阅读说明：本技术 基于电活性天然大分子胶束的超灵敏蛋白质分子印迹电化学传感器及其制备方法和应用 (Ultrasensitive protein molecular imprinting electrochemical sensor based on electroactive natural macromolecular micelle and preparation method and application thereof ) 是由 张荣莉 翁闽闽 杨晓良 霍朝飞 刘欢 崔婉颖 胡宇龙 过学军 黄中桂 于 2021-01-07 设计创作，主要内容包括：本发明提供基于电活性天然大分子胶束的超灵敏蛋白质分子印迹电化学传感器及其制备方法和应用,制备方法包括：电活性天然大分子的制备、蛋白质/电活性天然大分子胶束的制备、蛋白质/电活性天然大分子胶束修饰电极的制备和超灵敏蛋白质分子印迹电化学传感器的制备。本发明制备的电活性天然大分子胶束具有纳米结构和良好的生物相容性,将其应用于蛋白质分子印迹传感器,可以有效提高蛋白质在印迹聚合物中的传质速率,所构建的蛋白质印迹传感器具有良好的选择性、稳定性、灵敏性和较宽的检测范围；大分子自组装技术与蛋白质分子印迹技术、电化学生物传感技术的结合,可广泛应用于生物医药、食品安全以及环境监测等领域。(The invention provides an ultrasensitive protein molecular imprinting electrochemical sensor based on electroactive natural macromolecular micelles as well as a preparation method and application thereof, wherein the preparation method comprises the following steps: the method comprises the following steps of preparing electroactive natural macromolecules, preparing protein/electroactive natural macromolecular micelles, preparing modified electrodes of the protein/electroactive natural macromolecular micelles and preparing an ultrasensitive protein molecular imprinting electrochemical sensor. The electroactive natural macromolecular micelle prepared by the method has a nano structure and good biocompatibility, can effectively improve the mass transfer rate of protein in an imprinted polymer when being applied to a protein molecular imprinted sensor, and the constructed protein imprinted sensor has good selectivity, stability and sensitivity and a wider detection range; the combination of the macromolecule self-assembly technology, the protein molecular imprinting technology and the electrochemical biosensing technology can be widely applied to the fields of biological medicine, food safety, environmental monitoring and the like.)

1. The preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle is characterized by comprising the following steps of:

1) preparation of electroactive natural macromolecules: dissolving natural macromolecules in a solvent, sequentially adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide or 4-dimethylaminopyridine and water-soluble thiophene, stirring for reaction, and dialyzing, freezing and drying after the reaction is finished to obtain electroactive natural macromolecules;

2) preparing a protein/electroactive natural macromolecular micelle;

3) preparing a protein/electroactive natural macromolecular micelle modified electrode;

4) and (3) preparing an ultrasensitive protein molecular imprinting electrochemical sensor.

2. The method according to claim 1, wherein the natural macromolecule in step 1) is sodium alginate, chitosan, polyamino acid, starch or dextran.

3. The production method according to claim 1 or 2, characterized in that the water-soluble thiophene in step 1) is aminothiophene or carboxythiophene.

4. The production method according to claim 1 or 2, characterized in that the ratio of the amount of the natural macromolecular repeating unit to the amount of the substance of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in step 1) is 6:1 to 1: 6; the mass ratio of the natural macromolecule repeating unit to the N-hydroxysuccinimide or 4-dimethylaminopyridine is 6:1-1: 6; the ratio of the natural macromolecule repeating unit to the amount of the water-soluble thiophene monomer is 6:1-1: 6.

5. The method according to claim 1 or 2, wherein the natural macromolecule in step 1), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide or 4-dimethylaminopyridine, and the water-soluble thiophene monomer are mixed, reacted in an ice bath for 2 to 6 hours, and then reacted at room temperature for 6 to 48 hours.

6. The preparation method according to claim 1, wherein the step 2) is specifically: respectively dissolving the electroactive natural macromolecules prepared in the step 1) and the protein, dropwise adding the protein solution into the electroactive natural macromolecule solution, and assembling the electroactive natural macromolecule solution into the protein/electroactive natural macromolecule micelle through weak interaction force.

