CN109517170B

CN109517170B - Bovine serum albumin magnetic bionic immunoassay kit and application thereof

Info

Publication number: CN109517170B
Application number: CN201811296410.XA
Authority: CN
Inventors: 傅强; 陈国宁; 郭鹏琦; 舒花; 王燕; 王璐; 常春
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2018-11-01
Filing date: 2018-11-01
Publication date: 2020-06-19
Anticipated expiration: 2038-11-01
Also published as: CN109517170A

Abstract

The invention discloses a bovine serum albumin magnetic bionic immunoassay kit and application thereof, wherein the kit comprises a BSA magnetic molecularly imprinted polymer, Biotin-BSA, a BSA standard solution and HRP-SA. The BSA magnetic molecularly imprinted polymer is a bionic antibody which is synthesized by a surface polymerization method and has a function of identifying Bovine Serum Albumin (BSA), and is specifically obtained by the steps of preparing magnetic nano microspheres, coating a silicon dioxide layer, polymerizing dopamine to form a molecularly imprinted polymer layer, removing template molecules combined on the molecularly imprinted polymer and the like. The kit can be used for analyzing and determining the content of bovine serum albumin, and has the advantages of high accuracy, good stability, strong adaptability, convenient operation and low cost.

Description

Bovine serum albumin magnetic bionic immunoassay kit and application thereof

Technical Field

The invention belongs to the field of immunoadsorption analysis and detection based on a biotin-avidin system, and particularly relates to a Bovine Serum Albumin (BSA) magnetic bionic immunoassay kit.

Background

The immunoassay is a high-selectivity analysis method established by utilizing the characteristic of specific binding between a substance to be detected and a corresponding antibody, has the advantages of simple and convenient operation, sensitivity, rapidness, high-throughput measurement and the like, and can be widely applied to actual analysis and detection work. Traditional immunoassay is based on biological antibody as a recognition element, and the preparation process of the biological antibody is complicated, long in periodicity and poor in tolerance to external environment. The molecular imprinting technology can prepare the molecular imprinting polymer with high selective recognition effect on target molecules, and compared with biological antibodies, the molecular imprinting polymer has the advantages of simple and convenient preparation process, good stability, strong extreme environment resistance, low cost and the like, so that the molecular imprinting polymer serving as a bionic antibody has certain advantages when being used for recognizing and detecting substances to be detected.

Enzyme-linked immunoassays are the analytical methods commonly used in immunoassays, and enzymes generally used in immunoassays can only be used in aqueous phase. Most of the molecularly imprinted polymers are prepared in an organic phase by adopting common methods such as a bulk method, an in-situ method, a precipitation method and the like, and the obtained molecularly imprinted polymers are usually required to be identified in the organic phase, so that the application of the molecularly imprinted polymers to immunoassay instead of biological antibodies is limited.

At present, a BSA (bovine serum albumin) molecularly imprinted polymer functional material prepared by using an aqueous phase medium is not seen, and the BSA molecularly imprinted polymer functional material is used as a bionic antibody and is applied to an immunoadsorption test or detection report.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a bovine serum albumin magnetic bionic immunoassay kit and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the bionic antibody comprises a bovine serum albumin magnetic molecularly imprinted polymer microsphere, wherein the molecularly imprinted polymer microsphere comprises a magnetic nano microsphere (inner core), a silicon dioxide interface layer coated on the surface of the magnetic nano microsphere and a bovine serum albumin molecularly imprinted polymer layer coated on the silicon dioxide interface layer.

Preferably, the thickness of the bovine serum albumin molecularly imprinted polymer layer is 20-40 nm, and the particle size of the magnetic nano-microsphere is 300-500 nm. The invention considers the influence of the thickness of the molecularly imprinted polymer layer on the nuclear magnetism and also considers the recognition capability of BSA, so that the molecularly imprinted polymer microspheres can be rapidly enriched (convenient for fixed use) and separated under the action of an external magnetic field and ensure the effective recognition of BSA.

Preferably, the biomimetic antibody further comprises a functional protein (the source of the functional protein may be gelatin) for blocking a non-specific binding site of bovine serum albumin on the bovine serum albumin molecularly imprinted polymer layer.

