CN109490389B - Preparation method of imprinted polymer modified electrode for detecting myoglobin with high sensitivity - Google Patents

Preparation method of imprinted polymer modified electrode for detecting myoglobin with high sensitivity Download PDF

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CN109490389B
CN109490389B CN201811430538.0A CN201811430538A CN109490389B CN 109490389 B CN109490389 B CN 109490389B CN 201811430538 A CN201811430538 A CN 201811430538A CN 109490389 B CN109490389 B CN 109490389B
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孙越
杨艺菲
李思雨
王伟
冯雪薇
赵梦元
刘雨桐
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Liaoning Normal University
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Abstract

The invention discloses a preparation method of an imprinted polymer modified electrode for detecting myoglobin with high sensitivity, which comprises the steps of firstly preparing an Au/3DG/Cs/nAu electrode, and then preparing a GCE/GR/CTAB/Cs electrode; modifying an Au/3DG/Cs/nAu electrode by using a sulfhydryl-terminated bromine-containing compound to obtain a bromide modified electrode, then putting a PBS solution containing alpha-methacrylic acid, myoglobin and N, N-methylene bisacrylamide into an electrolytic cell, putting the obtained bromide modified electrode into the solution in the electrolytic cell at room temperature, using a GCE/GR/CTAB/Cs electrode as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode as a reference electrode, and applying a constant potential for 50min at 0.185V to obtain the bromide modified electrode on which a polymer is deposited; and taking out the bromide modified electrode deposited with the polymer, and removing the myoglobin template in the polymer.

Description

Preparation method of imprinted polymer modified electrode for detecting myoglobin with high sensitivity
Technical Field
The invention discloses a preparation method of an imprinted polymer modified electrode, and particularly relates to a preparation method of an imprinted polymer modified electrode which is simple to prepare, energy-saving, clean and environment-friendly and can detect myoglobin with high sensitivity.
Background
Myoglobin (Mb) is a protein that stores and distributes oxygen, is mainly present in the sarcoplasmic muscle of muscle tissue in an amount of about 0.2% to 2%, has an isoelectric point of 6.78, and is a complex chromoprotein. At present, many methods for detecting Mb include gold nanoparticle colorimetric analysis, local surface plasmon resonance of gold nanorods, microfluidic fluorescence chips, fluorescence immunochromatography, and electrochemical analysis methods based on graphene and ZnS modified nanocrystals, self-assembled monomolecular films, and the like. However, the above method has the disadvantages of low sensitivity, poor specificity, high price and high requirements for instruments.
The molecular imprinting technology is a subject technology developed in recent years, and specifically identifies template molecules by utilizing a molecular imprinting polymer to simulate the interaction between enzyme and a substrate. The core of the molecular imprinting technology is a molecular imprinted polymer, and the preparation of the molecular imprinted polymer generally comprises three processes: (1) selecting proper functional monomers, wherein the functional monomers and the template molecules form a certain reversible complex through the interaction (including covalent bond, hydrogen bond and other weak actions) between the functional groups of the functional monomers and the template molecules; (2) adding a cross-linking agent, initiating a monomer to polymerize under the action of an initiator, and freezing a reversible compound formed by template molecules and functional monomers to embed the template molecules in the formed rigid high polymer material; (3) the template molecules are eluted from the polymer material by physical or chemical means, leaving behind three-dimensional cavities in the spatial positions and structures occupied by the template molecules, which cavities match the template molecules in terms of size, shape and structure. Because the functional monomer has a functional group complementary to the functional group of the template molecule, the synthesized molecularly imprinted polymer can specifically identify and bind with the template molecule.
The electrochemical sensor is a three-electrode system consisting of a working electrode, a reference electrode and a counter electrode, is a device for detecting a detected sample by using the change of an electrochemical signal, has the characteristics of high sensitivity, simple and convenient preparation, low cost, easy miniaturization, suitability for field detection and the like, and is one of the most mature biosensing technologies so far. However, no relevant report exists so far about a preparation method of an imprinted polymer modified electrode which is simple to prepare, energy-saving, clean and environment-friendly and can detect myoglobin with high sensitivity.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a preparation method of the imprinted polymer modified electrode which is simple in preparation, energy-saving, clean and environment-friendly and can detect myoglobin with high sensitivity.
