Helicobacter pylori ureB molecular imprinting electrochemical sensor and preparation method thereof

Abstract

The invention discloses a helicobacter pylori ureB molecular imprinting electrochemical sensor and a preparation method thereof, and belongs to the technical field of biological analysis. The technical problems that in the prior art, helicobacter pylori detection time is long, operation is complex, and dependence on laboratory equipment is high are solved, an electrode substrate is plated with gold to obtain a gold-plated electrode, the gold-plated electrode is treated with ethyl alcohol of 4-aminothiophenol and a PBS buffer solution of a protein cross-linking agent DTSSP to obtain a cleavable covalent bond fixed support electrode, and the cleavable covalent bond fixed support electrode is used for detecting helicobacter pylori. The preparation method comprises the following steps: preparing an ePDA film covered electrode, treating the ePDA film covered electrode with a ureB solution and a DA functional monomer solution to obtain an ePDA film covered electrode, treating the ePDA film covered electrode with an ethanol solution of 2-mercaptoethanol and an acetic acid solution to obtain a molecularly imprinted polymer electrode, and taking the molecularly imprinted polymer electrode as a working electrode to obtain the helicobacter pylori ureB protein molecularly imprinted electrochemical sensor. When the helicobacter pylori ureB molecularly imprinted electrochemical sensor is used for detecting helicobacter pylori ureB, the current response is good, the stability is high, and the operability is high.

Description

Technical Field

[0001] The invention relates to the technical field of bioanalysis, and in particular to a Helicobacter pylori ureB molecular imprinting electrochemical sensor and a preparation method thereof. Background Art

[0002] With the continuous development of urbanization and industrialization, water pollution has become one of the most serious environmental problems in today's society. Pathogen pollutants in water bodies not only pose a potential threat to human health, but also have an impact on the balance of the ecosystem that cannot be ignored. Among these pathogen pollutants, the presence of Helicobacter pylori has attracted widespread attention from humans. It is a Gram-negative bacterium and the main pathogenic factor causing gastric and duodenal diseases, including chronic gastritis, peptic ulcer and gastric cancer. However, the existing Hp epidemiological analysis reports are all statistical analyses based on clinical diagnostic data collected in different time and space environments, and their results inevitably have time lags and data uncertainties. Therefore, the use of reasonable detection technology to accurately and quantitatively analyze Hp will help promote the theoretical research progress of the correlation between Hp epidemiological surveys and related induced diseases, and can provide relevant environmental health analysis technology support for cities to respond to future public health emergencies.

[0003] At present, traditional detection methods show obvious limitations in practical applications. Not only is the detection time long and the operation process complicated and cumbersome, but it is also highly dependent on laboratory equipment and professional skills. These factors have limited the efficiency and popularity of detection to varying degrees. Summary of the invention

[0004] This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are described in further detail in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all of the drawings, and each claim.

[0005] Based on the deficiencies of the prior art, the present invention provides a Helicobacter pylori ureB molecular imprinting electrochemical sensor and a preparation method thereof, the specific contents of which are as follows.

[0006] The present invention provides a method for preparing a Helicobacter pylori ureB protein molecular imprinting electrochemical sensor, comprising the following steps: Step 1: Incubate the gold-plated electrode with an ethanol solution of 4-aminothiophenol to obtain 4-ATP / Au / SPE, and then incubate it with a PBS buffer solution of a protein crosslinker DTSSP to obtain a cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE.

[0007] The present invention uses an ethanol solution of 4-aminothiophenol to incubate the gold-plated electrode, and the sulfur atom of the 4-aminothiophenol forms a chemical bond with gold to be fixed on the electrode surface, thereby increasing the conductivity; it adsorbs on the electrode surface at multiple sites to increase the coverage rate, reduce the corrosion and wear of the gold plating layer, and extend the life of the electrode; the amino group can also combine with the N-hydroxysuccinimide active esters at both ends of the protein cross-linking agent DTSSP to achieve the fixation of DTSSP on the electrode surface.

[0008] The N-hydroxysuccinimide active esters at both ends of the protein cross-linker DTSSP combine with the amino groups in 4-aminobenzenethiol on the electrode surface, thereby being fixed on the electrode surface; the DTSSP molecule contains disulfide bonds, and using DTSSP as a cleavage layer is conducive to the generation of ureB molecular imprinted polymers on the electrode surface.

[0009] Step 2: Incubate the cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE with ureB solution to obtain ureB / DTSSP / 4-ATP / Au / SPE, then drop-cast the DA functional monomer solution onto the surface of ureB / DTSSP / 4-ATP / Au / SPE, and form an ePDA film by electrochemical polymerization to obtain ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film.

[0010] The surface of ureB contains many amino acid residues, such as lysine residues containing primary amine groups, which can undergo nucleophilic substitution reaction with the N-hydroxysuccinimide active esters at both ends of DTSSP to form stable amide bonds, thereby fixing it on the electrode surface and realizing the specific recognition of ureB by the electrode.

[0011] DA was simulated as a suitable functional monomer through molecular docking because the binding energy of DA is the strongest at -5.1 kcal / mol compared with the binding energy of m-phenylenediamine, p-phenylenediamine and aniline with ureB; dopamine can assemble with ureB on the electrode surface to form a polymer, thereby achieving specific imprinting of ureB.

[0012] Step 3: Use 2-mercaptoethanol ethanol solution to cleave the S–S bonds on the surface of ePDA / ureB / DTSSP / 4-ATP / Au / SPE, and then rinse the cleaved ePDA / ureB / DTSSP / 4-ATP / Au / SPE with acetic acid solution to obtain molecularly imprinted polymer electrodes ureB-MIPs.