Preferably, the bovine serum albumin molecularly imprinted polymer layer is formed by using Bovine Serum Albumin (BSA) as a template molecule, performing polymerization reaction on Dopamine (DA) at an aqueous phase-silica interface, and removing the template molecule after polymerization.

Preferably, the aqueous phase-silica interface is obtained by dispersing the magnetic nanospheres coated with silica in a basic buffer solution (pH 8.5 or less, such as PBS buffer, hydrochloric acid-tris buffer, etc.). Through the control of pH, the effective recognition of the molecular imprinting polymer microspheres on BSA is ensured.

The preparation method of the bovine serum albumin magnetic molecularly imprinted polymer bionic antibody comprises the following steps:

1) preparing magnetic nano microspheres;

2) coating silicon dioxide (SiO) on the surface of the magnetic nano microsphere₂) To obtain SiO₂Magnetic nanoparticle composite (Fe)₃O₄-SiO₂)；

3) Polymerizing monomer, bovine serum albumin as template molecule and SiO dispersed in solvent₂Mixing the magnetic nano microsphere compound to obtain an aqueous phase reaction system, and performing prepolymerization on the aqueous phase reaction system and further performing polymerization reaction on the SiO by adding a catalyst₂Coating the surface of the magnetic nano microsphere compound with a molecularly imprinted polymer combined with template molecules to obtain SiO₂Magnetic molecularly imprinted nanospheres;

4) removal of SiO₂And (3) template molecules combined on the molecularly imprinted polymer on the surface of the magnetic molecularly imprinted nanospheres to obtain the bovine serum albumin magnetic molecularly imprinted polymer microspheres.

The preparation method also comprises the following steps:

5) blocking the non-specific adsorption residue of the bovine serum albumin on the surface of the magnetic molecularly imprinted polymer microsphere of the bovine serum albumin.

Preferably, in the step 1), the magnetic nano-microspheres are prepared by a hot solvent method; in the step 2), SiO₂The magnetic nano-microsphere composite is prepared by a sol-gel method. By preparing SiO coated on the surface of the magnetic nano microsphere₂And a layer which serves as a binding interface of the molecularly imprinted polymer and protects the core during elution of the template molecules.

Preferably, in the step 3), the polymerized monomer includes a functional monomer and a crosslinking monomer, both of which are dopamine, the catalyst is a persulfate oxidant (e.g., ammonium persulfate), the solvent is a more basic buffer solution with a pH of 8.5 or less, e.g., a PBS buffer solution (phosphate buffer solution) with a pH of 7.2 to 7.4, the mass ratio of the template molecule to the polymerized monomer is 1:4 to 1:12, and SiO is in a range of 1:4 to 1:12₂The mass ratio of the magnetic nano microsphere composite to the polymerized monomer is 1: 0.5-1: 1.5.

Preferably, in the step 4), an elution solvent is used for removing the template molecules, wherein the elution solvent is a methanol-hydrochloric acid solution mixture, the mixing volume ratio of methanol to hydrochloric acid is 4: 1-9: 1, and the concentration of hydrochloric acid is 1 mol/L.

Preferably, in the step 5), the non-specific adsorption residue is blocked by using gelatin.

Preferably, the preparation of the bovine serum albumin magnetic molecularly imprinted polymer biomimetic antibody specifically comprises the following steps: 50-200 mg of SiO₂Uniformly dispersing a magnetic nano microsphere compound in 20-50 mL of PBS buffer solution with the pH value of 7.2-7.4 to obtain microsphere aqueous phase dispersion liquid, adding 50-200 mg of dopamine into the microsphere aqueous phase dispersion liquid, stirring at 10-40 ℃ for 1-3 h, then adding 10-40 mg of bovine serum albumin into the microsphere aqueous phase dispersion liquid, continuously stirring for 1-3 h to obtain prepolymerization dispersion liquid, adding 5-20 mg of ammonium persulfate into the prepolymerization dispersion liquid, stirring at 10-40 ℃ for 6-24 h, and then separating to obtain SiO₂Magnetic molecular engram nano microsphere, washing SiO with template molecule eluting solvent₂Magnetic molecular imprinting nano-microspheres, washing to remove residual eluting solvent, and drying at 40-60 ℃ to obtain microspherical cattleSerum albumin magnetic molecularly imprinted polymer (namely bovine serum albumin magnetic molecularly imprinted polymer microsphere, Fe₃O₄-SiO₂MIPs) and sealing the non-specific adsorption residues of the bovine serum albumin on the surface of the polymer by using 1-5 mg/mL gelatin water solution (water bath at 20-40 ℃ for 30-60 min).

