of an antibody-antigen complex, an antigen labeled with a reporter molecule that binds to the antibody and is capable of producing a detectable signal is then added and incubated to allow time for the labeled antigen to form an antibody-labeled antigen complex with the antibody that has not formed an antibody-antigen complex. Any unreacted material is washed away, and the presence of the analyte is determined by observing the decrease in the signal produced by the reporter molecule. The results can be qualitative, by simply observing the visible signal, or can be quantitative by comparison with a control sample containing a known amount of the analyte. There are many different variations of the antigen or antibody described depending on the assay.

[0045] The term "metal-free photoinitiated atom transfer radical polymerization" is a living radical polymerization method that uses an organic catalyst system in a typical polymerization process to achieve polymerization of monomers such as (meth)acrylates and styrene by free radicals under light, room temperature or even lower temperatures. The molecular weight of the resulting polymer is controllable and the molecular weight distribution index is narrow.

[0046] The term "initiator (single-head, double-head)" refers to a halogenated hydrocarbon compound that can generate primary free radicals for initiating free radical polymerization through a redox process with a catalytic system in an atom transfer radical.

[0047] The term "monomer" refers to a small molecule compound that can be activated by active species such as free radicals during the modification process, and then undergo a chain polymerization reaction to produce a polymer. Its structure contains groups that can react with active species, such as double bonds.

[0048] The term "polymer" refers to a high molecular weight compound having a long chain structure of a large number of simple repeating units linked by covalent bonds.

[0049] The term "modification" refers to the introduction of small molecules or polymers with different functions and chain lengths onto the surface of a substance through covalent bonds using polymerization methods.

[0050] The 1H NMR spectrum was tested by using a Bruker 300MHz NMR spectrometer, with the sample to be tested dissolved in CDCl3 as solvent and tetramethylsilane (TMS) as internal standard.

[0051] Example 1: Preparation of single-head initiator

[0052] Dissolve β-amino acid (8.9 g, 100 mmol) and sodium bicarbonate (21 g, 250 mmol) in 200 mL of deionized water and cool to 0°C; dissolve 2-bromoisobutyryl bromide (12.4 mL, 100 mmol) in 50 mL of dichloromethane, and then slowly drop the solution into the above solution. Stir at room temperature for 2 h. After the reaction, wash the aqueous phase with dichloromethane (100 mL × 3) and adjust pH to 2 with 1 M hydrochloric acid at 0°C. Then extract with ethyl acetate (100 mL × 6), dry with magnesium sulfate, remove the solvent, and recrystallize the product in ether / n-hexane (1 / 9 volume ratio). The obtained product (4.8 g, 20 mmol) and N-hydroxysuccinimide (2.5 g, 22 mmol) were dissolved in 200 mL of dichloromethane, cooled to 0°C, N, N'-diisopropylcarbodiimide (2.8 g, 22 mmol) was slowly added, and stirred at room temperature for 4 h. After filtering, the solvent was removed to obtain a single-head initiator crude product, which was purified by recrystallization in isopropanol.

[0053] Example 2: Preparation of double-head initiator

[0054] Dicyclohexylcarbodiimide (10.9 g, 53 mmol) was dissolved in 10 mL of dichloromethane and slowly added dropwise to a 100 mL dichloromethane solution containing 2-bromoisobutyric acid (8 g, 48 mmol) and N-hydroxysuccinimide (6.1 g, 53 mmol) at 0°C. Stir at room temperature overnight. After filtration, the filtrate was free of solvent and the resulting product was recrystallized from isopropanol. The recrystallized product was slowly added to an acetonitrile solution containing diethylenetriamine (1 g, 9.7 mmol) and triethylamine (1.4 mL, 10 mmol) at 0°C and stirred at room temperature overnight. After filtration, the solvent was dried and 50 mL of ethyl acetate was added to the resulting product. The organic phase was washed with 50 wt% sodium carbonate solution (20 mL × 3) and then washed with saturated brine (20 mL × 3). The organic phase was dried with sodium carbonate and the solvent was removed to obtain a crude product, which was purified by column chromatography using acetonitrile as the eluent. The purified product (2.2 g, 5.5 mmol) was dissolved in 50 mL of acetonitrile, succinic anhydride (600 mg, 6 mmol) and triethylamine (840 μL, 6 mmol) were added, and the mixture was stirred at room temperature overnight. After removing the solvent, 50 mL of ethyl acetate was added. The organic phase was washed with 1 M hydrochloric acid (20 mL × 3) and saturated brine (20 mL × 3), respectively. The organic phase was dried over magnesium sulfate and the solvent was removed. The obtained product (2 g, 2 mmol) was dissolved in 50 mL of acetonitrile, and di(N-succinimidyl) carbonate (1.1 g, 4.3 mmol) and triethylamine (560 μL, 4 mmol) were added and stirred at room temperature overnight. After removing the solvent, a crude product of the double-head initiator was obtained. It was purified by column chromatography, and the eluent was acetone / chloroform = 1 / 4.