7. The preparation method according to claim 1, wherein the step 3) is specifically: modifying the protein/electroactive natural macromolecular micelle solution on the surface of the gold electrode, and airing at room temperature to obtain the protein/electroactive natural macromolecular micelle modified electrode.

8. The preparation method according to claim 1, wherein the step 4) is specifically: and (3) carrying out electropolymerization on the dried protein/electroactive natural macromolecular micelle modified electrode in an acetonitrile solution of lithium perchlorate by using a cyclic voltammetry method, eluting in a methanol/glacial acetic acid mixed solution after drying to remove template protein, and naturally drying to obtain the ultrasensitive protein molecular imprinting electrochemical sensor.

9. An ultrasensitive protein molecular imprinting electrochemical sensor based on an electroactive natural macromolecular micelle, prepared by the preparation method of any one of claims 1 to 8.

10. Use of an ultrasensitive protein molecularly imprinted electrochemical sensor based on electroactive natural macromolecular micelles prepared by the preparation method according to any one of claims 1 to 8 for detecting proteins.

Technical Field

The invention belongs to the field of functional polymer materials and the technical field of electrochemical biosensor preparation, and particularly relates to an ultrasensitive protein molecular imprinting electrochemical sensor based on electroactive natural macromolecular micelles, and a preparation method and application thereof.

Background

Proteomics is one of the most important research fields at present, and has a very close relationship with human health and social development. The deep research on the protein not only can provide a material basis for disclosing the life activity rule, but also can provide a theoretical basis and a solution way for the explanation and attack of a plurality of disease mechanisms. In proteomics research, there is a need to develop methods for selectively isolating, identifying and detecting specific target proteins from complex matrix samples. Therefore, the development of a new separation and identification method will play an important role in the field of proteomics.

The western blotting technique is a novel technique developed by integrating a plurality of disciplines such as macromolecule synthesis, protein molecule identification, bionic bioengineering and the like, and is an effective method for selectively separating and identifying target protein from a complex matrix sample. However, mass transfer and diffusion in imprinted polymers are difficult due to the large spatial size of the protein molecules. The macromolecular self-assembly micelle with the nano structure is beneficial to improving the mass transfer rate of protein in the imprinted polymer micelle, but the improvement of the sensitivity of the western blot sensor is limited by the insulating imprinted polymer micelle.

Thiophene is a cyclic unsaturated thioether compound and has electrochemical activity. Under the electrochemical action, the thiophene monomer is subjected to oxidation reaction to obtain the crosslinked reticular polythiophene. The polythiophene and the polythiophene derivative have a large pi conjugated structure on a molecular chain, so that the whole molecule has delocalization characteristics, show good charge transfer capacity, and are widely applied to a plurality of research fields such as biosensors, nerve probes, drug delivery, tissue engineering, organic photovoltaic cells and the like.

Disclosure of Invention

The invention aims to provide an ultrasensitive protein molecular imprinting electrochemical sensor based on electroactive natural macromolecular micelles and a preparation method thereof.

The invention also aims to provide the application of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle, which is used for detecting protein and has the advantages of high selectivity, high sensitivity, wide monitoring range, low detection lower limit and the like.

The specific technical scheme of the invention is as follows:

the preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle comprises the following steps:

1) preparation of electroactive natural macromolecules: dissolving natural macromolecules in a solvent, sequentially adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide or 4-dimethylaminopyridine and water-soluble thiophene, stirring for reaction, and dialyzing, freezing and drying after the reaction is finished to obtain electroactive natural macromolecules;

2) preparing a protein/electroactive natural macromolecular micelle;

3) preparing a protein/electroactive natural macromolecular micelle modified electrode;

4) and (3) preparing an ultrasensitive protein molecular imprinting electrochemical sensor.