The technical solution of the invention is as follows: a preparation method of an imprinted polymer modified electrode for detecting myoglobin with high sensitivity is sequentially carried out according to the following steps:
a. dissolving 5mg of chitosan in 2ml of 0.2mol/L acetic acid solution to obtain acetic acid solution of chitosan; dispersing 1-2 mg of foamed graphene in an acetic acid solution of chitosan, and adjusting the pH value to 6.0 by using NaOH or HCl to obtain a first mixed solution; dripping the first mixed solution on the surface of a clean bare gold electrode, standing in a shade for 12 hours, and preparing an Au/3DG/Cs electrode; au/3DG/Cs is used as a working electrode, a platinum wire is used as a counter electrode, a saturated calomel electrode is used as a reference electrode, and 0.1mol/L HAuCl is contained at room temperature6·6H2In the solution of O, electrodeposition is carried out for 400s under the potential of-0.9V by utilizing a chronoamperometry to obtain an Au/3DG/Cs/nAu electrode;
b. dissolving 1mg of graphene in 1ml of a PBS (phosphate buffer solution) solution of Cetyl Trimethyl Ammonium Bromide (CTAB) with the pH value of 7.0 and the mass fraction of 0.1 percent to form a black suspension, and then adding an acetic acid solution of chitosan to obtain a second mixed solution; dripping the second mixed solution on the surface of the glassy carbon electrode, and standing for 12 hours in a shade place to obtain a GCE/GR/CTAB/Cs electrode;
c. modifying an Au/3DG/Cs/nAu electrode by using a bromine-containing compound with a mercapto end capping to obtain a bromide modified electrode, then putting a PBS solution containing 0.0125-0.2 mol/L alpha-methacrylic acid, 0.05-0.8 mg/mL myoglobin and 0.0025-0.04 mol/L N, N-methylene bisacrylamide into an electrolytic cell, putting the obtained bromide modified electrode into the solution in the electrolytic cell at room temperature, using a GCE/GR/CTAB/Cs electrode as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode as a reference electrode, and applying a constant potential for 50min at 0.185V to obtain the bromide modified electrode on which a polymer is deposited;
d. and taking out the bromide modified electrode on which the polymer is deposited, cleaning the bromide modified electrode with ultrapure water, soaking the bromide modified electrode in 0.5mol/L oxalic acid solution for 0.5h, and washing the bromide modified electrode with the ultrapure water to obtain the polymer modified electrode Au/3DG/PIPS for detecting the myoglobin with high sensitivity.
The invention has the advantages of simple preparation, energy saving, cleanness, environmental protection and the like, and the prepared imprinted polymer modified electrode is applied to the electrochemical imprinted sensor and can detect the myoglobin rapidly and with high sensitivity, and the experimental result shows that the linear range of the myoglobin standard solution detection is 1.0 × 10-10~1.0×10-1mg/L, detection limit of 1.10076 × 10-11mg/L（LOD，S/N=3）。
Drawings
FIG. 1 shows a modified electrode containing 5mmol/L [ Fe (CN) ] at different stages in the electrode preparation process of example 1 of the present invention6]3-/4-Cyclic voltammograms in +0.1mol/L KCl (pH 7.0 PBS) solution.
FIG. 2 is a schematic diagram of the selectivity of the polymer-modified electrode of example 1 of the present invention.
FIG. 3 is a differential pulse voltammetry curve A and a working curve B for myoglobin detection by the polymer modified electrode of example 1 of the present invention.