[0055] Example 3: Preparation of polymer-modified avidin using a single-head initiator

[0056] (1) Preparation of avidin modified by single-head initiator: 10 mg of avidin was dissolved in 1.38 mL of 0.1 M, pH 8.0 sodium phosphate buffer solution (0.0125 mM avidin). 0.62 mL of 10 mM 2-(4-hydroxybenzoxazole)benzoic acid was added to the solution and stirred at 4°C for 10 min. Subsequently, 10.45 mg (0.03125 mmol) of single-head initiator was added to the solution and stirred at 4°C for 2 h to obtain avidin solution modified by single-head initiator. Excess small molecules in the avidin solution modified by single-head initiator were removed by dialyzing using a 15 kDa molecular weight dialysis bag. The buffer solution used during the dialysis process was 25 mM pH 8.0 sodium phosphate solution.

[0057] (2) Preparation of the catalytic system: 96.8 mg of 4-hydroxyazobenzene-2-carboxylic acid was dissolved in 39.2 mL of sodium phosphate buffer solution (0.1 mol / L) and 0.8 mL of 1 M sodium hydroxide solution was added to obtain a HABA solution. 21.59 mg of eosin Y, 125 uL of pentamethyldiethylenetriamine, and 0.6 mL of the HABA solution were added to 10 mL of water to prepare the catalytic system.

[0058] (3) Preparation of polymer-modified avidin: Add polyethylene glycol methyl acrylate (2.75 mL, 0.3 mmol) with a molecular weight of 480 to the avidin solution modified with a single-head initiator prepared in step (1) to obtain a polymer-containing avidin solution modified with a single-head initiator. Add the catalytic system prepared in step (2) to the avidin solution modified with a single-head initiator containing a polymer, and bubbling nitrogen for 30 min to deoxygenate. Place the reaction bottle under a green LED light source (wavelength of 520 nm, light intensity of 25 mw / cm -2 ) and irradiated at 10 cm, and reacted for 24 h in an inert gas atmosphere at room temperature. After the reaction, dialyzed using a 25 kDa molecular weight dialysis bag, and the buffer solution during the dialysis process was a 25 mM pH 8.0 sodium phosphate solution, and freeze-dried to obtain polymer-modified avidin.

[0059] Example 4: Preparation of polymer-modified avidin using a double-head initiator

[0060] (1) Preparation of avidin modified by a double-head initiator: 10 mg of avidin was dissolved in 1.38 mL of 0.1 M, pH 8.0 sodium phosphate buffer solution to obtain a 0.0125 mM avidin solution. 0.62 mL of 10 mM 2-(4-hydroxybenzoxazole)benzoic acid was added to the avidin solution and stirred at 4°C for 10 min. 18.7 mg (0.03125 mmol) of a double-head initiator was dissolved in 0.08 mL of DMSO to prepare a double-head initiator solution. The double-head initiator solution was added to the avidin solution containing 2-(4-hydroxybenzoxazole)benzoic acid and stirred at 4°C for 2 h to obtain a double-head initiator modified avidin solution. Excess small molecules in the double-head initiator modified avidin solution were removed by dialyzing using a 15 kDa molecular weight dialysis bag. The buffer solution used during the dialysis process was a 25 mM pH 8.0 sodium phosphate solution.

[0061] (2) Preparation of the catalytic system: 96.8 mg of 4-hydroxyazobenzene-2-carboxylic acid was dissolved in 39.2 mL of sodium phosphate buffer solution (0.1 mol / L) and 0.8 mL of 1 M sodium hydroxide solution was added to obtain a HABA solution. 21.59 mg of eosin Y, 125 uL of pentamethyldiethylenetriamine, and 0.6 mL of the HABA solution were added to 10 mL of water to prepare a catalytic system solution.