[0013] The thiol group in 2-mercaptoethanol has strong nucleophilicity and will attack the S-S bond of DTSSP, thereby breaking the S-S bond of DTSSP and releasing ureB; the self-polymerization reaction of DA is more likely to occur under neutral or alkaline conditions. The acetic acid solution is used to make the electrode surface in an acidic environment, thereby inhibiting the self-polymerization reaction of DA; the DA molecule is a polar molecule. The acetic acid solution, as a polar solvent, can reduce the degree of aggregation of DA molecules on the electrode surface, thereby reducing the collision of intermolecular groups and inhibiting the self-polymerization reaction of DA molecules.

[0014] Step 4: Using saturated potassium chloride as the reference electrode, a platinum wire electrode as the counter electrode, and molecularly imprinted polymer electrode ureB-MIPs as the working electrode, a Helicobacter pylori ureB protein molecular imprinting electrochemical sensor was prepared.

[0015] Preferably, in step 1, the method for preparing the gold-plated electrode comprises the following steps: A1. Prepare gold plating electrolyte: mix chloroauric acid aqueous solution, deionized water and sodium sulfate solution evenly to obtain gold plating electrolyte; A2. Rinse the electrode substrate with ultrapure water, treat the electrode substrate with a gold-plating electrolyte, and deposit gold on the surface of the electrode substrate by cyclic voltammetry to obtain a gold-plated electrode.

[0016] The mass ratio of the aqueous solution of chloroauric acid, deionized water, and sodium sulfate solution is 2.5-3.5:4-8:0.8-1.2; The concentration of the sodium sulfate solution is 80-120 mM; The electrode substrate is a screen-printed electrode or a glassy carbon electrode; The potential parameter range of the cyclic voltammetry method is -1 to 1 V, the reference electrode is Ag / AgCl, the number of scans is 8 to 12, and the scan rate is 50 mV / s.

[0017] Gold has high conductivity. By plating gold on the electrode substrate, electrons can be quickly transferred between the electrode and the electrolyte, making the electrochemical sensor more sensitive to the detected substance.

[0018] Preferably, in step 1, the concentration of the ethanol solution of 4-aminothiophenol is 80-120 mM, and the concentration of the PBS buffer of the protein crosslinking agent DTSSP is 8-20 mM; the incubation time of the ethanol solution of 4-aminothiophenol is 20-40 min, and the incubation time of the PBS buffer of the protein crosslinking agent DTSSP is 20-40 min.

[0019] Preferably, in step 2, the DA functional monomer solution is prepared by dissolving the DA functional monomer in Tris-HCl buffer.

[0020] Preferably, in step 2, the concentration of the ureB solution is 0.14-0.18 μM, the concentration of the DA functional monomer solution is 0.25-0.35 mM; and the incubation time of the ureB solution is 20-40 min.

[0021] Preferably, in step 2, the potential parameter range of the electrochemical polymerization method is -0.8 to 0.8 V, the reference electrode is Ag / AgCl, the number of scans is 8 to 12, and the scan rate is 50 mV / s.

[0022] Preferably, in step 3, the concentration of the 2-mercaptoethanol ethanol solution is 0.05-0.15 M, and the mass fraction of the acetic acid solution is 8%-12%.

[0023] The present invention also provides a Helicobacter pylori ureB protein molecular imprinting electrochemical sensor prepared by the above-mentioned Helicobacter pylori ureB protein molecular imprinting electrochemical sensor preparation method.

[0024] The present invention also provides a method for detecting Helicobacter pylori using the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor, comprising the following steps: B1. Rinse the surface of the molecularly imprinted polymer electrode ureB-MIPs with deionized water, and incubate the molecularly imprinted polymer electrode ureB-MIPs with ureB solutions of different concentrations for 10 min. B2, Fe(CN) 6 3- / 4- As a redox probe, the redox peak changes of the probe on the molecularly imprinted polymer electrode ureB-MIPs were measured by square wave anodic stripping voltammetry, thereby quantitatively analyzing the concentration of ureB in various liquid samples.

[0025] Preferably, the potential parameter range of the square wave anodic stripping voltammetry is -0.8 to 0.8 V, the reference electrode is Ag / AgCl, the potential amplitude is 70 mV, the potential increment is 11 mV, the sampling width is 5 ms, and the pulse period is 20 ms.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention uses Au electrodeposition to modify the electrode substrate to improve the signal transmission capability, forms an ePDA film by electrochemical polymerization, uses an eluent to form a molecular imprinting polymer electrode ureB-MIPs, and studies the ability of the sensor system to selectively recognize ureB by square wave voltammetry. The Helicobacter pylori ureB molecular imprinting electrochemical sensor prepared by the present invention has good current response, strong stability, high repeatability and operability, and the preparation method has low requirements on experimental equipment and conditions, is low cost, and is safe and simple to operate.

[0027] (2) The present invention uses an ethanol solution of 4-aminothiophenol to incubate the gold-plated electrode. The sulfur atom of 4-aminothiophenol forms a chemical bond with gold and is fixed to the electrode surface, thereby increasing the conductivity; it adsorbs to multiple sites on the electrode surface, thereby increasing the coverage rate, reducing corrosion and wear of the gold plating layer, and extending the life of the electrode; the amino group can also combine with the N-hydroxysuccinimide active esters at both ends of the protein cross-linking agent DTSSP to achieve the fixation of DTSSP on the electrode surface.

