CN115494138A - Novel monomolecular enzyme electrochemical phenol sensor and preparation method and application thereof - Google Patents

Abstract

The invention discloses a novel unimolecular enzyme electrochemical phenolic sensor, its preparation method and application. Specifically, the present invention utilizes bilirubin oxidase as a phenolic acceptor, uses the Faradaic current generated when it oxidizes phenolic compounds as the basis of sensing, constructs an electron transfer channel between the electrode and bilirubin oxidase, and The bilirubin oxidase is efficiently assembled on the electrode surface with the help of gold nanoparticles and basic proteins, thereby constructing a single-molecule enzyme electrochemical phenolic sensor. Compared with traditional enzyme electrochemical sensors, the present invention has the technical advantages of high detection sensitivity, high quantitative accuracy and high enzyme activity stability in the detection of phenolic pollutants, and can realize real-time online qualitative analysis of phenolic pollutants Quantitative detection.

Description

A novel unimolecular enzyme electrochemical phenolic sensor and its preparation method and application

technical field

The invention relates to the technical field of electrochemical detection, in particular to a novel unimolecular enzyme electrochemical phenolic sensor and its preparation method and application.

Background technique

Phenolic compounds are an important class of toxic organic pollutants in the ecological environment. Phenolic compounds in the environment come from a wide range of sources, mainly including the discharge of industrial wastewater and domestic sewage, as well as the degradation of organophosphorus pesticides, which seriously threaten ecological security and human health. It is urgent to strengthen the monitoring, early warning and treatment of phenolic pollutants.

Traditionally, the analysis and detection methods for phenolic compounds are mainly chromatography and mass spectrometry, which are difficult to achieve low-cost, high-throughput real-time online monitoring. Bioelectrochemical sensors can well solve the problem of real-time online detection of special pollutants. However, the current bioelectrochemical sensors convert the concentration of pollutants into a detectable current through the principle of protein coating and electronic media conduction in technology, and are faced with low detection sensitivity, fast inactivation of functional enzymes, poor anti-interference ability, and quantitative general reliability issues.

Contents of the invention

Aiming at the deficiencies of the prior art, the object of the present invention is to provide a novel unimolecular enzyme electrochemical phenolic sensor and its preparation method and application.

In order to construct a novel bioelectrochemical phenolic sensor with technical and economic value, the present invention selects bilirubin oxidase (BOD, the same below, derived from Myrotheca spp.) as the electrochemical acceptor of phenol, and utilizes gold nanoparticles and basic protein A unique single-molecule interface was designed and realized between the BOD and the electrode substrate to further improve the activity and stability of the enzyme electrode, and a single-molecule enzyme electrochemical phenolic sensor based on the "nano-gold-basic protein-BOD" system was constructed.

The technical idea of the present invention is: deposit gold nanoparticles on the surface of conductive and inert solid analysis electrodes by electrochemical methods; The monolayer immobilizes the basic protein on the surface of gold nanoparticles in a covalent manner, and obtains a monolayer of basic protein on the surface of the electrode; covalently connects bilirubin oxidase to the basic protein, that is, makes a single-molecule enzyme electrode. Chemical phenolic sensors. The activity and stability of the enzyme electrode can be improved by introducing the basic protein between the electrode surface and the enzyme, and oxidize the phenolic compound at an appropriate potential to generate a specific current signal, which can be used as a qualitative and quantitative indicator for the phenolic compound in the water sample. in accordance with.

Therefore, the first object of the present invention is to provide a novel unimolecular enzyme electrochemical phenolic sensor comprising an electrode substrate, and a layer of gold nanoparticles, basic protein and bilirubin oxidized in sequence assembled on the surface of the electrode substrate. Enzymes, the basic protein is bound to the surface of the gold nanoparticle in a covalent connection through a sulfhydryl-containing carboxylic acid or an amine compound, and the bilirubin oxidase is covalently bonded to the basic protein.

Preferably, the electrode substrate is made of gold, platinum, glassy carbon or graphite.

Preferably, the basic protein is a conductive protein or a non-conductive protein.

Preferably, the conductive protein is heme cytochrome c or iron-sulfur protein; the obtained non-conductive protein is bovine serum albumin.