The natural macromolecules in the step 1) are sodium alginate, chitosan, polyamino acid, starch or glucan;

the water-soluble thiophene is aminothiophene or carboxythiophene; the aminothiophene is preferably 3-aminoethylthiophene or 3-aminothiophene; the carboxythiophene is preferably 3-carboxymethylthiophene or 3-carboxyethylthiophene;

dissolving natural macromolecules in a solvent in step 1), wherein the solvent is distilled water, dimethyl sulfoxide or dichloromethane; furthermore, natural macromolecules are dissolved in the solvent, and the dissolving temperature is 10-100 ℃.

In the step 1), the natural macromolecules are dissolved in a solvent, and the concentration of the natural macromolecule repeating units is 0.01-10 mol/L.

When the natural macromolecules in the step 1) are sodium alginate or polyamino acid, carrying out amidation reaction on carboxyl of the natural macromolecules and amino on aminothiophene to obtain electroactive natural macromolecules; when the natural macromolecule is chitosan, carrying out amidation reaction on amino and carboxyl on carboxythiophene to obtain the electroactive natural macromolecule; when the natural macromolecule is starch or glucan, the hydroxyl of the natural macromolecule and the carboxyl on the carboxyl thiophene are subjected to esterification reaction to obtain the electroactive natural macromolecule.

The mass ratio of the natural macromolecule repeating unit in the step 1) to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 6:1-1: 6; the mass ratio of the natural macromolecule repeating unit to the N-hydroxysuccinimide or 4-dimethylaminopyridine is 6:1-1: 6; the ratio of the natural macromolecule repeating unit to the amount of the water-soluble thiophene monomer is 6:1-1: 6.

Mixing the natural macromolecules in the step 1), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide or 4-dimethylaminopyridine and a water-soluble thiophene monomer, reacting in an ice bath for 2-6h, and reacting at room temperature for 6-48 h.

And (3) dialyzing in the step 1), putting reactants into a dialysis bag (MW8000-10000), and dialyzing and purifying by using distilled water.

The freeze drying in the step 1) refers to the freezing temperature of-70 ℃ and the time of 24-72 h.

The step 2) is specifically as follows: respectively dissolving the electroactive natural macromolecules prepared in the step 1) and the protein, stirring and mixing, and self-assembling into the protein/electroactive natural macromolecule micelle through weak interaction force.

Preferably, the solvent used for dissolving the electroactive natural macromolecules prepared from the starch or the glucan in the step 2) is dimethyl sulfoxide; dissolving electroactive natural macromolecules prepared from sodium alginate or polyamino acid in an aqueous solution, and adding hydrochloric acid to adjust the pH value to 3-5; the electroactive natural macromolecules prepared from chitosan are dissolved in 1% acetic acid solution, and sodium hydroxide is added to adjust the pH to 4-6.

Dissolving the protein in the step 2) by adopting a phosphate buffer solution;

the protein in the step 2) is bovine serum albumin, myoglobin, bovine hemoglobin, horseradish peroxidase, lysozyme or complexing protein.

After the electroactive natural macromolecules in the step 2) are dissolved, the concentration of the electroactive natural macromolecules is 0.001-100 mg/mL;

the protein in the step 2) is dissolved in phosphate buffer solution, and the concentration of the protein is 0.001-100 mg/mL.

The volume ratio of the electroactive natural macromolecule solution to the protein solution in the step 2) is 10:1-1: 10.

The step 3) is specifically as follows: modifying the protein/electroactive natural macromolecular micelle solution on the surface of the gold electrode, and airing at room temperature to obtain the protein/electroactive natural macromolecular micelle modified electrode.

In the step 3), the gold electrode is cleaned before use, and the method specifically comprises the following steps: gold electrode was coated with 0.3 μm and 0.05 μm of α -Al in this order₂O₃Polishing the surface of the powder, respectively cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol, and naturally drying the powder;

the concentration of the protein/electroactive natural macromolecular micelle used in the step 3) is 0.001-100mg/mL, and the volume used is 1.0-20 muL.

The step 4) is specifically as follows: and (3) carrying out electropolymerization on the dried protein/electroactive natural macromolecular micelle modified electrode in an acetonitrile solution of lithium perchlorate by using a cyclic voltammetry method, after drying, eluting to remove template protein, and naturally drying to obtain the ultrasensitive protein molecular imprinting electrochemical sensor.