[0062] (3) Preparation of polymer-modified avidin: Add polyethylene glycol methyl acrylate (2.75 mL, 0.3 mmol) with a molecular weight of 480 to the avidin solution modified with a double-head initiator to obtain a double-head initiator-modified avidin solution containing monomers. Add the catalytic system solution prepared in step (2) to the double-head initiator-modified avidin solution containing monomers, and bubbling nitrogen for 30 min to deoxygenate. Place the reaction bottle under a green LED light source (wavelength of 520 nm, light intensity of 25 mw / cm -2 ) and irradiated at 10 cm, and reacted for 24 h in an inert gas atmosphere at room temperature. After the reaction, dialyzed using a 25 kDa molecular weight dialysis bag, and the buffer solution during the dialysis process was a 25 mM pH 8.0 sodium phosphate solution, and freeze-dried to obtain polymer-modified avidin.

[0063] Example 5: Determination of the effect of avidin modified with a single-head initiator

[0064] 1. Detection steps

[0065] (1) adding different concentrations of biotin to samples with two procalcitonin concentration levels (sample 1 having a low procalcitonin concentration and sample 2 having a high procalcitonin concentration) so that the final concentrations of biotin are 0, 100, 300, 500, and 1000 (ng / mL), respectively, to obtain samples 1 and 2 containing different biotin concentrations;

[0066] (2) adding different concentrations of the polymer-modified avidin prepared in Example 3 to the marker reagent solution of the BRAHMS procalcitonin detection kit (chemiluminescent immunoassay), wherein the marker reagent includes a biotinylated procalcitonin antibody and an acridinium-esterified procalcitonin antibody;

[0067] (3) mixing the samples 1 and 2 with different biotin concentrations with the labeling reagent to which the polymer-modified avidin protein is added in step (2), and detecting the mixture on an automatic chemiluminescence immunoassay analyzer AE180;

[0068] (4) According to the calibrator provided by the BRAHMS procalcitonin detection kit, the standard curve of procalcitonin concentration and luminescence value was fitted to calculate the procalcitonin concentration in the samples under each condition. The concentration of the sample without biotin was used as the standard to calculate the interference rate of biotin concentration on procalcitonin detection at different concentrations.

[0069] 2. Test results

[0070] The BRAHMS procalcitonin detection kit uses the sandwich method principle for detection. The procalcitonin in the sample combines with the biotinylated procalcitonin antibody and the acridinium ester (luminescent substance) procalcitonin antibody in the kit to form a biotinylated antibody-procalcitonin-acridinium ester antibody complex. The biotinylated antibody-procalcitonin-acridinium ester antibody complex will be captured by avidin magnetic particles, thereby reflecting the content of procalcitonin by detecting the luminescent signal of the avidin magnetic particles. The higher the procalcitonin concentration, the stronger the luminescent signal. When biotin is present in the sample, the biotin in the sample will compete with the biotinylated antibody-procalcitonin-acridinium ester antibody complex to bind to the avidin magnetic particles, resulting in insufficient capture of the biotinylated antibody-procalcitonin-acridinium ester antibody complex by the avidin magnetic particles. During the optical signal detection, although the biotin in the sample is captured by the avidin magnetic beads, it cannot emit light because it does not carry the luminescent substance, and part of the biotinylated antibody-procalcitonin-acridinium esterified antibody complex is not captured by the avidin magnetic beads and is washed away during the washing, resulting in a decrease in the measurable optical signal. Since the concentration of the analyte and the optical signal level are positively correlated in the sandwich method mode, the sandwich method results are falsely reduced.

[0071] According to Table 1 and Figure 4 It can be seen that when the biotin content in the sample increases, the detection result will be significantly reduced when the polymer-modified anti-biotin protein prepared in Example 3 is not added, and the interference of biotin on the result is relatively obvious (interference rate -15.8% to -93.5%). When the polymer-modified anti-biotin protein prepared in Example 3 is added, the interference of biotin is significantly reduced (when the additive is 200 mg / L, the interference rate is -5.6% to -18.5%).

[0072] Table 1 Interference rate detection results of Example 5

[0073]

[0074] Example 6: Determination of the effect of avidin modified with a double-head initiator

[0075] 1. Experimental steps

[0076] (1) adding different concentrations of biotin to samples with three progesterone concentration levels (sample 1 represents a low progesterone concentration level, sample 2 represents a medium progesterone concentration level, and sample 3 represents a high progesterone concentration level), such that the biotin concentrations in the samples are 0, 50, 100, 300, and 500 (ng / mL), respectively;

[0077] (2) adding different concentrations of the polymer-modified avidin prepared in Example 4 to the marker reagent solution of the progesterone detection kit (chemiluminescent immunoassay), wherein the marker reagent includes a biotinylated progesterone antigen and an acridinium-esterified progesterone antibody;

[0078] (3) Mixing samples 1, 2 and 3 with different biotin concentrations with the labeling reagent to which polymer-modified avidin is added in step (2), and performing experiments on an automatic chemiluminescence immunoassay analyzer AE180, while performing a control group experiment;

[0079] (4) According to the calibration material provided in the progesterone detection kit, a standard curve of progesterone concentration and luminescence value was fitted to calculate the concentration of progesterone in the samples under various conditions. The concentration of the sample without biotin was used as the standard to calculate the interference rate of progesterone detection under different biotin concentrations.