The second object of the present invention is to provide a method for preparing the above-mentioned novel unimolecular enzyme electrochemical phenolic sensor, comprising the following steps: depositing gold nanoparticles on the surface of a conductive, inert solid analysis electrode; Carboxylic acid or amine compounds modify the self-assembled monolayer on the surface of gold nanoparticles; the self-assembled monolayer is used to fix the basic protein on the surface of the gold nanoparticle in a covalent manner, and a monolayer of basic protein is obtained on the electrode surface ; The bilirubin oxidase is covalently linked to the basic protein, that is, the unimolecular enzyme electrochemical phenolic sensor is prepared.

Preferably, the preparation method of the novel unimolecular enzyme electrochemical phenolic sensor comprises the following steps:

(1) In the three-electrode electrochemical system, the conductive and inert solid analysis electrode is used as the working electrode, the platinum wire is used as the counter electrode, and the silver/silver chloride is used as the reference electrode, and the electrodes are jointly immersed in the electrode containing chloroauric acid. In the deposition solution, the chloroaurate is reduced by cyclic voltammetry, so that the gold nanoparticles are deposited on the surface of the working electrode;

(2) immerse the working electrode on which the gold nanoparticles are deposited in the carboxylation modification solution for 6 to 48 hours to obtain a surface carboxylated gold nanoparticle electrode;

(3) The surface carboxylated gold nanoparticle electrode is treated with EDC-NHS solution, rinsed with water, soaked in the basic protein solution, so that the basic protein is covalently fixed on the surface of the gold nanoparticle, and then treated with EDC-NHS The solution is processed, rinsed with water, and then soaked in the bilirubin oxidase solution to prepare a single-molecule enzyme electrochemical sensor.

Preferably, in step (1), the conductive and inert solid analysis electrode is made of gold, platinum, glassy carbon or graphite.

Preferably, in step (1), the composition of the electrodeposition solution is: 1mM chloroauric acid, 50mM sodium sulfate, 0.5M sulfuric acid, and the solvent is water; the potential range of the cyclic voltammetry is -1.4V ~+0.6V, the scan rate is 10~200mV/s, and the number of scan cycles is 5~20.

Preferably, in step (2), the composition of the carboxylation modification solution is: β-mercaptoethanol 9mM, thioglycolic acid 1mM, and the solvent is water.

Preferably, in step (3), the composition of the EDC-NHS solution is: 0.1M EDC, 0.5M Sulfo-NHS, 0.1M morpholineethanesulfonic acid, and the solvent is water.

Preferably, in step (3), the base protein is multi-heme cytochrome c, iron-sulfur protein or bovine serum albumin; the concentration of the base protein solution is 1 μM, and the bilirubin oxidase solution The concentration is 740U BOD/mL PBS buffer.

The third object of the present invention is to provide the application of the above-mentioned novel single-molecule enzyme electrochemical phenolic sensor in detecting phenolic pollutants.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention utilizes bilirubin oxidase as a phenolic acceptor, uses the faradaic current generated when it oxidizes phenolic compounds as the basis of sensing, constructs an electron transfer channel between the electrode and bilirubin oxidase, and uses nano-gold Particles and basic proteins efficiently assemble bilirubin oxidase on the electrode surface, thereby constructing a single-molecule enzyme electrochemical phenolic sensor. Compared with traditional enzyme electrochemical sensors, the present invention has the technical advantages of high detection sensitivity, high quantitative accuracy and high enzyme activity stability in the detection of phenolic pollutants, and can realize real-time online qualitative analysis of phenolic pollutants Quantitative detection has high economic and technical value.

Description of drawings

Figure 1 is a schematic diagram of the "nano gold-CctA-BOD" electrode.

Figure 2 is the cyclic voltammograms of "nano-gold-BOD" and "nano-gold-CctA-BOD" systems sensing 2,4-dichlorophenol.

Fig. 3 is a fitting linear diagram of the concentration of 2,4-dichlorophenol and the test current of the sensor.

Fig. 4 shows the signal changes of the sensor lacking CctA (“nanogold-BOD”) and the sensor adding CctA (“nanogold-CctA-BOD”) to 2,4-dichlorophenol during multiple scans.

Fig. 5 shows the response signal change of the BSA-added sensor (“nano gold-BSA-BOD”) to 2,4-dichlorophenol during multiple scans.

detailed description

The following examples are to further illustrate the present invention, rather than limit the present invention.