The kit comprises a bionic antibody with a recognition function for Bovine Serum Albumin (BSA), and a detection marker which can be competitively combined with the bionic antibody in a water phase, wherein the bionic antibody comprises the above-mentioned BSA magnetic molecularly imprinted polymer microspheres, and the kit can be applied to analysis of BSA in samples such as Bovine Serum Albumin (BSA) by sealing the BSA non-specific binding sites on the surfaces of the BSA magnetic molecularly imprinted polymer microspheres.

Preferably, the detection marker is Biotin-labeled bovine serum albumin (Biotin-BSA, available by home-made or purchased), and the kit further comprises horseradish peroxidase-labeled streptavidin (HRP-SA, shanghai Bintian biotechnology limited, with an initial concentration of 1mg/mL), a bovine serum albumin standard (solution), a magnet, a disposable EP tube, a color developing solution, a sample diluent, a washing solution and a stop solution.

Preferably, the color developing solution comprises a color developing substrate solution A and a color developing substrate solution B, wherein the color developing substrate solution A is a solution containing hydrogen peroxide, and the color developing substrate solution B is a solution containing Tetramethylbenzidine (TMB); the stop solution is a 2mol/L sulfuric acid solution; the washing solution is PBS buffer solution (pH is 7.2-7.4) containing Tween-20; the sample diluent is PBS buffer solution (pH 7.2-7.4).

The invention has the beneficial effects that:

the bionic antibody recognition element adopted by the kit is a magnetic molecularly imprinted polymer, and compared with a biological antibody, the kit has the characteristics of good stability, strong adaptability, good extreme resistance, simple and convenient preparation and separation processes, easiness in fixation, low cost and the like, is favorable for reducing the preparation cost of the immunoadsorption analysis kit, and is favorable for storage and transportation of the kit. The kit can be used for analyzing and determining the content of bovine serum albumin, and has the advantages of high accuracy, good stability, strong adaptability, convenient operation and low cost.

Furthermore, the invention takes dopamine as a functional monomer and a cross-linking agent, and adopts a dopamine auto-polymerization method to synthesize the molecular imprinting bionic antibody with the recognition function to BSA in an aqueous phase environment, thereby not only simplifying the preparation process of the molecular imprinting polymer, but also specifically recognizing and combining target molecules in the aqueous phase environment by the prepared BSA magnetic molecular imprinting polymer, and replacing BSA biological antibodies.

Drawings

FIG. 1 is a schematic diagram of a process for preparing a BSA magnetic molecularly imprinted polymer biomimetic antibody and performing immunoassay detection.

FIG. 2 is Fe₃O₄Magnetic nano-microsphere, Fe₃O₄-SiO₂Scanning (a, b) and transmission (c, d) electron micrographs of MIPs.

FIG. 3 is Fe₃O₄Magnetic nano-microsphere (a), Fe₃O₄-SiO₂(b) And Fe₃O₄-SiO₂XPS characterization results of MIPs (c).

FIG. 4 is Fe₃O₄Magnetic nano-microsphere (a), Fe₃O₄-SiO₂(b) And Fe₃O₄-SiO₂Hysteresis loop diagram of mips (c).

FIG. 5 is Fe₃O₄Magnetic nano-microsphere (a), Fe₃O₄-SiO₂(b) And Fe₃O₄-SiO₂-X-ray diffraction pattern of mips (c).

FIG. 6 is Fe₃O₄-SiO₂MIPs and Fe₃O₄-SiO₂Graphs of the results of the adsorption of NIPs to Biotin-BSA after blocking.

FIG. 7 is Fe₃O₄-SiO₂Graph of MIPs versus Biotin-BSA adsorption.

FIG. 8 is a graph showing the binding of HRP-SA to Biotin-BSA.