[0080] 2. Test results

[0081] The progesterone test kit uses the competitive method principle for detection. The progesterone in the sample and the biotinylated progesterone antigen in the kit compete with the acridinium ester (luminescent substance) progesterone antibody to form a progesterone (or biotinylated progesterone antigen)-acridium ester progesterone antibody complex. The higher the progesterone content in the sample, the less biotinylated progesterone antigen-acridium ester progesterone antibody complex will be formed, and the formed biotinylated progesterone antigen-acridium ester progesterone antibody complex will be captured by avidin magnetic particles, thereby reflecting the progesterone content by detecting the luminescent signal of avidin magnetic particles. The higher the progesterone concentration, the lower the luminescent signal. When biotin is present in the sample, the biotin in the sample will compete with the biotinylated progesterone antigen-acridium ester progesterone antibody complex to bind to the avidin magnetic particles, resulting in insufficient capture of the biotinylated progesterone antigen-acridium ester progesterone antibody complex by the avidin magnetic particles. During the optical signal detection, although the biotin in the sample is captured by the avidin magnetic beads, it cannot emit light because it does not carry a luminescent substance, and some biotinylated progesterone-acridinium esterified progesterone antibodies are not captured by the avidin magnetic beads and are washed away during washing, resulting in a weakening of the measurable optical signal. Since the concentration of the analyte is negatively correlated with the optical signal level in the competition method mode, the competition method result is falsely increased.

[0082] From Table 2 and Figure 5It can be seen that when the biotin content in the sample increases, the failure to add the polymer-modified avidin of Example 4 will lead to a significant increase in the test result, and the interference of biotin on the result is more obvious; when the polymer-modified avidin of Example 3 is added, the interference of biotin is significantly reduced.

[0083] Table 2 Interference rate detection results of Example 6

[0084]

[0085] Comparative Example 1: Determination of the effect of polymer-modified avidin catalyzed by metal catalyst

[0086] 1. Preparation of polymer-modified avidin catalyzed by metal catalysts

[0087] (1) Dissolve 10 mg of avidin in 1.38 mL of 0.1 M, pH 8.0 sodium phosphate buffer solution (0.0125 mM avidin). Add 0.62 mL of 10 mM 2-(4-hydroxybenzoxazole)benzoic acid to the solution and stir at 4°C for 10 min. Then add 10.45 mg (0.03125 mmol) of single-head initiator to the solution and continue stirring at 4°C for 2 h to obtain a single-head initiator-modified avidin solution. Excess small molecules in the single-head initiator-modified avidin solution are removed by dialyzing using a 15 kDa molecular weight dialysis bag. The buffer solution during the dialysis process is 25 mM pH 8.0 sodium phosphate solution.

[0088] (2) Preparation of the catalytic system: 60 μL of 0.1 mM sodium ascorbate solution and 100 μL (0.06 mmol) of 1,1,4,7,10,10-hexamethyltriethylenetetramine were added to 1.2 mL of 50 mM copper chloride solution to prepare the catalytic system.

[0089] (3) Preparation of polymer-modified avidin: Add polyethylene glycol methyl acrylate (2.75 mL, 0.3 mmol) with a molecular weight of 480 to the avidin solution modified with a single-head initiator prepared in step (1) to obtain a single-head initiator-modified avidin solution containing polyethylene glycol methyl acrylate. Add the catalytic system prepared in step (2) to the avidin solution modified with a single-head initiator containing polyethylene glycol methyl acrylate, bubbling nitrogen for 30 minutes for deoxygenation, and then reacting for 2 hours under an inert gas atmosphere at room temperature. After the reaction is completed, dialyze using a 25 kDa molecular weight dialysis bag, use a 25 mM pH 8.0 sodium phosphate solution as the buffer solution, and freeze-dry to obtain polymer-modified avidin.