[0028] (3) The present invention uses an ethanol solution of 4-aminothiophenol to incubate a gold-plated electrode. The sulfur atom in 4-aminothiophenol forms a stable chemical bond with gold, thereby fixing it on the electrode surface and increasing the conductivity of the electrode; 4-aminothiophenol forms multi-site adsorption on the electrode surface, increasing the coverage of the electrode surface, reducing corrosion and wear of the gold plating, and thus increasing the service life of the electrode; the amino group in 4-aminothiophenol can combine with the N-hydroxysuccinimide active ester at both ends of the protein crosslinker DTSSP, thereby achieving fixation of the protein crosslinker DTSSP on the electrode surface; the N-hydroxysuccinimide active ester at both ends of the protein crosslinker DTSSP combines with the amino group in 4-aminothiophenol on the electrode surface, thereby fixing it on the electrode surface; the DTSSP molecule contains a disulfide bond, and using DTSSP as a cleavage layer is conducive to the generation of ureB molecular imprinted polymers on the electrode surface.

[0029] (4) The present invention selects DA as a suitable functional monomer for molecular docking simulation because, compared with the binding energies of m-phenylenediamine, p-phenylenediamine and aniline with HpureB, DA has the strongest binding energy of -5.1 kcal / mol; dopamine can assemble with ureB on the electrode surface to form a polymer, thereby achieving specific imprinting of ureB; by reacting ureB and DTSSP, the surface of ureB contains many amino acid residues, such as lysine residues containing primary amine groups, which can undergo nucleophilic substitution reaction with the N-hydroxysuccinimide active esters at both ends of DTSSP to form stable amide bonds, thereby being fixed on the electrode surface, thereby achieving specific recognition of ureB by the electrode.

[0030] (5) The present invention uses an ethanol solution of 2-mercaptoethanol to cleave the S-S bond of DTSSP. The thiol group in 2-mercaptoethanol has strong nucleophilicity and will attack the S-S bond of DTSSP, thereby causing the S-S bond of DTSSP to break and release ureB. The self-polymerization reaction of DA is more likely to occur under neutral or alkaline conditions. The electrode surface is placed in an acidic environment using acetic acid solution, thereby inhibiting the self-polymerization reaction of DA. The DA molecule is a polar molecule. The acetic acid solution, as a polar solvent, can reduce the degree of aggregation of DA molecules on the electrode surface, thereby reducing the collision of intermolecular groups and inhibiting the self-polymerization reaction of DA molecules.

[0031] (6) The present invention uses chloroauric acid solution to plate gold on the electrode substrate. Gold has a high conductivity. By plating gold on the electrode substrate, electrons can be quickly transferred between the electrode and the electrolyte, thereby making the electrochemical sensor more sensitive to the detection substance.

[0032] (7) The Helicobacter pylori ureB molecular imprinting electrochemical sensor prepared by the present invention has the advantages of low cost, high detection sensitivity, fast detection speed and convenient carrying. It is also easy to operate, quick to respond and highly specific when detecting different concentrations of ureB. The detection range of the Helicobacter pylori ureB molecular imprinting electrochemical sensor provided by the present invention can reach 10 -6 Therefore, its development is more suitable for detecting various urban water environment samples with large volume and low concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A schematic diagram of a process for preparing a Helicobacter pylori ureB molecular imprinting electrochemical sensor according to an embodiment of the present invention; Figure 2 A schematic diagram of a process for preparing a Helicobacter pylori ureB molecular imprinting electrochemical sensor according to an embodiment of the present invention; Figure 3 This is a scanning electron microscope image of the screen-printed electrode substrate and the gold-plated electrode in Example 2 of the present invention; Figure 4 Cyclic voltammograms of the screen-printed electrode substrate of Example 2 of the present invention, the gold-plated electrode of Example 2, the 4-ATP / Au / SPE of Example 4-1, the DTSSP / 4-ATP / Au / SPE of Example 4-1, the ureB / DTSSP / 4-ATP / Au / SPE of Example 6-1, the ePDA / ureB / DTSSP / 4-ATP / Au / SPE of Example 6-1, and the molecularly imprinted polymer electrode ureB-MIPs of Example 7-1; Figure 5 Scanning electron micrographs of the ePDA / ureB / DTSSP / 4-ATP / Au / SPE of Example 6-1 and the molecularly imprinted polymer electrode ureB-MIPs of Example 7-1 of the present invention; Figure 6 Energy dispersive X-ray spectroscopy characterization diagrams of ePDA / ureB / DTSSP / 4-ATP / Au / SPE of Example 6-1 of the present invention and molecular imprinting polymer electrode ureB-MIPs of Example 7-1; Figure 7Square wave voltammogram, electrochemical impedance diagram, and nonlinear logarithmic calibration curve diagram of the Helicobacter pylori ureB molecular imprinting electrochemical sensor prepared in Example 8 of the present invention when detecting samples with ureB concentrations of 100fg / mL-10ng / mL. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present application, but should not be construed as limiting the present application. The following describes the Helicobacter pylori ureB molecular imprinting electrochemical sensor and its preparation method in the embodiments of the present application with reference to the accompanying drawings.

[0036] Specifically, Figure 1 This is a schematic flow chart of a method for preparing a Helicobacter pylori ureB molecular imprinting electrochemical sensor provided in an embodiment of the present application.

[0037] like Figure 1 As shown, the preparation method of Helicobacter pylori ureB molecular imprinting electrochemical sensor includes the following steps: In step S101, the electrode substrate is treated with a gold-plating electrolyte to obtain a gold-plated electrode.

[0038] The electrode substrate may be a glassy carbon electrode or a screen-printed electrode.

[0039] It is understandable that gold has a high conductivity, and using a gold plating electrolyte to plate gold on the surface of the electrode substrate can enable electrons to be quickly transferred between the electrode and the electrolyte, thereby making the electrochemical sensor more sensitive to the detection substance.