Further, performing electropolymerization by using cyclic voltammetry, wherein the scanning range is from 0V to 1.5V; the scanning rate is 100 mV/s; the number of scanning turns is 5-30.

Further, the concentration of the lithium perchlorate acetonitrile solution in the step 4) is 0.01-2.0 mol/L.

The eluent used in the elution in the step 4) is a methanol/glacial acetic acid mixed solution, wherein the volume ratio of methanol to glacial acetic acid is 10:1-1: 10.

The specific technological parameter of elution in the step 4) is elution for 1-24h at room temperature.

The invention provides an ultrasensitive protein molecular imprinting electrochemical sensor based on electroactive natural macromolecular micelles, which is prepared by adopting the method.

The invention provides an application of an ultrasensitive protein molecular imprinting electrochemical sensor based on electroactive natural macromolecular micelles, which is used for detecting protein, and the specific method comprises the following steps:

s1, placing the ultra-sensitive protein molecular imprinting electrochemical sensor in a phosphate buffer solution (blank solution) with the protein concentration of 0, standing, placing in an electrolyte solution, and measuring the electrochemical response value of the blank solution by using a differential conventional pulse voltammetry method;

s2, placing the protein molecular imprinting electrochemical sensor in a protein solution with a certain concentration, standing and adsorbing, placing in an electrolyte solution, measuring an electrochemical response value by using a differential conventional pulse voltammetry, and taking out the sensor to elute proteins;

s3, placing the eluted protein molecular imprinting electrochemical sensor into a protein solution with another concentration, and repeating the step S2;

s4, constructing a linear relation by taking the absolute value of the difference between the electrochemical response value measured under a certain protein concentration and the electrochemical response value of the blank solution as a ordinate and the protein concentration as an abscissa.

Further, the pH value of the phosphate buffer solution is 7.2, and the concentration is 0.1 mol/L; differential conventional pulsed voltammetry (DNPV) measurements, scan ranging from-0.1V to 0.9V; the scanning rate is 20 mV/s; the electrolyte solution is a phosphate buffer solution with pH value of 7.2 and concentration of 0.1mol/L, which contains 5mmol/L potassium ferricyanide, 5mmol/L potassium ferrocyanide and 0.1mol/L potassium chloride.

And respectively placing the prepared electrochemical sensors in a blank solution and a protein solution with the concentration to be measured for adsorption, respectively measuring electrochemical response values of the blank solution and the protein solution with the concentration to be measured in an electrolyte solution by using a differential conventional pulse voltammetry method, calculating an absolute value of an electrochemical response difference value, and obtaining the concentration of the protein solution to be measured according to the obtained absolute value of the electrochemical response difference value and the linear relation.

According to the invention, natural macromolecules with electric activity are dissolved in a solvent, proteins are dissolved in a phosphate buffer solution, the electric activity natural macromolecules embed protein molecules through weak interaction force to form protein/electric activity natural macromolecule micelles, then the protein/electric activity natural macromolecule micelles are modified on the surface of a gold electrode, and after the gold electrode is naturally dried, electric polymerization is carried out in an acetonitrile solution of lithium perchlorate to form a conductive polythiophene network, so that the conductivity of a self-assembled micelle membrane is increased, and the detection sensitivity is improved; and naturally airing, and eluting in a methanol/glacial acetic acid mixed solution to remove template molecules, thereby preparing the high-sensitivity protein molecular imprinting sensor. The prepared protein molecular imprinting sensor has the advantages of high selectivity, high sensitivity, wide detection range, low detection lower limit and the like, and is expected to be widely applied to the fields of biological medicine, food safety, environmental monitoring and the like.

Compared with the prior art, the invention has the main advantages that:

1) the protein/electroactive natural macromolecular micelle adopted in the preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle has good biocompatibility, and is beneficial to maintaining the activity of the protein in the preparation process of the sensor;

2) the protein/electroactive natural macromolecular micelle adopted in the preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle has a nano structure, and is favorable for improving the mass transfer rate of the protein in the molecular imprinting polymer micelle.