[0090] 2. Detection steps

[0091] (1) adding different concentrations of biotin to samples containing procalcitonin, so that the concentrations of biotin in the samples are 500, 1200, and 1500 (ng / mL), respectively;

[0092] (2) Adding the polymer-modified avidin obtained in Example 3 and the polymer-modified avidin obtained in this comparative example to the marker reagent solution of the BRAHMS procalcitonin detection kit (chemiluminescent immunoassay) respectively;

[0093] (3) mixing and incubating the marker reagent prepared in step (2) and the sample in step (1), respectively, and performing detection on a fully automatic chemiluminescence immunoassay analyzer AE180;

[0094] (4) The interference rate at each biotin concentration was calculated by taking the luminescence value of the sample without biotin as the control, interference rate = [sample (B) / sample - 1] * 100%. The reagents were a reagent without adding polymer-modified avidin, a reagent with adding polymer-modified avidin prepared in Example 3, and a reagent with adding polymer-modified avidin prepared in this comparative example. When the biotin content in the sample increases, the smaller the interference rate, indicating that the anti-biotin interference ability of the reagent is stronger and the performance of the polymer-modified avidin is better.

[0095] The luminescence value of the sample without biotin and without adding the polymer-modified avidin reagent was used as a control to calculate the blocking rate of the sample without biotin and adding the polymer-modified avidin reagent prepared in Example 3 and this comparative example on the biotinylated marker. Blockage rate = [sample (Avi*) / sample - 1] * 100%. The smaller the blocking rate, the more effective the polymer modification method can be in blocking the binding of avidin to the biotinylated marker, and the better the performance of the polymer-modified avidin.

[0096] 3. Test results

[0097] The results of the biotin interference rate test are shown in Table 3, where "Avi* photoinitiator" represents the polymer-modified avidin prepared in Example 3; "Avi* metal catalysis" represents the polymer-modified avidin prepared in this comparative example. It can be seen that when the biotin content in the sample increases, the detection result of procalcitonin will decrease significantly, and the interference of biotin on the result is relatively obvious (interference rate -53.3% to -93.7%); when the polymer-modified avidin of Example 3 is added, the interference of biotin is significantly reduced (interference rate -3.2% to -0.7%); when the polymer-modified avidin of this comparative example is added, the interference of biotin is also reduced (interference rate -7.5% to -28.7%), but the effect of reducing the interference rate is not as good as that of Example 3.

[0098] In addition, the polymer-modified avidin prepared in Example 3 and this comparative example also has different blocking rates for biotinylated markers. In the absence of biotin interference, the luminescence value of the polymer-modified avidin of Example 3 decreases less than that of the comparative example (-6.2% vs -27.8%). The smaller the decrease in the luminescence value, the less binding of avidin to the biotinylated marker, indicating that the polymer-modified avidin of Example 3 has a stronger blocking effect on the biotinylated marker than the polymer-modified avidin of this comparative example.

[0099] In summary, the polymer-modified avidin of Example 3 can more significantly reduce the interference of biotin and more effectively block the binding of avidin to the biotinylated marker compared to the polymer-modified avidin of this comparative example. The polymer modification method of Example 3 has better performance.

[0100] Table 3 Comparison of interference rate detection results between comparative example 1 and embodiment 3

[0101]

[0102] Comparative Example 2: Determination of the effect of avidin modified with eosin Y and triethylamine

[0103] 1. Preparation of polymer-modified avidin catalyzed by eosin Y and triethylamine

[0104] (1) Preparation of avidin solution modified by single-head initiator: 10 mg of avidin was dissolved in 1.38 mL of 0.1 M, pH 8.0 sodium phosphate buffer solution (0.0125 mM avidin). 0.62 mL of 10 mM 2-(4-hydroxybenzoxazole)benzoic acid was added to the solution and stirred at 4°C for 10 min. Subsequently, 10.45 mg (0.03125 mmol) of single-head initiator was added to the solution and stirred at 4°C for 2 h to obtain avidin solution modified by single-head initiator. Excess small molecules in the avidin solution modified by single-head initiator were removed by dialyzing using a 15 kDa molecular weight dialysis bag. The buffer solution used during the dialysis process was 25 mM pH 8.0 sodium phosphate solution.

[0105] (2) Preparation of the catalyst system: 96.8 mg of 4-hydroxyazobenzene-2-carboxylic acid was dissolved in 39.2 mL of sodium phosphate buffer solution (0.1 mol / L) and 0.8 mL of 1 M sodium hydroxide solution was added to obtain a HABA solution. 21.59 mg of eosin Y, 60.6 mg of triethylamine, and 0.6 mL of the HABA solution were added to 10 mL of water to prepare a catalyst system solution.