[0040] In step S102, the gold-plated electrode is treated with an ethanol solution of 4-aminothiophenol and a PBS buffer solution of a protein cross-linking agent DTSSP to obtain a cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE.

[0041] It can be understood that when the gold-plated electrode is incubated with an ethanol solution of 4-aminothiophenol, the sulfur atom in 4-aminothiophenol forms a stable chemical bond with gold, thereby fixing it on the electrode surface and increasing the conductivity of the electrode; 4-aminothiophenol forms multi-site adsorption on the electrode surface, increasing the coverage of the electrode surface, reducing corrosion and wear of the gold plating, and thus increasing the service life of the electrode; the amino group in 4-aminothiophenol can combine with the N-hydroxysuccinimide active esters at both ends of the protein crosslinker DTSSP, thereby achieving fixation of the protein crosslinker DTSSP on the electrode surface; the N-hydroxysuccinimide active esters at both ends of the protein crosslinker DTSSP combine with the amino group in 4-aminothiophenol on the electrode surface, thereby fixing it on the electrode surface; the DTSSP molecule contains disulfide bonds, and using DTSSP as a cleavage layer is conducive to the generation of ureB molecular imprinted polymers on the electrode surface.

[0042] In step S103, the cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE is treated with ureB solution and DA functional monomer solution to obtain ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film.

[0043] It is understandable that the surface of ureB contains many amino acid residues, such as lysine residues containing primary amine groups, which can undergo nucleophilic substitution reactions with the N-hydroxysuccinimide active esters at both ends of DTSSP to form stable amide bonds, thereby fixing it on the electrode surface and realizing the specific recognition of ureB by the electrode.

[0044] DA was simulated as a suitable functional monomer through molecular docking because the binding energy of DA is the strongest at -5.1 kcal / mol compared with the binding energy of m-phenylenediamine, p-phenylenediamine and aniline with ureB; dopamine can assemble with ureB on the electrode surface to form a polymer, thereby achieving specific imprinting of ureB.

[0045] In step S104, ePDA / ureB / DTSSP / 4-ATP / Au / SPE is treated with 2-mercaptoethanol ethanol solution and acetic acid solution to obtain molecularly imprinted polymer electrodes ureB-MIPs.

[0046] It is understandable that the thiol group in 2-mercaptoethanol has strong nucleophilicity and will attack the S-S bond of DTSSP, thereby causing the S-S bond of DTSSP to break and release ureB; the self-polymerization reaction of DA is more likely to occur under neutral or alkaline conditions, and the acetic acid solution is used to make the electrode surface in an acidic environment, thereby inhibiting the self-polymerization reaction of DA; the DA molecule is a polar molecule, and the acetic acid solution, as a polar solvent, can reduce the degree of aggregation of DA molecules on the electrode surface, thereby reducing the collision of intermolecular groups and inhibiting the self-polymerization reaction of DA molecules.

[0047] In step S105, saturated potassium chloride is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and a molecularly imprinted polymer electrode ureB-MIPs is used as a working electrode to obtain a Helicobacter pylori ureB protein molecularly imprinted electrochemical sensor.

[0048] According to a method for preparing a Helicobacter pylori ureB molecular imprinting electrochemical sensor provided in an embodiment of the present application, after gold is plated on the surface of the electrode substrate, it is treated with an ethanol solution of 4-aminothiophenol and a PBS buffer solution of a protein cross-linking agent DTSSP to obtain a cleavable covalent bond fixed bracket DTSSP / 4-ATP / Au / SPE, which is then treated with a ureB solution and a DA functional monomer solution to obtain an ePDA film-covered ePDA / ureB / DTSSP / 4-ATP / Au / SPE, which is then treated with an ethanol solution of 2-mercaptoethanol and an acetic acid solution to obtain a molecular imprinting polymer electrode ureB-MIPs, and then a saturated potassium chloride is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and a molecular imprinting polymer electrode ureB-MIPs is used as a working electrode to obtain a Helicobacter pylori ureB protein molecular imprinting electrochemical sensor. The present invention overcomes the technical problems of traditional detection methods in actual detection, such as long detection time, complicated operation process and high dependence on laboratory equipment. The Helicobacter pylori ureB protein molecular imprinting electrochemical sensor prepared by the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor preparation method of the present invention has the advantages of low cost, high detection sensitivity, fast detection speed and convenient carrying, and is easy to operate, quick to respond and strong specificity when detecting different concentrations of ureB.

[0049] The following is a specific example to illustrate the preparation method of Helicobacter pylori ureB molecular imprinting electrochemical sensor. Figure 2 As shown, the preparation method of the Helicobacter pylori ureB molecular imprinting electrochemical sensor comprises the following steps: S201. Mix chloroauric acid aqueous solution, deionized water, and 100 mM sodium sulfate solution in a mass ratio of 2.5-3.5:4-8:0.8-1.2, and mix evenly using an oscillator to obtain a gold-plated electrolyte. Rinse the electrode substrate with ultrapure water 2-3 times, transfer the gold-plated electrolyte to the surface of the electrode substrate, and deposit gold on the surface of the electrode substrate by cyclic voltammetry to obtain a gold-plated electrode.

[0050] In the cyclic voltammetry, the potential parameter is -1 to 1 V, the reference electrode is Ag / AgCl, the number of scans is 10, and the scan rate is 50 mV / s.