Detailed Description
Example 1:
the preparation method of the imprinted polymer modified electrode for detecting myoglobin with high sensitivity is sequentially carried out according to the following steps:
a. dissolving 5mg of chitosan (Cs) in 2ml of 0.2mol/L acetic acid solution to obtain acetic acid solution of chitosan; dispersing 2mg of foamed graphene (3 DG) in an acetic acid solution of chitosan, and adjusting the pH value to 6.0 by using NaOH or HCl to obtain a first mixed solution; dripping the first mixed solution on the surface of a clean bare gold electrode, standing in a shade and dark place for 12 hours to prepare an Au/3DG/Cs electrode; au/3DG/Cs is used as a working electrode, a platinum wire is used as a counter electrode, a saturated calomel electrode is used as a reference electrode, and 0.1mol/L HAuCl is contained at room temperature6·6H2In the solution of O, the solution is carried out at a potential of-0.9V by a chronoamperometryPerforming electrodeposition for 400s to obtain an Au/3DG/Cs/nAu electrode;
b. dissolving 1mg of Graphene (GR) in 1ml of PBS (phosphate buffer solution) of Cetyl Trimethyl Ammonium Bromide (CTAB) with the pH value of 7.0 and the mass fraction of 0.1 percent to form a black suspension, and then adding acetic acid solution of chitosan to obtain a second mixed solution; dripping the second mixed solution on the surface of a glassy carbon electrode, and standing for 12 hours in a shade dark place to obtain a GCE/GR/CTAB/Cs electrode;
c. modifying an Au/3DG/Cs/nAu electrode by using a sulfhydryl-terminated bromine-containing compound to obtain a bromide modified electrode, then putting a PBS solution containing 0.05mol/L alpha-methacrylic acid, 0.2 mg/mL myoglobin and 0.01mol/L N, N-methylene bisacrylamide into an electrolytic cell, putting the obtained bromide modified electrode into the solution in the electrolytic cell at room temperature, using a GCE/GR/CTAB/Cs electrode as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode as a reference electrode, and applying a constant potential for 50min at 0.185V to obtain the bromide modified electrode deposited with a polymer;
d. and taking out the bromide modified electrode on which the polymer is deposited, cleaning the bromide modified electrode with ultrapure water, soaking the bromide modified electrode in 0.5mol/L oxalic acid (OX) solution for 0.5h to remove the myoglobin template in the polymer, and washing the washed myoglobin template with the ultrapure water to obtain the polymer modified electrode Au/3DG/PIPS for detecting the myoglobin with high sensitivity.
Example 1 in the preparation process of the electrode, the modified electrode at different stages contains 5mmol/L [ Fe (CN)6]3-/4-The cyclic voltammogram in a solution of +0.1 ml/L KCl (pH 7.0 PBS) is shown in FIG. 1.
FIG. 1 is a CV curve of a modified electrode, in which a bare gold electrode in curve 1 has a pair of reversible redox peaks near 0.2V, and the peaks are on the surface of the bare gold electrode [ FeCN ]6]3-/4- Curve 2 is the oxidation reduction peak of the probe ion, the peak current is higher than curve 1, indicating that the conductivity of the modified electrode increases sharply after the deposition of nanogold, curve 3 is the CV curve of the initiator modified gold electrode, the peak current is lower than curve 1, because the self-assembly initiator prevents the probe ion from reaching the electrode surface, the CV curve when polymerized with α -methacrylic acid is as shown in curve 4Curve 5 shows that the redox peak current increases after myoglobin elutes from the polymer modified electrode due to myoglobin elution and increased conductivity, resulting in the formation of imprinted voids.
To examine the selectivity of the Au/3DG/PIPS modified electrode obtained in the example of the present invention, lysozyme (Lyz) (MW14.4 kDa), hemoglobin (Hb) (MW 64 kDa), Bovine Serum Albumin (BSA) (MW 66.44 kDa), and mouse antibody (IgG) (MW 150 kDa) were used as the interferents for the experiment. Using differential pulse voltammetry by measuring the concentration to be 10-3The selectivity of the electrode obtained by the invention is studied by the difference of response signals of different proteins to the imprinted electrode (polymer modified electrode) and the non-imprinted electrode (the preparation method is the same as the imprinted electrode, except that myoglobin is not added in the process) at mg/L, and the result is shown in figure 2. FIG. 2 shows that the Au/3DG/PIPs modified electrode of the invention detects hemoglobin response signal deltaI21.70 μ A, 6.36, 10.64, 10.83 and 12.35 times higher than Lyz, Hb, BSA and IgG, respectively. The result shows that the Au/3DG/PIPS modified electrode has better selectivity on the target protein (Mb). The selectivity of the electrode was evaluated by the imprinting factor (K), calculated as K = ΔI(PIPs/Au)/ΔI(NIPs/Au), whereinI(NIPs) is the response signal of the Au/3DG/NIPs modified electrode to the protein, and ΔI(PIPs) are response signals of Au/3DG/PIPs modified electrodes to proteins. The K values by calculation of Lyz, Hb, BSA, IgG and Mb were 2.21, 1.40, 1.43, 1.46 and 8.23, respectively. The K value of Mb is the largest, which indicates that the capacity of detecting Mb of the Au/3DG/PIPS modified electrode is far higher than that of the Au/3DG/NIPs modified electrode.
FIG. 3 is a differential pulse voltammetry curve A and a working curve B for myoglobin detection by the polymer modified electrode of example 1 of the present invention.