[0106] (3) Preparation of polymer-modified avidin: Add polyethylene glycol methyl acrylate (2.75 mL, 0.3 mmol) with a molecular weight of 480 to the avidin solution modified with the single-head initiator prepared in step (1) to obtain an avidin solution containing polyethylene glycol methyl acrylate. Add the catalyst system solution prepared in step (2) to the avidin solution containing polyethylene glycol methyl acrylate, and bubbling nitrogen for 30 min to deoxygenate. Place the reaction bottle under a green LED light source (wavelength of 520 nm, light intensity of 25 mw / cm -2 ) and irradiated at 10 cm, and reacted for 24 h in an inert gas atmosphere at room temperature. After the reaction, dialyzed using a 25 kDa molecular weight dialysis bag, and the buffer solution during the dialysis process was a 25 mM pH 8.0 sodium phosphate solution, and the polymer-modified avidin was obtained after freeze drying.

[0107] 2. Detection steps

[0108] (1) adding different concentrations of biotin to samples containing procalcitonin to obtain samples with biotin concentrations of 0, 500, 1200, and 1500 (ng / mL), respectively;

[0109] (2) Adding the polymer-modified avidin of Example 3 and the polymer-modified avidin of this comparative example to the marker reagent solution of the BRAHMS procalcitonin detection kit (chemiluminescent immunoassay) respectively;

[0110] (3) respectively mixing and incubating the samples to which different concentrations of biotin have been added in step (1) and the labeling reagents to which different avidin proteins have been added in step (2), and performing detection on an automatic chemiluminescence immunoassay analyzer AE180;

[0111] (4) The interference rate at each biotin concentration was calculated by taking the luminescence value of the sample without biotin as the control, interference rate = [sample (B) / sample - 1] * 100%. The reagents were a reagent without adding polymer-modified avidin, a reagent with adding polymer-modified avidin prepared in Example 3, and a reagent with adding polymer-modified avidin prepared in this comparative example. When the biotin content in the sample increases, the smaller the interference rate, indicating that the anti-biotin interference ability of the reagent is stronger and the performance of the polymer-modified avidin is better.

[0112] The luminescence value of the sample without biotin and without adding the polymer-modified avidin reagent was used as a control to calculate the blocking rate of the sample without biotin and adding the polymer-modified avidin reagent prepared in Example 3 and this comparative example on the biotinylated marker. Blockage rate = [sample (Avi*) / sample - 1] * 100%. The smaller the blocking rate, the more effective the polymer modification method can be in blocking the binding of avidin to the biotinylated marker, and the better the performance of the polymer-modified avidin.

[0113] 3. Test results

[0114] For the polymer-modified avidin of this comparative example, since it uses eosin Y and triethylamine for polymer-modified avidin, the initiation efficiency is low, which leads to insufficient polymer modification. Both biotin and biotinylated procalcitonin can pass through the gaps formed by the polymer. Not only biotin can bind to avidin, but biotinylated procalcitonin can also bind to avidin, thereby affecting the detection of procalcitonin.

[0115] The results of the biotin interference rate test are shown in Table 4, where "Avi*eosin Y and pentamethyldiethylenetriamine" represent the polymer-modified avidin prepared in Example 3; "Avi*eosin Y and triethylamine" represent the polymer-modified avidin prepared in this comparative example. It can be seen that when the biotin content in the sample increases, the detection result of procalcitonin will decrease significantly, and the interference of biotin on the result is relatively obvious (interference rate -60.2% to -95.1%); when the polymer-modified avidin of Example 3 is added, the interference of biotin is significantly reduced (interference rate -12.0% to -17.2%); when the polymer-modified avidin of this comparative example is added, the interference of biotin is also reduced (interference rate -22.0% to -56.9%), but the effect of reducing the interference rate is not as good as that of Example 3.

[0116] In addition, the polymer-modified avidin prepared in Example 3 and this comparative example also has different blocking rates for biotinylated markers. In the absence of biotin interference, the luminescence value of the polymer-modified avidin of Example 3 decreases less than that of the comparative example (-2.7% vs -97.6%). The smaller the decrease in the luminescence value, the less binding of avidin to the biotinylated marker, indicating that the polymer-modified avidin of Example 3 has a stronger blocking effect on the biotinylated marker than the polymer-modified avidin of this comparative example.

[0117] In summary, the polymer-modified avidin of Example 3 can more significantly reduce the interference of biotin and more effectively block the binding of avidin to the biotinylated marker compared to the polymer-modified avidin of this comparative example. The polymer modification method of Example 3 has better performance.