[0051] S202. After rinsing the gold-plated electrode with ultrapure water, add 80-120 mM 4-aminobenzenethiol ethanol solution to the surface of the gold-plated electrode, incubate for 20-40 min, and vortex in ethanol to remove unbound 4-ATP to obtain electrode 4-ATP / Au / SPE, drop-cast 8-20 mM PBS solution of protein cross-linker DTSSP on the top of 4-ATP / Au / SPE, incubate for 20-40 min, and rinse the incompletely bound DTSSP with PBS to obtain a cleavable covalent bond fixed scaffold DTSSP / 4-ATP / Au / SPE.

[0052] S203. Drop-cast 0.14~0.18μM ureB solution on the electrode (DTSSP / 4-ATP / Au / SPE) and incubate for 20~40min. Rinse off the incompletely bound ureB with PBS. Drop-cast 0.25~0.35mM DA functional monomer solution on the top of ureB / DTSSP / 4-ATP / Au / SPE. Form ePDA film by electrochemical polymerization to obtain ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film, which is placed on a clean bench for 8~15h to stabilize.

[0053] Among them, in the electrochemical polymerization method, the potential parameter range is -0.8 to 0.8 V, the reference electrode is Ag / AgCl, the number of scans is 10, and the scan rate is 50 mV / s.

[0054] S204. Place ePDA / ureB / DTSSP / 4-ATP / Au / SPE in a 0.05~0.15M 2-mercaptoethanol ethanol solution to cleave the S–S bond of DTSSP and promote the release of ureB. Then rinse the electrode with 8%~12% acetic acid solution to inhibit the self-polymerization reaction of DA, thereby generating ureB cavities in the ePDA film to obtain molecularly imprinted polymer electrodes ureB-MIPs.

[0055] S205. Using saturated potassium chloride as the reference electrode, a platinum wire electrode as the counter electrode, and a molecularly imprinted polymer electrode ureB-MIPs as the working electrode, a Helicobacter pylori ureB protein molecularly imprinted electrochemical sensor is obtained.

[0056] In summary, in this embodiment, after gold plating on the surface of the electrode substrate, the electrode substrate is treated with an ethanol solution of 4-aminothiophenol and a PBS buffer solution of a protein crosslinker DTSSP to obtain a cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE, and then treated with a ureB solution and a DA functional monomer solution to obtain an ePDA film-covered ePDA / ureB / DTSSP / 4-ATP / Au / SPE, and then treated with an ethanol solution of 2-mercaptoethanol and an acetic acid solution to obtain a molecularly imprinted polymer electrode ureB-MIPs, and then saturated potassium chloride is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and a molecularly imprinted polymer electrode ureB-MIPs is used as a working electrode to obtain a Helicobacter pylori ureB protein molecularly imprinted electrochemical sensor. The Helicobacter pylori ureB molecularly imprinted electrochemical sensor prepared by the present invention has good current response, strong stability, high repeatability and operability, and the preparation method has low requirements on experimental equipment and conditions, low cost, and safe and simple operation.

[0057] The present application is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present application in any way.

[0058] The chloroauric acid stock solution used in Example 1 is sourced from Shanghai McLean Biochemical Technology Co., Ltd., with a CAS number of 16903-35-8 and a gold content of 23.5% to 23.8%; the DA functional monomer used in Example 5 is sourced from Shanghai McLean Biochemical Technology Co., Ltd., with a product number of A902400-1g, a CAS number of 51-61-6, and a purity of 98%.

[0059] Example 1 This embodiment provides a method for preparing a gold plating electrolyte 3.079 mL of chloroauric acid stock solution and 5.921 mL of deionized water were added to 1 mL of 100 mM sodium sulfate solution, and the mixture was mixed evenly using an oscillator to obtain a gold plating electrolyte.

[0060] Example 2 This embodiment provides a method for preparing a gold-plated electrode The gold plating electrolyte used in this example comes from the gold plating electrolyte prepared in Example 1.

[0061] The screen-printed electrodes were rinsed with ultrapure water (SEM images as shown in Figure 3 As shown in the cyclic voltammogram Figure 4After the surface of the screen-printed electrode was prepared, 150 μL of the gold-plated electrolyte was transferred to the surface of the screen-printed electrode to uniformly deposit gold on the electrode surface by cyclic voltammetry to form a gold-plated electrode (SEM image as shown in Figure 3 As shown in the cyclic voltammogram Figure 4 As shown), the gold-plated electrode after deposition is placed on a clean bench for 12 hours for use.

[0062] In the cyclic voltammetry, the potential parameter is -1 to 1 V, the reference electrode is Ag / AgCl, the number of scans is 10, and the scan rate is 50 mV / s.

[0063] Example 3 This embodiment provides a method for preparing a gold-plated electrode The gold plating electrolyte used in this example comes from the gold plating electrolyte prepared in Example 1.

[0064] After washing the surface of the glassy carbon electrode with ultrapure water, 150 μL of gold-plated electrolyte was transferred to the surface of the screen-printed electrode, and gold was uniformly deposited on the electrode surface by cyclic voltammetry to form a gold-plated electrode. The gold-plated electrode after deposition was placed on a clean bench for 12 hours for use.

[0065] In the cyclic voltammetry, the potential parameter is -1 to 1 V, the reference electrode is Ag / AgCl, the number of scans is 10, and the scan rate is 50 mV / s.

[0066] Example 4-1 This embodiment provides a method for preparing a cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE The gold-plated electrode used in this example comes from the gold-plated electrode prepared in Example 2.