In FIG. 3A, the myoglobin concentrations corresponding to curves 1-11 are 0 and 10, respectively-10，10-9，10-8，10-7，10-6，10-5，10-4，10-3，10-2，10-1mg/L. As can be seen from fig. 3, as the Mb concentration increases, the DPV peak current decreases instead. From the difference in peak current (response signal, Δ) of the DPVI) The work curve (shown in FIG. 3B) of the modified electrode for detecting Mb is obtained by plotting the logarithm of the Mb concentration, and the result shows that the linear response range of the Au/3DG/PIPS modified electrode to the Mb concentration is 1.0 × 10-10~1.0×10-1mg/L, linear regression equation of ΔI(μA)=3.07972logC(mg/L) +32.45557, correlation coefficient 0.99972, detection limit 1.10076 × 10-11mg/L（LOD，S/N=3）。
Example 2:
the preparation method of the imprinted polymer modified electrode for detecting myoglobin with high sensitivity is sequentially carried out according to the following steps:
a. dissolving 5mg of chitosan (Cs) in 2ml of 0.2mol/L acetic acid solution to obtain acetic acid solution of chitosan; dispersing 1mg of foamed graphene (3 DG) in an acetic acid solution of chitosan, and adjusting the pH value to 6.0 by using NaOH or HCl to obtain a first mixed solution; dripping the first mixed solution on the surface of a clean bare gold electrode, standing in a shade and dark place for 12 hours to prepare an Au/3DG/Cs electrode; au/3DG/Cs is used as a working electrode, a platinum wire is used as a counter electrode, a saturated calomel electrode is used as a reference electrode, and 0.1mol/L HAuCl is contained at room temperature6·6H2In the solution of O, electrodeposition is carried out for 400s under the potential of-0.9V by utilizing a chronoamperometry to obtain an Au/3DG/Cs/nAu electrode;
b. dissolving 1mg of Graphene (GR) in 1ml of PBS (phosphate buffer solution) of Cetyl Trimethyl Ammonium Bromide (CTAB) with the pH value of 7.0 and the mass fraction of 0.1 percent to form a black suspension, and then adding acetic acid solution of chitosan to obtain a second mixed solution; dripping the second mixed solution on the surface of a glassy carbon electrode, and standing for 12 hours in a shade dark place to obtain a GCE/GR/CTAB/Cs electrode;
c. modifying an Au/3DG/Cs/nAu electrode by using a sulfhydryl-terminated bromine-containing compound to obtain a bromide modified electrode, then putting a PBS solution containing 0.1mol/L alpha-methacrylic acid, 0.4 mg/mL myoglobin and 0.02mol/L N, N-methylene bisacrylamide into an electrolytic cell, putting the obtained bromide modified electrode into the solution in the electrolytic cell at room temperature, using a GCE/GR/CTAB/Cs electrode as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode as a reference electrode, and applying a constant potential for 50min at 0.185V to obtain the bromide modified electrode deposited with a polymer;
d. and taking out the bromide modified electrode on which the polymer is deposited, cleaning the bromide modified electrode with ultrapure water, soaking the bromide modified electrode in 0.5mol/L oxalic acid (OX) solution for 0.5h to remove the myoglobin template in the polymer, and washing the washed myoglobin template with the ultrapure water to obtain the polymer modified electrode Au/3DG/PIPS for detecting the myoglobin with high sensitivity.