FIG. 9 shows the specific selectivity of the biomimetic immunoassay kit for BSA and other proteins, wherein 1 is bovine serum albumin, 2 is pepsin, 3 is lysozyme, 4 is β -galactosidase, 5 is β -glucuronidase, and 6 is bromelain.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Synthesis of Biotin-labeled BSA (Biotin-BSA)

BSA was dissolved in a carbonate buffer solution of pH 9.2 in advance to a concentration of 1mg mL^-1(ii) a Biotin-N-succinimidyl ester was dissolved in DMF to give a concentration of 5mg mL^-1(ii) a Uniformly mixing Biotin-N-succinimidyl ester and BSA (bovine serum albumin) according to the mass ratio of 1:7, stirring at room temperature for 4 hours to obtain a connecting product (containing Biotin-BSA), putting the connecting product into a dialysis bag, dialyzing with PBS (pH 7.2-7.4) at 2-8 ℃ for 24 hours, changing the solution for 3-4 times during the dialysis, adding glycerol with the same volume after dialysis, and subpackaging at-20 ℃ for later use.

(II) BSA magnetic molecularly imprinted polymer (Fe)₃O₄-SiO₂Preparation of MIPs)

1)Fe₃O₄Preparation of magnetic Nano-microsphere (Hot solvent method)

Weighing FeCl₃·6H₂O5.328 g was dissolved in 160mL of ethylene glycol with stirring to give a dark brown solution. 5.328g of polyethylene glycol (specifically PEG2000) is added into the solution, and after stirring and dissolving at 60 ℃, 14.4g of sodium acetate is added, and stirring is continued for 1 hour, so as to obtain a mixed solution. Evenly subpackaging the mixed solution into two reaction kettles with the volume of 200mL, reacting for 12h at 200 ℃ in a vacuum drying oven, cooling to room temperature, collecting the product with a magnet, washing with absolute ethyl alcohol for 4-6 times, and vacuum drying to obtain Fe₃O₄The diameter of the magnetic nano microsphere is about 400 nm.

2)SiO₂Magnetic nano microsphere (Fe for short)₃O₄-SiO₂) Preparation of (sol-gel method)

600mg of Fe are weighed₃O₄Magnetic nano-microsphere is added into 88mL ethanol-water (10:1, v/v) mixed solution for ultrasonic treatmentDispersing the mixture. Then, 10mL of aqueous ammonia (25 wt%) and 4mL of Tetraethylorthosilicate (TEOS) were added, respectively, and the reaction was mechanically stirred at room temperature for 10 hours. Collecting the product with magnet, washing the product with water and ethanol in sequence, and vacuum drying to obtain Fe₃O₄-SiO₂。

3) Preparation of BSA magnetic molecularly imprinted polymer

Weighing 100mg SiO₂The magnetic nano-microspheres are uniformly dispersed in 30mL of PBS buffer (pH 7.4) by ultrasonic, 120mg of dopamine is added into the obtained dispersion system, the mixture is stirred for 1 hour at room temperature, 20mg of BSA is added, and the reaction is continued for 2 hours; thereafter, 10mg of ammonium persulfate was added, the reaction was stirred at room temperature for 12 hours, and the product was collected with a magnet. Methanol-1 mol L is adopted^-1Eluting (eluting) template molecules (24h) by hydrochloric acid solution (4:1, v/v), then washing the template molecules to be neutral by water and methanol in sequence, and drying the template molecules in vacuum at 40 ℃ to obtain Fe₃O₄-SiO₂-MIPs。

Preparation of Fe₃O₄-SiO₂-NIPs: except that no template molecules BSA, Fe₃O₄-SiO₂Method for preparing-NIPs and Fe as above₃O₄-SiO₂The preparation method of the MIPs is the same.

Referring to FIG. 2, it can be seen from FIG. 2 that Fe is prepared after surface modification (i.e., coating silica) and polymerization₃O₄-SiO₂MIPs still remain spherical, around 400nm in diameter. Fe can be seen from FIG. 2(d)₃O₄-SiO₂MIPs surface form a uniform polymeric layer around about 30nm, indicating successful encapsulation by Molecularly Imprinted Polymers (MIPs).