[0067] 20 μL of 100 mM 4-aminothiophenol ethanol solution was transferred to the gold-plated electrode and incubated for 30 min to form 4-ATP / Au / SPE, and then vortexed in 10 mL of ethanol for 2 min to remove unbound 4-aminothiophenol to obtain 4-ATP / Au / SPE (cyclic voltammogram as shown in Figure 4 shown); 20 μL of 10 mM protein cross-linker DTSSP in PBS buffer was drop-casted on the top of 4-ATP / Au / SPE and incubated for 30 min to produce a cleavable linker monolayer, while the incompletely bound DTSSP was rinsed with 1 mL PBS to obtain a cleavable covalent bond fixed scaffold DTSSP / 4-ATP / Au / SPE (cyclic voltammogram as shown in Figure 4 as shown).

[0068] Example 4-2 This embodiment provides a method for preparing a cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE The gold-plated electrode used in this example comes from the gold-plated electrode prepared in Example 3.

[0069] 20 μL of 100 mM 4-aminothiophenol ethanol solution was transferred to the gold-coated electrode and incubated for 30 min to form 4-ATP / Au / SPE, and vortexed in 10 mL of ethanol for 2 min to remove unbound 4-aminothiophenol to obtain 4-ATP / Au / SPE; 20 μL of 10 mM protein cross-linker DTSSP in PBS buffer was drop-casted on top of 4-ATP / Au / SPE and incubated for 30 min to produce a cleavable linker monolayer, while the incompletely bound DTSSP was rinsed with 1 mL PBS to obtain the cleavable covalently fixed scaffold DTSSP / 4-ATP / Au / SPE.

[0070] Example 4-3 This embodiment provides a method for preparing a cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE The gold-plated electrode used in this example comes from the gold-plated electrode prepared in Example 2.

[0071] 20 μL of 80 mM 4-aminothiophenol ethanol solution was transferred into the gold-plated electrode and incubated for 20 min to form 4-ATP / Au / SPE, and vortexed in 80 mL of ethanol for 2 min to remove unbound 4-aminothiophenol to obtain 4-ATP / Au / SPE; 8 μL of 10 mM protein cross-linker DTSSP in PBS buffer was drop-cast on the top of 4-ATP / Au / SPE and incubated for 20 min to produce a cleavable linker monolayer, and the incompletely bound DTSSP was rinsed with 1 mL of PBS to obtain a cleavable covalently fixed scaffold DTSSP / 4-ATP / Au / SPE.

[0072] Example 4-4 This embodiment provides a method for preparing a cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE The gold-plated electrode used in this example comes from the gold-plated electrode prepared in Example 2.

[0073] 20 μL of 120 mM 4-aminothiophenol ethanol solution was transferred into the gold-plated electrode and incubated for 40 min to form 4-ATP / Au / SPE, and vortexed in 120 mL of ethanol for 2 min to remove unbound 4-aminothiophenol to obtain 4-ATP / Au / SPE; 12 μL of 15 mM protein cross-linker DTSSP in PBS buffer was drop-cast on the top of 4-ATP / Au / SPE and incubated for 40 min to produce a cleavable linker monolayer, and the incompletely bound DTSSP was rinsed with 1 mL of PBS to obtain a cleavable covalently fixed scaffold DTSSP / 4-ATP / Au / SPE.

[0074] Example 5 This embodiment provides a method for preparing a DA functional monomer solution 0.015 g of DA functional monomer was added into 100 ml of Tris-HCl buffer and fully dissolved to obtain a 0.3 mM DA functional monomer solution.

[0075] Example 6-1 This embodiment provides a method for preparing ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film The cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE used in this example comes from the cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE prepared in Example 4-1, and the DA functional monomer solution used comes from the DA functional monomer solution prepared in Example 5.

[0076] 20 μL of 0.16 μM ureB solution was drop-casted on the cleavable linker-modified electrode DTSSP / 4-ATP / Au / SPE and incubated for 30 min. 1 mL of PBS was used to wash away the incompletely bound ureB to obtain ureB / DTSSP / 4-ATP / Au / SPE (cyclic voltammogram as shown in Figure 2). Figure 4 150 μL of DA functional monomer solution was drop-casted on the top of ureB / DTSSP / ATP / Au / SPE, and an ePDA film was formed by electrochemical polymerization to obtain ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with an ePDA film (cyclic voltammogram as shown in Figure 4 As shown in the scanning electron microscope image Figure 5 As shown), place it on the clean bench for 12 hours to allow it to stabilize.

[0077] Among them, in the electrochemical polymerization method, the potential parameter range is -0.8 to 0.8 V, the reference electrode is Ag / AgCl, the number of scans is 10, and the scan rate is 50 mV / s.

[0078] Example 6-2 This embodiment provides a method for preparing ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film The cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE used in this example comes from the cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE prepared in Example 4-1, and the DA functional monomer solution used comes from the DA functional monomer solution prepared in Example 5.

[0079] 20 μL of 0.14 μM ureB solution was drop-casted on the electrode DTSSP / 4-ATP / Au / SPE modified with a cleavable linker and incubated for 20 min. The incompletely bound ureB was also rinsed off with 1 mL of PBS to obtain ureB / DTSSP / 4-ATP / Au / SPE. 150 μL of DA functional monomer solution was drop-casted on the top of ureB / DTSSP / ATP / Au / SPE, and an ePDA film was formed by electrochemical polymerization to obtain ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with an ePDA film, which was placed on a clean bench for 12 h to stabilize.

[0080] Among them, in the electrochemical polymerization method, the potential parameter range is -0.8 to 0.8 V, the reference electrode is Ag / AgCl, the number of scans is 10, and the scan rate is 50 mV / s.

[0081] Example 6-3 This embodiment provides a method for preparing ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film The cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE used in this example comes from the cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE prepared in Example 4-1, and the DA functional monomer solution used comes from the DA functional monomer solution prepared in Example 5.