3) The protein/electroactive natural macromolecular micelle adopted in the preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle forms a conductive crosslinking network through electropolymerization and crosslinking, and simultaneously fixes a binding site, thereby being beneficial to improving the electron transfer rate between the protein to be detected and a substrate electrode.

4) The preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle has the advantages of high selectivity, high sensitivity, wide detection range, low detection lower limit and the like.

5) The invention combines the macromolecule self-assembly technology, the electrochemical biosensing technology and the protein imprinting technology to construct the high-sensitivity protein molecular imprinting sensor, and is expected to be applied to the fields of biological medicine, food safety, environmental monitoring and the like.

Drawings

FIG. 1 is a synthetic route of thiophene modified gamma-polyglutamic acid polymer; wherein 4600< n <6700, 200< m < 2000;

FIG. 2 shows the NMR spectrum (a) and IR spectrum (b) of gamma-polyglutamic acid and thiophene-modified gamma-polyglutamic acid polymer;

FIG. 3 is a scanning electron microscope image of thiophene modified gamma-polyglutamic acid self-assembled micelle (a) and thiophene modified gamma-polyglutamic acid/bovine serum albumin composite assembled micelle (b);

FIG. 4 is a cyclic voltammetry spectrum of different modification stages of the preparation process of the Western blot sensor of the present invention;

FIG. 5 shows the differential conventional pulse voltammogram (a) and linear voltammogram (b) of the Western blot sensor in different concentrations of protein solution.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.

Example 1

The preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle comprises the following steps:

1) preparing an electroactive natural macromolecule; dissolving 2mmol of polyglutamic acid (gamma-PGA, calculated by a repeating unit) in 40mL of distilled water, placing the mixture in an ice bath for 1h after the polyglutamic acid (gamma-PGA) is completely dissolved At room temperature, adding 2mmol of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, adding 4mmol of N-hydroxysuccinimide after the polyglutamic acid (gamma-PGA) is completely dissolved, adding 2mmol of 3-aminothiophene (At) after the polyglutamic acid (gamma-PGA) is completely dissolved, reacting for 2h under the conditions of ice bath and magnetic stirring, reacting for 24h At room temperature, placing the mixture in a dialysis bag (MW8000-10000) for dialysis, and freeze-drying the mixture At-70 ℃ for 72h to obtain the thiophene modified gamma-polyglutamic acid (At-gamma-PGA) polymer. The synthetic route is shown in figure 1.

FIG. 2 shows the hydrogen nuclear magnetic spectrum of γ -PGA and At- γ -PGA (¹H NMR) (a) and infrared (b). It can be seen from (a) in FIG. 2 that the nuclear magnetic spectrum of the At- γ -PGA polymer has 3 more vibrational peaks than that of γ -PGA; it can be seen from FIG. 2 (b) that the infrared spectrum of the At- γ -PGA polymer clearly shows an absorption peak more than that of γ -PGA, which is the absorption peak of thiophene, indicating the success of the grafting reaction.

2) Preparation of protein/electroactive natural macromolecular micelles: dissolving At-gamma-PGA in water to form 0.1mg/mL solution; dissolving bovine serum albumin in 1mol/L phosphate buffer solution with pH of 5.0 to form 0.1mg/mL solution; and dropwise adding the prepared 20mL of bovine serum albumin solution into 100mL of At-gamma-PGA solution, adding the solution after the bovine serum albumin solution is completely dispersed, dropwise adding the solution, uniformly mixing, adjusting the pH value of the solution to 4.0 by using hydrochloric acid, and carrying out induced assembly to obtain the bovine serum albumin/thiophene modified gamma-polyglutamic acid (BSA/At-gamma-PGA) micelle.

FIG. 3 is a scanning electron microscope photograph of At- γ -PGA polymer and BSA/At- γ -PGA micelle At pH 4.0. As can be seen from the figure: the At-gamma-PGA polymer is in a slightly irregular spherical shape At the pH of 4.0, the particle size is about 100nm, and the particle size distribution is wider; the BSA/At-gamma-PGA micelle has a regular spherical morphology At pH 4.0 and a particle size of about 150 nm. The particle size was increased compared to At- γ -PGA polymer micelles because bovine serum albumin was supported.