Example 1

1. Preparation of unimolecular enzyme electrochemical phenolic sensor ("nano gold-CctA-BOD" electrode)

(1) Preparation of nano-gold modified electrodes

In this case, the gold plate electrode was used as the substrate, and the aluminum oxide powder of 1 μm and 50 nm was used for wet polishing in sequence before use. When the surface of the electrode was polished to a smooth and bright surface, it was ultrasonically cleaned three times with ethanol and pure water. Assemble a conventional three-electrode electrolysis system, with the polished and cleaned gold plate electrode as the working electrode, the platinum wire as the counter electrode, and the silver/silver chloride as the reference electrode and jointly immerse in the electrodeposition solution containing chloroauric acid (1mM gold chloride acid, 50mM sodium sulfate, 0.5M sulfuric acid, the solvent is water), and connected to the electrochemical workstation. Scanning cyclic voltammetry under vigorous stirring (potential range is -1.4V～+0.6V, scanning rate is 50mV/s, and scanning cycle number is 10 times), and chloroaurate is reduced, and gold nano-deposition particles are deposited on the electrode surface, The results showed that the surface of the electrode changed from smooth and golden yellow to frosted and purplish red, which proved the formation of a dense nano-gold layer.

(2) Carboxylation of gold nanoparticles

Rinse the gold nanoparticle-deposited working electrode obtained in step (1) with pure water, air-dry and soak in carboxylation modification solution (β-mercaptoethanol 9mM, thioglycolic acid 1mM, solvent is water) for 18h, take it out and rinse with water again , to obtain surface carboxylated gold nanoparticles electrode.

(3) Acquisition of the expression of multiheme cytochrome c

In this case, multiheme cytochrome c—CctA from Shewanella oneidensis MR-1 was used as the basic protein in the electrode. In order to extract the protein, the DNA of CctA (GenBank: AAN55755.1) was recombined into the plasmid pBAD202/D-TOPO (purchased from ThermoFisher, and the circular plasmid was subcultured in the laboratory to preserve the circular plasmid in E. coli) and 6× His tag, and then reintroduced into the bacteria. The recombinant strain was activated to the middle of the logarithmic growth phase by using LB medium, and L-arabinose (1 mM) was added for regular induction expression. After inducing expression for 10 h, the bacterial cells were collected and the bacterial cells were lysed by sonication. The supernatant was collected by ultracentrifugation, then passed through a nickel affinity resin, which was washed as usual and the CctA protein was eluted from the resin using imidazole. The ultrafiltration cup was used for buffer replacement, and the protein was dissolved in PBS buffer (20 mM NaH ₂ PO ₄ , 80 mM Na ₂ HPO ₄ , pH 7.4, the solvent was water) to obtain a CctA protein solution with a concentration of 1 μM.

(4) CctA is covalently assembled onto the surface of gold nanoparticles

Soak the surface carboxylated gold nanoparticle electrode obtained in step (2) in EDC-NHS solution (containing 0.1MEDC, 0.5M Sulfo-NHS, 0.1M morpholineethanesulfonic acid, pH 6.0, solvent is water) for 1h, Then remove and rinse with pure water, air dry. The transfer was immersed in a CctA protein solution with a concentration of 1 μM for 1 h, so that the conductive protein CctA was covalently immobilized on the surface of the gold nanoparticles.

(5) BOD is covalently assembled to the surface of CctA

Gently rinse the surface of the electrode obtained in step (4) with pure water, then soak in the EDC-NHS solution for 1 hour again, take it out and rinse with pure water. Transfer and soak in BOD solution (740 U BOD/mL PBS buffer) for 1 hour, and then gently rinse the electrode to obtain the "nano gold-CctA-BOD" electrode (Figure 1).

The preparation steps of the "nano-gold-BOD" electrode are similar to the "nano-gold-CctA-BOD" electrode, specifically: the electrode deposited with gold nanoparticles obtained in step (1) is rinsed with pure water and soaked in EDC-NHS solution 1h, take it out and rinse with pure water. Transfer and soak in BOD solution (740 U BOD/mL PBS buffer) for 1 hour, and then gently rinse the electrode to obtain a "nano-gold-BOD" electrode.