[0082] 20 μL of 0.18 μM ureB solution was drop-casted on the electrode DTSSP / 4-ATP / Au / SPE modified with a cleavable linker and incubated for 40 min. The incompletely bound ureB was also rinsed off with 1 mL of PBS to obtain ureB / DTSSP / 4-ATP / Au / SPE. 150 μL of DA functional monomer solution was drop-casted on the top of ureB / DTSSP / ATP / Au / SPE, and an ePDA film was formed by electrochemical polymerization to obtain ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with an ePDA film, which was placed on a clean bench for 12 h to stabilize.

[0083] Among them, in the electrochemical polymerization method, the potential parameter range is -0.8 to 0.8 V, the reference electrode is Ag / AgCl, the number of scans is 10, and the scan rate is 50 mV / s.

[0084] Example 7-1 This embodiment provides a method for preparing molecularly imprinted polymer electrodes ureB-MIPs The ePDA / ureB / DTSSP / ATP / Au / SPE covered with ePDA film used in this example is derived from the ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film prepared in Example 6-1.

[0085] The ePDA film-covered ePDA / ureB / DTSSP / ATP / Au / SPE was placed in 15 mL of 0.1 M 2-mercaptoethanol ethanol solution and stirred at 200 r for 5 min to treat the imprinted polymer membrane to cleave the S–S bond of DTSSP and promote the release of ureB. The electrode was then rinsed with 1 mL of 10% acetic acid solution to inhibit the self-polymerization reaction of DA, thereby generating ureB cavities in the ePDA film to obtain molecularly imprinted polymer electrodes ureB-MIPs (cyclic voltammograms are shown in Figure 2). Figure 4 As shown in the scanning electron microscope image Figure 5 as shown).

[0086] Example 7-2 This embodiment provides a method for preparing molecularly imprinted polymer electrodes ureB-MIPs The ePDA / ureB / DTSSP / ATP / Au / SPE covered with ePDA film used in this example is derived from the ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film prepared in Example 6-1.

[0087] The ePDA / ureB / DTSSP / ATP / Au / SPE covered with ePDA film was placed in 15 mL of 0.05 M 2-mercaptoethanol ethanol solution and the imprinted polymer membrane was treated with stirring at a rate of 200 r for 5 min to cleave the S–S bond of DTSSP and promote the release of ureB. The electrode was then rinsed with 1 mL of 8% acetic acid solution to inhibit the self-polymerization reaction of DA, thereby generating ureB cavities in the ePDA film to obtain molecularly imprinted polymer electrodes ureB-MIPs.

[0088] Example 7-3 This embodiment provides a method for preparing molecularly imprinted polymer electrodes ureB-MIPs The ePDA / ureB / DTSSP / ATP / Au / SPE covered with ePDA film used in this example is derived from the ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film prepared in Example 6-1.

[0089] The ePDA / ureB / DTSSP / ATP / Au / SPE covered with ePDA film was placed in 15 mL of 0.15 M 2-mercaptoethanol ethanol solution and the imprinted polymer membrane was treated by stirring at a rate of 200 r for 5 min to cleave the S–S bond of DTSSP and promote the release of ureB. The electrode was then rinsed with 1 mL of 12% acetic acid solution to inhibit the self-polymerization reaction of DA, thereby generating ureB cavities in the ePDA film to obtain molecularly imprinted polymer electrodes ureB-MIPs.

[0090] Example 8 This embodiment provides a method for preparing a Helicobacter pylori ureB protein molecular imprinting electrochemical sensor The molecular imprinted polymer electrode ureB-MIPs used in this example is the molecular imprinted polymer electrode ureB-MIPs prepared in Example 7-1.

[0091] A Helicobacter pylori ureB protein molecular imprinting electrochemical sensor was prepared using saturated potassium chloride as the reference electrode, a platinum wire electrode as the counter electrode, and molecularly imprinted polymer electrode ureB-MIPs as the working electrode.

[0092] Performance Testing Measurement of the redox probe Fe(CN) using square wave voltammetry 6 3- / 4- The change in redox potential peak indicates the interaction between the MIPs layer and ureB, thereby quantitatively analyzing the ureB concentration in various liquid samples. The electrochemical measurement process includes pre-washing, incubation and detection.

[0093] Six test groups were set up. In each test group, 1 mL of deionized water was used to rinse the surface of the molecularly imprinted polymer electrode ureB-MIPs. The Helicobacter pylori ureB protein molecularly imprinted electrochemical sensor was treated with a ureB-containing sample for 10 min. The ureB concentrations in each test group were 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, and 100 fg, respectively. Other conditions remained unchanged. Detection: Square wave anodic stripping voltammetry was used to detect the presence of 50 mM K 4 [Fe(CN) 6 ] / K 3 [Fe(CN) 6] was used to plot the signal response of KCl solution of Helicobacter pylori ureB molecular imprinting electrochemical sensor when detecting ureB concentration of 100fg / mL-10ng / mL, square wave voltammogram, electrochemical impedance diagram and nonlinear logarithmic calibration curve.

[0094] Data analysis The logarithmic value of ureB concentration was used as the independent variable x, and the change of redox potential peak measured by square wave voltammetry was used as the dependent variable y. The response signal values ​​obtained in the detection of ureB samples with different concentrations were collected, and the collected data were subjected to regression analysis using Origin software to obtain the correlation coefficient R 2 .

[0095] Test results like Figure 7 As shown, from the square wave voltammogram, it can be seen that the redox potential peak of the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor detecting the ureB-containing sample is around 0.25V.