3) Preparing a protein/electroactive natural macromolecular micelle modified electrode: gold electrode was coated with 0.3 μm and 0.05 μm of α -Al in this order₂O₃And (3) polishing the surface of the powder, respectively cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol, naturally airing the powder, and detecting the powder on an electrochemical workstation by using a cyclic voltammetry method to ensure that the difference of the oxidation reduction peaks of the powder and the ethanol is less than 0.1V, namely the cleaning of the bare electrode is finished. And modifying 5 mu L of 0.1mg/mL BSA/At-gamma-PGA micelle dispersion liquid to the surface of the gold electrode, and airing At room temperature to obtain the BSA/At-gamma-PGA micelle modified electrode.

4) Preparing an ultrasensitive protein molecular imprinting electrochemical sensor: electropolymerizing the air-dried BSA/At-gamma-PGA micelle modified electrode in 0.1mol/L of lithium perchlorate acetonitrile solution by using a cyclic voltammetry method, wherein the scanning range is from 0V to 1.5V; the scanning rate is 100 mV/s; and scanning for 20 circles, taking out the electrode, drying in the air, eluting in a methanol/glacial acetic acid (V: 9:1) mixed solution for 10 hours to remove the bovine serum albumin as a template, and naturally drying in the air to obtain the ultrasensitive bovine serum molecular imprinting sensor.

Fig. 4 is a cyclic voltammetry spectrogram of bovine serum albumin molecularly imprinted sensor in example 1 At different modification stages, where the cyclic voltammetry curve of the bare gold electrode in step 3) in example 1 corresponds to the bare gold electrode curve in fig. 4, the cyclic voltammetry curve of the BSA/At- γ -PGA micelle modified electrode prepared in step 3) in example 1 corresponds to the micelle modified electrode curve in fig. 4, and the cyclic voltammetry curve of the electrode after electropolymerization in step 4) in example 1 corresponds to the curve after electropolymerization in fig. 4; example 1 the post-elution cyclic voltammogram of the electrode in step 4) corresponds to the post-elution curve in figure 1; the post-adsorption curve in fig. 4 is a curve after the sensor is statically placed to adsorb bovine serum albumin. The CV spectrogram of the bare gold electrode in the electrolyte solution has a pair of relatively symmetrical redox peaks; after the insulating BSA/At-gamma-PGA glue is dripped, compared with a bare electrode, the resistance value is increased, the redox peak current value is reduced, namely the conductivity is reduced; the oxidation-reduction peak current value in a CV spectrogram after electropolymerization is increased, because thiophene groups form a conjugated structure, and the conductivity of a micelle membrane is increased; after the template bovine serum albumin is removed by elution, the oxidation-reduction peak current value in the CV spectrogram is further increased, because a cavity site is left after the bovine serum albumin is eluted, so that small molecules in the electrolyte are easy to enter and exit; after the adsorption of the template bovine serum albumin, the redox peak current value in the CV spectrogram is reduced because the holes are occupied again by the bovine serum albumin, which hinders the entrance and exit of small molecules in the electrolyte.

The application of the prepared sensor is used for detecting the concentration of bovine serum albumin in a solution to be detected, and specifically comprises the following steps:

respectively placing the prepared ultrasensitive protein molecular imprinting electrochemical sensor in a position of 0 and a position of 1 multiplied by 10^-9、1×10^-8、1×10^-7、1×10^-6、1×10^-5And (3) in mol/L phosphate buffer solution of bovine serum albumin with different concentrations, wherein the pH of the phosphate buffer solution is 7.2, the concentration is 0.1mol/L, standing and adsorbing for 5min, and placing the phosphate buffer solution in an electrolyte solution to measure an electrochemical response value by using a differential conventional pulse voltammetry. The sweep ranged from-0.1V to 0.9V, the sweep rate was 20mV/s, and the electrolyte was a phosphate buffer solution at a pH of 7.2 and a concentration of 0.1mol/L containing 5mmol/L potassium ferricyanide, 5mmol/L potassium ferrocyanide, and 0.1mol/L potassium chloride. And taking out and eluting the solution for 3min after each measurement, then carrying out the next concentration detection, and constructing a linear relation by taking the absolute value of the difference value between the electrochemical response value measured under a certain concentration and the electrochemical response value measured by a blank solution (the concentration of bovine serum albumin is 0mol/L) as a vertical coordinate and the protein concentration as a horizontal coordinate.