Referring to FIG. 3, XPS was used for Fe₃O₄Magnetic nano-microsphere, Fe₃O₄-SiO₂And Fe₃O₄-SiO₂Analysis of the surface elemental composition of MIPs, results show: fe₃O₄The surface of the magnetic nano microsphere has C, O and the absorption peak of Fe element (figure 3a), Fe₃O₄-SiO₂The surface had C, O and the absorption peak of Si element (FIG. 3b), together with Fe₃O₄Surface element composition ratio of magnetic nano microsphere, Fe₃O₄-SiO₂The surface has no Fe element absorption peak, and a stronger Si element absorption peak appears, which shows that SiO₂Successfully coated on Fe₃O₄Magnetic nano microsphere surface. Fe₃O₄-SiO₂MIPs surface with C, O, N and absorption peak of trace Si element, with Fe₃O₄Magnetic nanospheres and Fe₃O₄-SiO₂Surface elemental composition ratio, Fe₃O₄-SiO₂The MIPs have no Fe element absorption peak on the surface, the intensity of the Si element absorption peak is greatly reduced, an N element absorption peak appears, and the N element absorption peak can be attributed to the N element absorption peak on dopamine, so that the dopamine polymer is successfully coated on Fe₃O₄-SiO₂The surface, namely a molecularly imprinted polymer layer prepared by self polymerization of dopamine is successfully coated on Fe₃O₄Magnetic nano microsphere surface.

See FIG. 4, from Fe₃O₄Magnetic nanospheres (FIG. 4a), Fe₃O₄-SiO₂(FIG. 4b) and Fe₃O₄-SiO₂As can be seen in the hysteresis loop of MIPs (FIG. 4c), all three substances have superparamagnetism and strong magnetic field inductivity, indicating Fe₃O₄Surface-coated SiO₂The layer and the polymer layer do not affect Fe₃O₄Superparamagnetism of the magnetic nano-microsphere, thereby ensuring the prepared Fe₃O₄-SiO₂MIPs are capable of rapid separation under the action of an applied magnetic field and can be immobilized on a carrier surface and applied to immunoassays.

See FIG. 5, made of Fe₃O₄As can be seen from the XRD pattern of the magnetic nanosphere (fig. 5a), Fe appears at 2 θ ═ 30.08 °, 35.47 °, 43.05 °, 53.38 °, 56.83 ° and 62.43 °₃O₄Characteristic diffraction peaks of the crystal structure, which correspond one-to-one to Fe₃O₄The cubic crystal plane of (a): (220) (311), (400), (422), (511) and (440). Thus, the prepared magnetic MIPs microspheres (Fe) are proved₃O₄-SiO₂MIPs) core of Fe₃O₄，Fe₃O₄-SiO₂(FIG. 5b), Fe₃O₄-SiO₂XRD pattern of MIPs (FIG. 5c) with Fe₃O₄Comparison of XRD patterns of magnetic nanospheres (FIG. 5a) can be seen at Fe₃O₄SiO is wrapped on the surface of the magnetic nano microsphere layer by layer₂And after MIPs, the XRD spectrogram still contains Fe₃O₄Characteristic diffraction peaks in the crystal structure. Thus illustrating Fe₃O₄The crystal structure of the microsphere wraps SiO layer by layer₂And MIPs functional shells (polymeric layers) are unchanged, except that as the thickness of the wrapping layer is increased, the intensity of the diffraction peak is relatively weakened, and the peak width is slightly widened.

(III) evaluation of imprinting Effect of BSA molecularly imprinted polymer

See FIG. 6, from Fe₃O₄-SiO₂MIPs and Fe₃O₄-SiO₂The chromogenic effect of NIPs (non-imprinted polymers) on Biotin-BSA after adsorption was observed, Fe₃O₄-SiO₂The adsorption capacity of MIPs to Biotin-BSA is obviously stronger than that of Fe₃O₄-SiO₂-adsorption capacity of NIPs to Biotin-BSA, indicating that the molecularly imprinted polymer (Fe) prepared by the invention₃O₄-SiO₂MIPs) has better imprinting effect. The molecular imprinting polymer layer is provided with three-dimensional holes which are highly matched with the template molecules in the aspects of size, space structure, action sites and the like due to the addition of the template molecules.