[0096] Table 1 shows the detection results of Helicobacter pylori ureB molecular imprinting electrochemical sensor

[0097] As shown in Table 1, the detection results of Helicobacter pylori ureB molecular imprinting electrochemical sensor in the range of 100fg / mL-10ng / mL, after regression analysis, the logarithmic correlation coefficient R between the response signal of Helicobacter pylori ureB protein molecular imprinting electrochemical sensor and ureB concentration was obtained. 2 It is 0.99015, which shows that the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor prepared by the present invention has high accuracy and reliability, and can be effectively used for quantitative analysis of ureB concentration.

[0098] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that all equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for preparing a Helicobacter pylori ureB protein molecular imprinting electrochemical sensor, characterized in that: The following steps are involved: Step 1: Incubate the gold-plated electrode with an ethanol solution of 4-aminothiophenol to obtain 4-ATP / Au / SPE, and then incubate it with a PBS buffer solution of a protein crosslinker DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)) to obtain a cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE; Step 2: Incubate the cleavable covalent bond fixed support DTSSP / 4-ATP / Au / SPE with ureB solution to obtain ureB / DTSSP / 4-ATP / Au / SPE, then drop-cast the DA functional monomer solution onto the surface of ureB / DTSSP / 4-ATP / Au / SPE, and form an ePDA film by electrochemical polymerization to obtain ePDA / ureB / DTSSP / 4-ATP / Au / SPE covered with ePDA film; Step 3: Use 2-mercaptoethanol ethanol solution to cleave the S–S bond on the surface of ePDA / ureB / DTSSP / 4-ATP / Au / SPE, and then rinse the cleaved ePDA / ureB / DTSSP / 4-ATP / Au / SPE with acetic acid solution to obtain molecularly imprinted polymer electrode ureB-MIPs; Step 4: Using saturated potassium chloride as the reference electrode, a platinum wire electrode as the counter electrode, and a molecularly imprinted polymer electrode ureB-MIPs as the working electrode, a Helicobacter pylori ureB protein molecularly imprinted electrochemical sensor is obtained.

2. The method for preparing the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor according to claim 1, characterized in that: In step 1, the method for preparing the gold-plated electrode comprises the following steps: A1. Prepare gold plating electrolyte: mix chloroauric acid aqueous solution, deionized water and sodium sulfate solution evenly to obtain gold plating electrolyte; A2. After rinsing the electrode substrate with ultrapure water, the electrode substrate is treated with a gold-plating electrolyte, and gold is deposited on the surface of the electrode substrate by cyclic voltammetry to obtain a gold-plated electrode; The mass ratio of the aqueous solution of chloroauric acid, deionized water, and sodium sulfate solution is 2.5-3.5:4-8:0.8-1.2; The concentration of the sodium sulfate solution is 80-120 mM; The electrode substrate is a screen-printed electrode or a glassy carbon electrode; The potential parameter range of the cyclic voltammetry method is -1 to 1 V, the reference electrode is Ag / AgCl, the number of scans is 8 to 12, and the scan rate is 50 mV / s.

3. The method for preparing the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor according to claim 1, characterized in that: In the step 1, the concentration of the ethanol solution of 4-aminothiophenol is 80-120 mM, and the concentration of the PBS buffer solution of the protein cross-linking agent DTSSP is 8-20 mM; the incubation time of the ethanol solution of 4-aminothiophenol is 20-40 min, and the incubation time of the PBS buffer solution of the protein cross-linking agent DTSSP is 20-40 min.

4. The method for preparing the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor according to claim 1, characterized in that: In step 2, the DA functional monomer solution is prepared by dissolving the DA functional monomer in Tris-HCl buffer.

5. The method for preparing the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor according to claim 1, characterized in that: In the step 2, the concentration of the ureB solution is 0.14-0.18 μM, and the concentration of the DA functional monomer solution is 0.25-0.35 mM; the incubation time of the ureB solution is 20-40 min.

6. The method for preparing the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor according to claim 1, characterized in that: In step 2, the potential parameter range of the electrochemical polymerization method is -0.8 to 0.8 V, the reference electrode is Ag / AgCl, the number of scans is 8 to 12, and the scan rate is 50 mV / s.

7. The method for preparing the Helicobacter pylori ureB protein molecular imprinting electrochemical sensor according to claim 1, characterized in that: In the step 3, the concentration of the 2-mercaptoethanol ethanol solution is 0.05-0.15 M, and the mass fraction of the acetic acid solution is 8%-12%. 8 . A Helicobacter pylori ureB protein molecular imprinting electrochemical sensor prepared according to the method for preparing a Helicobacter pylori ureB protein molecular imprinting electrochemical sensor according to any one of claims 1 to 6 .

9. A detection method of Helicobacter pylori ureB protein molecular imprinting electrochemical sensor according to claim 8, characterized in that: The following steps are involved: B1. Rinse the surface of the molecularly imprinted polymer electrode ureB-MIPs with deionized water, and incubate the molecularly imprinted polymer electrode ureB-MIPs with ureB solutions of different concentrations for 10 min. B2, Fe(CN)6 3- / 4- As a redox probe, the redox peak changes of the probe on the molecularly imprinted polymer electrode ureB-MIPs were measured by square wave anodic stripping voltammetry, thereby quantitatively analyzing the concentration of ureB in various liquid samples.

10. The detection method according to claim 9, characterized in that: The potential parameter range of the square wave anodic stripping voltammetry is -0.8 to 0.8 V, the reference electrode is Ag / AgCl, the potential amplitude is 70 mV, the potential increment is 11 mV, the sampling width is 5 ms, and the pulse period is 20 ms.

Images