And constructing a linear relation by taking the absolute value of the difference value between the electrochemical response value measured under a certain concentration and the electrochemical response value measured by the blank solution as a vertical coordinate and the protein concentration as a horizontal coordinate. The linear equation obtained was Δ I11.459 +0.8387 × (log C)_BSA) Δ I is the current difference in units of μ A, C_BSAThe concentration of bovine serum albumin is expressed in mol/L; the square of the correlation coefficient was 0.997. The linear relationship is shown in fig. 5.

And respectively placing the prepared electrochemical sensors in a blank solution and a protein solution with the concentration to be measured for adsorption, respectively measuring electrochemical response values of the blank solution and the protein solution with the concentration to be measured in an electrolyte solution by using a differential conventional pulse voltammetry method, calculating an absolute value of an electrochemical response difference value, and obtaining the concentration of the protein solution to be measured according to the obtained absolute value of the electrochemical response difference value and the linear relation.

Example 2

The preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle comprises the following steps:

1) preparation of electroactive native macromolecules

Dissolving 2mmol of sodium alginate (counted by a repeating unit) in 40mL of distilled water, placing the solution in an ice bath for 2h after complete dissolution, adding 2mmol of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, adding 4mmol of N-hydroxysuccinimide after complete dissolution, adding 2mmol of 3-aminothiophene after complete dissolution, reacting for 2h under ice bath and magnetic stirring conditions, reacting for 24h at room temperature, placing the solution in a dialysis bag, dialyzing and purifying distilled water, and freeze-drying to obtain the thiophene modified sodium alginate polymer.

2) Preparation of protein/electroactive natural macromolecular micelle

Dissolving thiophene modified alginic acid in a phosphate buffer solution with the pH value of 5.0 to form a 0.1mg/mL solution; dissolving bovine hemoglobin in phosphate buffer solution with pH of 5.0 to form 0.1mg/mL solution; dropwise adding 20mL of bovine hemoglobin solution into 100mL of thiophene modified sodium alginate solution, mixing, adjusting the pH value of the solution to 4.0 by hydrochloric acid, and self-assembling to obtain the bovine hemoglobin/thiophene modified alginic acid micelle through weak interaction force.

3) Preparation of protein/electroactive natural macromolecular micelle modified electrode

Gold electrode was coated with 0.3 μm and 0.05 μm of α -Al in this order₂O₃And (3) polishing the surface of the powder, respectively cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol, naturally airing, modifying 5 mu L of 0.1mg/mL bovine hemoglobin/thiophene modified alginic acid micelle solution on the surface of the gold electrode, and airing at room temperature to obtain the bovine hemoglobin/thiophene modified alginic acid micelle modified electrode.

4) Preparation of ultrasensitive protein molecular imprinting electrochemical sensor

Electropolymerizing the dried bovine hemoglobin/thiophene modified alginic acid micelle modified electrode in 0.1mol/L acetonitrile solution of lithium perchlorate by using a cyclic voltammetry method, wherein the scanning range is from 0V to 1.5V; the scanning rate is 100 mV/s; and (3) scanning for 20 circles, after polymerization, drying the electrode in the air, eluting in a methanol/glacial acetic acid (V: V ═ 9:1) mixed solution for 24 hours to remove the bovine hemoglobin as a template, and naturally drying in the air to obtain the ultrasensitive bovine hemoglobin molecular imprinting sensor.

Example 3

The preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle comprises the following steps:

1) preparation of electroactive native macromolecules

Dissolving 2mmol of starch (counted by a repeating unit) in 40mL of dimethyl sulfoxide, cooling to room temperature after complete dissolution at 90 ℃, adding 2mmol of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, adding 4mmol of 4-dimethylaminopyridine after complete dissolution, adding 2mmol of 3-carboxymethyl thiophene after complete dissolution, reacting for 2 hours under ice bath and magnetic stirring conditions, reacting for 24 hours at room temperature, filling into a dialysis bag, dialyzing and purifying distilled water, and freeze-drying to obtain the thiophene modified starch polymer.