(IV) preparation of molecular imprinting bionic immunoassay kit

1. The molecular imprinting bionic immunoassay kit comprises the following components: the kit comprises a magnet, a disposable EP tube, a bionic antibody (BSA magnetic molecularly imprinted polymer) capable of identifying BSA, a ligation-BSA, HRP-SA, a BSA standard solution, a developing solution, a sample diluent, a washing solution and a stop solution.

2. Preparation of the used reagent

1) Preparation of sample dilutions (PBS, pH 7.4) (final concentration): NaCl 137mM, KCl 2.7mM, Na₂HPO₄·12H₂O10 mM, and KH₂PO₄2mM, and fixing the volume to 1 by using triple distilled waterAnd L, storing at room temperature.

2) Preparation of wash (PBST, pH 7.4) (final concentration): NaCl 137mM, KCl 2.7mM, Na₂HPO₄·12H₂O 10mM、KH₂PO₄2mM and 0.5mL of Tween-20, and adding triple distilled water to a constant volume of 1L, fully mixing, and storing at room temperature.

3) The preparation method of the chromogenic substrate solution A comprises the following steps: 2.72g of anhydrous sodium acetate, 0.32g of citric acid and 60 mu L of 30% hydrogen peroxide, using triple-distilled water to fix the volume to 100mL, and storing at 2-8 ℃ in a dark place.

4) The preparation method of the chromogenic substrate solution B comprises the following steps: disodium EDTA 40mg, citric acid 0.19g, Glycerol 10mL, and 10mg mL^-14mL of 3,3',5,5' -tetramethylbenzidine (dissolved in dimethyl sulfoxide), constant volume to 100mL by using triple distilled water, and keeping away from light at 2-8 ℃.

5) Stop solution (2mol L)^-1H₂SO₄Solution) preparation: 400mL of triple distilled water and 55.6mL of concentrated sulfuric acid (slowly dropwise adding and continuously stirring), and adding the triple distilled water to reach a constant volume of 500 mL.

3. Molecular imprinting bionic recognition detection step

1) 500. mu.g mL of the suspension was taken^-1BSA magnetic molecularly imprinted polymer (Fe)₃O₄-SiO₂MIPs) suspension 100. mu.L (PBST dispersion), adding into EP tube, fixing with magnet adsorption, discarding supernatant solution for subsequent operation;

2) 2 mg/tube^-1Gelatin solution of 200 μ L is reacted for 30min at room temperature, and then the gelatin solution is removed to seal the non-specific adsorption residue on the surface of the polymer;

3) adding 100 μ L of standard solution or sample solution and 100 μ L of Biotin-BSA solution into each tube, performing room temperature competitive reaction for 40min, and washing the tubes with 200 μ L of PBST for 3 times;

4) adding 150 mu L of HRP-SA solution into each tube, reacting at room temperature for 30min, and washing the tube with PBST for 3 times;

5) restoring the chromogenic substrate solutions A and B to room temperature, and mixing in equal volume to obtain chromogenic substrate mixed solution; adding 150 mu L of chromogenic substrate mixed solution into an EP tube, developing for 30min at room temperature, and adding 50 mu L of stop solution;

6) the 160. mu.L of solution in the EP tube was removed in a 96-well plate and the absorbance was read at 450nm using a microplate reader.

(V) establishment of molecular imprinting bionic recognition analysis method

Dilution determination of Biotin-BSA and HRP-SA: taking 50 mu g of BSA magnetic molecularly imprinted polymer as a bionic antibody, selecting the concentrations of Biotin-BSA and HRP-SA by adopting a chessboard titration method, diluting Biotin-BSA by adopting sample diluent for 500 times, 1000 times, 2000 times and 4000 times respectively, diluting HRP-SA by adopting sample diluent for 5000 times, 10000 times and 20000 times respectively, determining absorbance values (the result is shown in table 1), and selecting the optimal working concentration.

TABLE 1 dilution optimization of Biotin-BSA and HRP-SA

The results of the above matrix experiments show that the optimal dilution of Biotin-BSA is 1:1000 and the optimal dilution of HRP-SA is 1: 10000.