Example 3:
the preparation method of the imprinted polymer modified electrode for detecting myoglobin with high sensitivity is sequentially carried out according to the following steps:
a. dissolving 5mg of chitosan (Cs) in 2ml of 0.2mol/L acetic acid solution to obtain acetic acid solution of chitosan; dispersing 2mg of foamed graphene (3 DG) in an acetic acid solution of chitosan, and adjusting the pH value to 6.0 by using NaOH or HCl to obtain a first mixed solution; dripping the first mixed solution on the surface of a clean bare gold electrode, standing in a shade and dark place for 12 hours to prepare an Au/3DG/Cs electrode; au/3DG/Cs is used as a working electrode, a platinum wire is used as a counter electrode, a saturated calomel electrode is used as a reference electrode, and 0.1mol/L HAuCl is contained at room temperature6·6H2In the solution of O, electrodeposition is carried out for 400s under the potential of-0.9V by utilizing a chronoamperometry to obtain an Au/3DG/Cs/nAu electrode;
b. dissolving 1mg of Graphene (GR) in 1ml of PBS (phosphate buffer solution) of Cetyl Trimethyl Ammonium Bromide (CTAB) with the pH value of 7.0 and the mass fraction of 0.1 percent to form a black suspension, and then adding acetic acid solution of chitosan to obtain a second mixed solution; dripping the second mixed solution on the surface of a glassy carbon electrode, and standing for 12 hours in a shade dark place to obtain a GCE/GR/CTAB/Cs electrode;
c. modifying an Au/3DG/Cs/nAu electrode by using a sulfhydryl-terminated bromine-containing compound to obtain a bromide modified electrode, then putting a PBS solution containing 0.2mol/L alpha-methacrylic acid, 0.8mg/mL myoglobin and 0.04mol/L N, N-methylene bisacrylamide into an electrolytic cell, putting the obtained bromide modified electrode into the solution in the electrolytic cell at room temperature, using a GCE/GR/CTAB/Cs electrode as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode as a reference electrode, and applying a constant potential for 50min at 0.185V to obtain the bromide modified electrode deposited with a polymer;
d. and taking out the bromide modified electrode on which the polymer is deposited, cleaning the bromide modified electrode with ultrapure water, soaking the bromide modified electrode in 0.5mol/L oxalic acid (OX) solution for 0.5h to remove the myoglobin template in the polymer, and washing the washed myoglobin template with the ultrapure water to obtain the polymer modified electrode Au/3DG/PIPS for detecting the myoglobin with high sensitivity.
Example 4:
the preparation method of the imprinted polymer modified electrode for detecting myoglobin with high sensitivity is sequentially carried out according to the following steps:
a. dissolving 5mg of chitosan (Cs) in 2ml of 0.2mol/L acetic acid solution to obtain acetic acid solution of chitosan; dispersing 2mg of foamed graphene (3 DG) in an acetic acid solution of chitosan, and adjusting the pH value to 6.0 by using NaOH or HCl to obtain a first mixed solution; dripping the first mixed solution on the surface of a clean bare gold electrode, standing in a shade and dark place for 12 hours to prepare an Au/3DG/Cs electrode; au/3DG/Cs is used as a working electrode, a platinum wire is used as a counter electrode, a saturated calomel electrode is used as a reference electrode, and 0.1mol/L HAuCl is contained at room temperature6·6H2In the solution of O, electrodeposition is carried out for 400s under the potential of-0.9V by utilizing a chronoamperometry to obtain an Au/3DG/Cs/nAu electrode;
b. dissolving 1mg of Graphene (GR) in 1ml of PBS (phosphate buffer solution) of Cetyl Trimethyl Ammonium Bromide (CTAB) with the pH value of 7.0 and the mass fraction of 0.1 percent to form a black suspension, and then adding acetic acid solution of chitosan to obtain a second mixed solution; dripping the second mixed solution on the surface of a glassy carbon electrode, and standing for 12 hours in a shade dark place to obtain a GCE/GR/CTAB/Cs electrode;
c. modifying an Au/3DG/Cs/nAu electrode by using a sulfhydryl-terminated bromine-containing compound to obtain a bromide modified electrode, then putting a PBS solution containing 0.025mol/L alpha-methacrylic acid, 0.1 mg/mL myoglobin and 0.005mol/L N, N-methylene bisacrylamide into an electrolytic cell, putting the obtained bromide modified electrode into the solution in the electrolytic cell at room temperature, using a GCE/GR/CTAB/Cs electrode as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode as a reference electrode, and applying a constant potential for 50min at 0.185V to obtain the bromide modified electrode deposited with a polymer;
d. and taking out the bromide modified electrode on which the polymer is deposited, cleaning the bromide modified electrode with ultrapure water, soaking the bromide modified electrode in 0.5mol/L oxalic acid (OX) solution for 0.5h to remove the myoglobin template in the polymer, and washing the washed myoglobin template with the ultrapure water to obtain the polymer modified electrode Au/3DG/PIPS for detecting the myoglobin with high sensitivity.