[0118] Table 4 Comparison of interference rate detection results between comparative example 2 and embodiment 3

[0119]

[0120]

[0121] Comparative Example 3: Determination of the effect of avidin modified by catalysis of tris(phenylpyridine)iridium complex and pentamethyldiethylenetriamine

[0122] 1. Preparation of polymer-modified avidin catalyzed by tris(phenylpyridine)iridium complex and pentamethyldiethylenetriamine

[0123] (1) Preparation of avidin modified by single-head initiator: 10 mg of avidin was dissolved in 1.38 mL of 0.1 M, pH 8.0 sodium phosphate buffer solution (0.0125 mM avidin). 0.62 mL of 10 mM 2-(4-hydroxybenzoxazole)benzoic acid was added to the solution and stirred at 4°C for 10 min. Subsequently, 10.45 mg (0.03125 mmol) of single-head initiator was added to the solution and stirred at 4°C for 2 h to obtain avidin solution modified by single-head initiator. Excess small molecules in the avidin solution modified by single-head initiator were removed by dialyzing using a 15 kDa molecular weight dialysis bag. The buffer solution used during the dialysis process was 25 mM pH 8.0 sodium phosphate solution.

[0124] (2) Preparation of the catalytic system: 96.8 mg of 4-hydroxyazobenzene-2-carboxylic acid was dissolved in 39.2 mL of sodium phosphate buffer solution (0.1 mol / L) and 0.8 mL of 1 M sodium hydroxide solution was added to obtain a HABA solution. 20.43 mg of tris(phenylpyridine)iridium complex, 125 uL of pentamethyldiethylenetriamine, and 0.6 mL of HABA solution were added to 10 mL of water to prepare the catalytic system.

[0125] (3) Preparation of polymer-modified avidin: Next, polyethylene glycol methyl acrylate (2.75 mL, 0.3 mmol) with a molecular weight of 480 was added to the solution. The catalyst solution was added to the avidin solution, nitrogen was bubbled in for 30 min for deoxygenation, and the reaction bottle was placed under a blue LED light source (wavelength 460 nm, light intensity 30 mw / cm -2) and irradiated at 10 cm, and reacted for 24 h at room temperature in an inert gas atmosphere. After the reaction, dialyzed using a 25 kDa molecular weight dialysis bag, using a 25 mM pH 8.0 sodium phosphate solution as the buffer solution, and freeze-dried to obtain polymer-modified avidin.

[0126] 2. Detection steps

[0127] (1) adding different concentrations of biotin to samples containing procalcitonin to obtain samples containing biotin at concentrations of 0, 500, 1200, and 1500 (ng / mL), respectively;

[0128] (2) Adding the polymer-modified avidin of Example 3 and the polymer-modified avidin of this comparative example to the marker reagent solution of the BRAHMS procalcitonin detection kit (chemiluminescent immunoassay) respectively;

[0129] (3) respectively mixing and incubating the samples to which different concentrations of biotin have been added in step (1) and the labeling reagents to which different avidin proteins have been added in step (2), and performing detection on an automatic chemiluminescence immunoassay analyzer AE180;

[0130] (4) The luminescence value of the sample without biotin concentration is used as the control group, and the interference rate at each biotin concentration is calculated. Interference rate = ([sample (B) / sample-1)]*100%. The reagents are respectively a reagent without adding polymer-modified avidin, a reagent with adding polymer-modified avidin prepared in Example 3, and a reagent with adding polymer-modified avidin prepared in this comparative example. When the biotin content in the sample increases, the smaller the interference rate, indicating that the anti-biotin interference ability of the reagent is stronger, and the performance of the polymer-modified avidin product is better.

[0131] The luminescence value of the sample without biotin and without adding the polymer-modified avidin reagent was used as a control to calculate the blocking rate of the sample without biotin and adding the polymer-modified avidin reagent prepared in Example 3 and this comparative example on the biotinylated marker. Blockage rate = ([sample (Avi*) / sample-1)]*100%. The smaller the blocking rate, the more effective the polymer-modified product can be in blocking the binding of avidin to the biotinylated marker, and the better the performance of the polymer-modified avidin product.