2. Determination of optimal competition reaction time: the kit takes the adsorption equilibrium time of the BSA magnetic molecularly imprinted polymer to the Biotin-BSA as the subsequent competitive adsorption time, detects the absorbance values of the BSA magnetic molecularly imprinted polymer after adsorption to the Biotin-BSA at different time points (1min, 2min, 5min, 10min, 20min, 30min, 40min, 50min, 60min, 90min and 120min) respectively, and selects the optimal competitive adsorption time. FIG. 7 shows that the optimal competition time is 40 min.

3. Determination of optimal binding time of biotin to avidin: the kit takes HRP-SA and Biotin-BSA combined equilibrium time as the adsorption time of a subsequent experiment, and respectively detects the absorbance values of HRP-SA and Biotin-BSA after adsorption at different time points (1min, 2min, 5min, 10min, 20min, 30min, 40min, 50min and 60min) and selects the optimal adsorption time. FIG. 8 shows that the optimal binding time is 30 min.

4. Establishment of a standard curve: are respectively prepared into the concentrations of 1000 mug and mL^-1、500μg·mL^-1、100μg·mL^-1、50μg·mL^-1、10μg·mL^-1And 5. mu.g.mL^-1The standard working solution of (1) is repeated for 6 times at each concentration, and the absorbance value of the obtained standard product is divided by the absorbance value of the blank and then multiplied by 100, thus obtaining the inhibition rate. And drawing a standard curve by taking the inhibition rate as an ordinate and taking the logarithm value of the concentration of the standard substance as the ordinate. And calculating the content of BSA in the sample to be detected according to the standard curve.

(VI) examination of kit Performance indicators

1. The detection specificity inspection of the kit comprises the steps of selecting several proteins of bromelain, pepsin, lysozyme, β -glucuronidase and β -galactosidase as reference substances of an adsorption selectivity experiment, respectively calculating the inhibition rate of each reference substance on the signal intensity, and displaying that the result (shown in figure 9) shows that the specificity of the kit on bovine serum albumin is strong and the selectivity on other proteins is poor.

2. Accuracy experiment and precision experiment: respectively preparing high, medium and low (500 mug. multidot.mL)^-1、100μg·mL^-1、10μg·mL^-1) The method comprises the following steps of adding standard bovine serum albumin with three concentrations into a standard bovine serum solution, wherein each concentration is prepared into 6 parts, obtaining the absorbance values of the standard bovine serum solutions with different concentrations according to the experimental operation steps of the kit, respectively calculating the inhibition rates of the standard bovine serum solutions with different concentrations, and calculating the recovery rate and the relative standard deviation by combining the drawn standard curve, so as to investigate the analysis accuracy and precision. The results (see Table 2) show that under the above-mentioned standard concentration, the recovery rate is between 89.0% and 102.3%, and the precision RSD value is below 4.6%.

TABLE 2 accuracy and precision of the molecularly imprinted biomimetic immunoassay kit for BSA in bovine serum (n ═ 6)

Claims

1. A bionic antibody of a magnetic molecularly imprinted polymer of bovine serum albumin is characterized in that: the bionic antibody comprises bovine serum albumin magnetic molecularly imprinted polymer microspheres, wherein the molecularly imprinted polymer microspheres comprise magnetic nano-microspheres, a silicon dioxide interface layer coated on the surfaces of the magnetic nano-microspheres and a bovine serum albumin molecularly imprinted polymer layer coated on the silicon dioxide interface layer; the bovine serum albumin molecularly imprinted polymer layer is formed by taking bovine serum albumin as a template molecule, performing catalytic polymerization reaction on dopamine on a water phase-silicon dioxide interface, and removing the template molecule after polymerization.

2. The bovine serum albumin magnetic molecularly imprinted polymer biomimetic antibody according to claim 1, characterized in that: the thickness of the bovine serum albumin molecularly imprinted polymer layer is 20-40 nm, and the particle size of the magnetic nano-microspheres is 300-500 nm.