Example 5:
the preparation method of the imprinted polymer modified electrode for detecting myoglobin with high sensitivity is sequentially carried out according to the following steps:
a. dissolving 5mg of chitosan (Cs) in 2ml of 0.2mol/L acetic acid solution to obtain acetic acid solution of chitosan; dispersing 1mg of foamed graphene (3 DG) in an acetic acid solution of chitosan, and adjusting the pH value to 6.0 by using NaOH or HCl to obtain a first mixed solution; dripping the first mixed solution on the surface of a clean bare gold electrode, standing in a shade and dark place for 12 hours to prepare an Au/3DG/Cs electrode; au/3DG/Cs is used as a working electrode, a platinum wire is used as a counter electrode, a saturated calomel electrode is used as a reference electrode, and 0.1mol/L HAuCl is contained at room temperature6·6H2In the solution of O, electrodeposition is carried out for 400s under the potential of-0.9V by utilizing a chronoamperometry to obtain an Au/3DG/Cs/nAu electrode;
b. dissolving 1mg of Graphene (GR) in 1ml of PBS (phosphate buffer solution) of Cetyl Trimethyl Ammonium Bromide (CTAB) with the pH value of 7.0 and the mass fraction of 0.1 percent to form a black suspension, and then adding acetic acid solution of chitosan to obtain a second mixed solution; dripping the second mixed solution on the surface of a glassy carbon electrode, and standing for 12 hours in a shade dark place to obtain a GCE/GR/CTAB/Cs electrode;
c. modifying an Au/3DG/Cs/nAu electrode by using a sulfhydryl-terminated bromine-containing compound to obtain a bromide modified electrode, then putting a PBS solution containing 0.0125mol/L alpha-methacrylic acid, 0.05 mg/mL myoglobin and 0.0025mol/L N, N-methylene bisacrylamide into an electrolytic cell, putting the obtained bromide modified electrode into the solution in the electrolytic cell at room temperature, using a GCE/GR/CTAB/Cs electrode as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode as a reference electrode, and applying a constant potential for 50min at 0.185V to obtain the bromide modified electrode on which a polymer is deposited;
d. and taking out the bromide modified electrode on which the polymer is deposited, cleaning the bromide modified electrode with ultrapure water, soaking the bromide modified electrode in 0.5mol/L oxalic acid (OX) solution for 0.5h to remove the myoglobin template in the polymer, and washing the washed myoglobin template with the ultrapure water to obtain the polymer modified electrode Au/3DG/PIPS for detecting the myoglobin with high sensitivity.

Claims (1)

1. A preparation method of an imprinted polymer modified electrode for detecting myoglobin with high sensitivity is sequentially carried out according to the following steps:
dissolving 5mg of chitosan in 2ml of 0.2mol/L acetic acid solution to obtain acetic acid solution of chitosan; dispersing 1-2 mg of foamed graphene in an acetic acid solution of chitosan, and adjusting the pH value to 6.0 by using NaOH or HCl to obtain a first mixed solution; dripping the first mixed solution on the surface of a clean bare gold electrode, standing in a shade for 12 hours, and preparing an Au/3DG/Cs electrode; au/3DG/Cs is used as a working electrode, a platinum wire is used as a counter electrode, a saturated calomel electrode is used as a reference electrode, and the solution is prepared by adding 0.1mol/LHAuCl into the solution at room temperature6·6H2In the solution of O, electrodeposition is carried out for 400s under the potential of-0.9V by utilizing a chronoamperometry to obtain an Au/3DG/Cs/nAu electrode;
dissolving 1mg of graphene in 1ml of PBS (phosphate buffer solution) solution of hexadecyl trimethyl ammonium bromide with the pH value of 7.0 and the mass fraction of 0.1% to form a black suspension, and then adding acetic acid solution of chitosan to obtain a second mixed solution; dripping the second mixed solution on the surface of the glassy carbon electrode, and standing for 12 hours in a shade place to obtain a GCE/GR/CTAB/Cs electrode;
modifying an Au/3DG/Cs/nAu electrode by using a bromine-containing compound with a mercapto end capping to obtain a bromide modified electrode, then putting a PBS solution containing 0.0125-0.2 mol/L alpha-methacrylic acid, 0.05-0.8 mg/mL myoglobin and 0.0025-0.04 mol/L N, N-methylene bisacrylamide into an electrolytic cell, putting the obtained bromide modified electrode into the solution in the electrolytic cell at room temperature, using a GCE/GR/CTAB/Cs electrode as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode as a reference electrode, and applying a constant potential for 50min at 0.185V to obtain the bromide modified electrode on which a polymer is deposited;
d. and taking out the bromide modified electrode on which the polymer is deposited, cleaning the bromide modified electrode with ultrapure water, soaking the bromide modified electrode in 0.5mol/L oxalic acid solution for 0.5h, and washing the bromide modified electrode with the ultrapure water to obtain the polymer modified electrode Au/3DG/PIPS for detecting the myoglobin with high sensitivity.
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