[0132] 3. Test results

[0133] When the polymer-modified avidin of Example 3 is added, it can resist the interference of 1500 ng / mL of biotin (the working concentration of avidin is 2 mg / L, and the interference rate is -39.2% when the biotin concentration is 1500 ng / mL); when the polymer-modified avidin of this comparative example is increased to 2 mg / L, although it can resist the interference of 1500 ng / mL of biotin (109.0%), it still has a great impact on the luminescence value of the kit (-98.1%); when applied to the kit, if the concentration of avidin is low, it cannot resist the interference of 1500 ng / mL of biotin, and when the avidin is increased, the luminescence value of the kit is significantly reduced, resulting in insufficient sensitivity, so it is impossible to select a suitable concentration of avidin, indicating that this solution is not feasible. In this comparative example, the polymer-modified avidin obtained by using the catalyst iridium complex and PMDETA is insufficiently modified with the polymer, so that not only biotin can bind to the avidin, but also the biotinylated analyte can bind to the avidin, thereby affecting the detection of the analyte.

[0134] The results of the biotin interference rate test are shown in Table 5, where "Avi*eosin Y and pentamethyldiethylenetriamine" represent the polymer-modified avidin prepared in Example 3; "Avi*tri(phenylpyridine)iridium complex and pentamethyldiethylenetriamine" represent the polymer-modified avidin prepared in this comparative example. It can be seen that when the biotin content in the sample increases, the detection result of procalcitonin will decrease significantly, and the interference of biotin on the result is relatively obvious (interference rate -60.2% to -95.1%); when the polymer-modified avidin of Example 3 is added, the interference of biotin is significantly reduced (interference rate -12.0% to -17.2%); when the polymer-modified avidin of this comparative example is added, the interference of biotin is also reduced (interference rate -25.3% to -65.1%), but the effect of reducing the interference rate is not as good as that of Example 3.

[0135] In addition, the polymer-modified avidin prepared in Example 3 and this comparative example also has different blocking rates for biotinylated markers. In the absence of biotin interference, the luminescence value of the polymer-modified avidin of Example 3 decreases less than that of the comparative example (-2.7% vs -98.1%). The smaller the decrease in the luminescence value, the less binding of avidin to the biotinylated marker, indicating that the polymer-modified avidin of Example 3 has a stronger blocking effect on the biotinylated marker than the polymer-modified avidin of this comparative example.

[0136] In summary, the polymer-modified avidin of Example 3 can more significantly reduce the interference of biotin and more effectively block the binding of avidin to the biotinylated marker compared to the polymer-modified avidin of this comparative example. The polymer modification method of Example 3 has better performance.

[0137] Table 5 Comparison of interference rate detection results between comparative example 3 and embodiment 3

[0138]

[0139] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing a polymer-modified avidin, characterized in that: The following steps are involved: S1. Mixing avidin and an initiator in the presence of an organic solvent to react to obtain an initiator-modified avidin; S2, mixing the initiator-modified avidin with a monomer in the presence of a photocatalyst and a reducing agent, and reacting them under light conditions to obtain the polymer-modified avidin; Wherein, the structural formula of the initiator is as shown in Formula I or Formula II: The structural formula of the monomer is shown in Formula III or Formula IV: Wherein, n is an integer of 10-200, R is a C1-C6 alkylene group, the photocatalyst is eosin Y, and the reducing agent is pentamethyldiethylenetriamine.

2. The preparation method according to claim 1, characterized in that: The organic solvent in step S1 is 2-(4-hydroxybenzoxazole)benzoic acid.

3. The preparation method according to claim 1, characterized in that: The reaction temperature in step S1 is 0-10°C.

4. The preparation method according to claim 1, characterized in that: The molar ratio of the monomer, the initiator, eosin Y and pentamethyldiethylenetriamine is (100-500):1:1:

10.

5. The preparation method according to claim 1, characterized in that: The illumination conditions in step S2 are as follows: the illumination wavelength is 500-540nm, and the illumination intensity is 20-30mw / cm -2 .

6. The preparation method according to claim 1, characterized in that: The reaction in step S2 is carried out under an inert atmosphere, and the reaction temperature is 20-28°C.

7. The preparation method according to claim 1, characterized in that: In step S2, a monomer is added to the initiator-modified avidin in the presence of a photocatalyst, a reducing agent and a developer, and the reaction is carried out under light conditions to obtain a polymer-modified avidin; wherein the developer includes 4-hydroxyazobenzene-2-carboxylic acid.

8. The polymer-modified avidin obtained by the preparation method according to any one of claims 1 to 7.

9. A method for capturing biotin from a system containing biotin and a biotinylated analyte, characterized in that: The method comprises adding the polymer-modified avidin according to claim 8 to a system containing biotin and a biotinylated analyte for reaction.

10. The method according to claim 9, characterized in that The biotinylated analyte is a protein or nucleic acid covalently linked to biotin.

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