3. The method for preparing the bionic antibody of the magnetic molecularly imprinted polymer of the bovine serum albumin as claimed in claim 1, which is characterized in that: the method comprises the following steps:

1) preparing magnetic nano microspheres;

2) coating silicon dioxide on the surface of the magnetic nano microsphere to obtain SiO₂A magnetic nanoparticle composite;

3) polymerizing monomer, bovine serum albumin as template molecule and SiO dispersed in solvent₂Mixing the magnetic nano microsphere compound to obtain an aqueous phase reaction system, and performing prepolymerization on the aqueous phase reaction system and polymerization reaction on the aqueous phase reaction system after adding a catalyst to obtain the SiO₂Coating the surface of the magnetic nano microsphere compound with a molecularly imprinted polymer combined with template molecules to obtain SiO₂Magnetic molecularly imprinted nanospheres;

4) removal of SiO₂And (3) template molecules combined on the surface of the magnetic molecularly imprinted nanospheres to obtain the bovine serum albumin magnetic molecularly imprinted polymer microspheres.

4. The method of claim 3, wherein: in the step 3), the polymerization monomer is dopamine, the solvent is a slightly alkaline buffer solution with the pH value less than or equal to 8.5, and catalysis is carried outThe agent is persulfate, the mass ratio of the template molecules to the polymerized monomers is 1: 4-1: 12, and SiO is₂The mass ratio of the magnetic nano microsphere composite to the polymerized monomer is 1: 0.5-1: 1.5.

5. The method of claim 3, wherein: in the step 4), an elution solvent is adopted to remove the template molecules, wherein the elution solvent is a methanol-hydrochloric acid mixture, the mixing volume ratio of methanol to hydrochloric acid is 4: 1-9: 1, and the concentration of hydrochloric acid is 1 mol/L.

6. The method of claim 3, wherein: the preparation of the bovine serum albumin magnetic molecularly imprinted polymer biomimetic antibody further comprises the following steps: blocking the non-specific adsorption residue of the bovine serum albumin on the surface of the magnetic molecularly imprinted polymer microsphere of the bovine serum albumin.

7. The method according to claim 3 or 6, characterized in that: the preparation method of the bovine serum albumin magnetic molecularly imprinted polymer bionic antibody specifically comprises the following steps: 50-200 mg of SiO₂Uniformly dispersing a magnetic nano microsphere compound in 20-50 mL of PBS buffer solution with the pH value of 7.2-7.4 to obtain microsphere aqueous phase dispersion liquid, adding 50-200 mg of dopamine into the microsphere aqueous phase dispersion liquid, stirring for 1-3 h at 10-40 ℃, then adding 10-40 mg of bovine serum albumin, continuously stirring for 1-3 h to obtain prepolymerization dispersion liquid, adding 5-20 mg of ammonium persulfate into the prepolymerization dispersion liquid, stirring for 6-24 h at 10-40 ℃, and then separating to obtain SiO₂Magnetic molecular engram nano microsphere, washing SiO with template molecule eluting solvent₂And (3) washing the magnetic molecularly imprinted nanospheres to remove the residual elution solvent, and drying at 40-60 ℃ to obtain the microspherical bovine serum albumin magnetic molecularly imprinted polymer.

8. A bionic immunoassay kit for bovine serum albumin is characterized in that: the kit comprises a bionic antibody with a function of identifying bovine serum albumin and a detection marker which can be competitively combined with the bionic antibody in a water phase, wherein the bionic antibody comprises bovine serum albumin magnetic molecularly imprinted polymer microspheres, and the molecularly imprinted polymer microspheres comprise magnetic nano-microspheres, a silicon dioxide interface layer coated on the surfaces of the magnetic nano-microspheres and a bovine serum albumin molecularly imprinted polymer layer coated on the silicon dioxide interface layer; the bovine serum albumin molecularly imprinted polymer layer is formed by taking bovine serum albumin as a template molecule, performing catalytic polymerization reaction on dopamine on a water phase-silicon dioxide interface, and removing the template molecule after polymerization.

9. The bovine serum albumin biomimetic immunoassay kit according to claim 8, wherein: the detection marker is Biotin-BSA, and the kit further comprises HRP-SA, a developing solution, a sample diluent, a washing solution and a stop solution.

10. The use of the bovine serum albumin biomimetic immunoassay kit according to claim 8 for the analysis of BSA content in bovine serum.