2) Preparation of protein/electroactive natural macromolecular micelle

Dissolving thiophene modified starch in dimethyl sulfoxide to form 0.1mg/mL solution; dissolving lysozyme in phosphate buffer solution with pH of 5.0 to form 0.1mg/mL solution; and (3) dropwise adding 1mL of lysozyme solution into 1mL of thiophene modified starch solution, mixing, and assembling the lysozyme/thiophene modified starch micelle through weak interaction force.

3) Preparation of protein/electroactive natural macromolecular micelle modified electrode

Gold electrode was coated with 0.3 μm and 0.05 μm of α -Al in this order₂O₃And (3) polishing the surface of the powder, respectively cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol, naturally drying the powder, modifying 5 mu L of 0.1mg/mL lysozyme/thiophene modified starch micelle solution on the surface of the gold electrode, and drying the modified gold electrode at room temperature to obtain the lysozyme/thiophene modified starch micelle modified electrode.

4) Preparation of ultrasensitive protein molecular imprinting electrochemical sensor

Electropolymerizing the dried lysozyme/thiophene modified starch micelle modified electrode in 0.1mol/L acetonitrile solution of lithium perchlorate by using a cyclic voltammetry method, wherein the scanning range is from 0V to 1.5V; the scanning rate is 100 mV/s; scanning for 20 circles, drying, eluting in a methanol/glacial acetic acid (V: V ═ 9:1) mixed solution to remove the template lysozyme, and naturally drying to obtain the ultrasensitive lysozyme molecularly imprinted sensor.

Example 4

The preparation method of the ultrasensitive protein molecular imprinting electrochemical sensor based on the electroactive natural macromolecular micelle comprises the following steps:

1) preparation of electroactive native macromolecules

Dissolving 2mmol of glucan (calculated by a repeating unit) in 40mL of dimethyl sulfoxide, adding 2mmol of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride after complete dissolution, adding 4mmol of 4-dimethylaminopyridine after complete dissolution, adding 2mmol of 3-carboxyethyl thiophene after complete dissolution, reacting for 2 hours under ice bath and magnetic stirring conditions, reacting for 24 hours at room temperature, filling into a dialysis bag, dialyzing and purifying by distilled water, and freeze-drying to obtain the thiophene modified glucan polymer.

2) Preparation of protein/electroactive natural macromolecular micelle

Dissolving thiophene modified glucan in dimethyl sulfoxide to form a 0.1mg/mL solution; dissolving myoglobin in phosphate buffer solution with pH 5.0 to form 0.1mg/mL solution; and adding 20mL of myoglobin solution into 100mL of thiophene modified glucan solution drop by drop, mixing, and assembling into myoglobin/thiophene modified glucan micelles through weak interaction force.

3) Preparation of protein/electroactive natural macromolecular micelle modified electrode

Gold electrode was coated with 0.3 μm and 0.05 μm of α -Al in this order₂O₃And (3) polishing the surface of the powder, respectively cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol, naturally airing, modifying 10 mu L of 0.2mg/mL myoglobin/thiophene modified glucan micelle solution on the surface of the gold electrode, and airing at room temperature to obtain the myoglobin/thiophene modified glucan micelle modified electrode.

4) Preparation of ultrasensitive protein molecular imprinting electrochemical sensor

Electropolymerizing the dried myoglobin/thiophene modified glucan micelle modified electrode in 0.1mol/L acetonitrile solution of lithium perchlorate by using a cyclic voltammetry method, wherein the scanning range is from 0V to 1.5V; the scanning rate is 100 mV/s; scanning for 20 circles, air-drying, eluting in a methanol/glacial acetic acid (V: V ═ 9:1) mixed solution to remove the template myoglobin, and naturally air-drying to obtain the ultrasensitive myoglobin molecular imprinting sensor.