2. Electrochemical sensing of 2,4-dichlorophenol

Using 2,4-dichlorophenol as a representative analyte, the electrochemical sensing test was carried out in a three-electrode electrolysis system under the control of an electrochemical workstation. The "nano-gold-BOD" or "nano-gold-CctA-BOD" electrode prepared above was used as the working electrode, the platinum wire electrode was used as the counter electrode, and the saturated silver/silver chloride electrode was used as the reference electrode for the experiment. The electrolyte used was PBS with 50 mM NaCl added as a supporting electrolyte, and 2,4-dichlorophenol was added at a concentration of 2 mM. The redox test of 2,4-dichlorophenol was carried out by the method of cyclic voltammetry scanning (the potential range is -0.6V～+0.8V, the scanning rate is 10mV/s, and the number of scanning cycles is 2 times).

In the cyclic voltammogram (Figure 2) of the reduction-oxidation transformation of 2,4-dichlorophenol, an obvious oxidation peak was observed in the high potential region (+0.575V), as a characteristic signal of its oxidation by BOD. This result proves that the "nano-gold-CctA-BOD" electrode designed in the present invention has the ability to sense 2,4-dichlorophenol.

3. Standard curve of 2,4-dichlorophenol concentration

In the system of step 2, cyclic voltammetry was scanned for 2,4-dichlorophenol solutions with working concentrations of 20 μM, 50 μM, 100 μM, 150 μM, 200 μM, 400 μM, 600 μM, 800 μM, 1 mM, 1.5 mM, and 2 mM, respectively. Record the peak current value at the position of +0.575V for each cyclic voltammetry, and establish the linear regression equation of concentration-peak current, as shown in Figure 3. The results show that the concentration of 2,4-dichlorophenol solution has a good correlation with the peak current, which meets the requirements of concentration quantification.

4. Effect of cytochrome c on sensor stability

In the electrolyte solution containing 2mM 2,4-dichlorophenol, the "nano-gold-BOD" and "nano-gold-CctA-BOD" electrodes were used as working electrodes respectively, and multiple sensing tests were carried out in the three-electrode system. The change of the characteristic oxidation peak current signal of 2,4-dichlorophenol reflects the change of sensor activity (as shown in Figure 4). The oxidation peak current signal of the sensor "nano gold-BOD" lacking the auxiliary protein CctA gradually decreased and inactivated in multiple rounds of testing, and the sensor "nano gold-CctA-BOD" added with the auxiliary protein CctA significantly improved the activity and stability of BOD sex.

Through example tests, the single-molecule enzyme electrochemical phenolic sensor for phenolic pollutants in the present invention has the advantages of high sensitivity and high enzyme activity stability, and is suitable for on-line industrial detection and environmental monitoring of phenolic pollutants. economic and technical value.

Example 2

1. Preparation of unimolecular enzyme electrochemical phenolic sensor ("nano gold-BSA-BOD" electrode)

In this case, the preparation steps of the "nano-gold-bovine serum albumin (BSA)-BOD" electrode are the same as in Example 1, except that the basic protein in the "nano-gold-BSA-BOD" electrode is the non-conductive protein BSA.

2. Electrochemical sensing of 2,4-dichlorophenol

The stability of the "nano-gold-BSA-BOD" electrode was tested in an electrolyte containing 2mM 2,4-dichlorophenol, and multiple sensing tests were performed in a three-electrode system, with the characteristics of 2,4-dichlorophenol The change of the oxidation peak current signal reflects the change of the sensor activity. The specific steps are the same as in Example 1, and the results are shown in FIG. 5 . It can be seen from Figure 5 that the sensor "nano gold-BSA-BOD" added with the basic protein BSA also significantly improved the activity and stability of BOD.

After the above example tests, the single-molecule enzyme electrochemical phenolic sensor for phenolic pollutants in the present invention can well improve the activity and stability of the enzyme electrode, effectively prolong the service life of the enzyme electrode, and make the enzyme electrode have better performance. The reproducibility and long-term monitoring ability make the enzyme electrode have high economic and technical value.

The above are only preferred implementations of the present invention. It should be noted that the above preferred implementations should not be regarded as limiting the present invention, and the scope of protection of the present invention should be based on the scope defined in the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (10)

1. A novel monomolecular enzyme electrochemical phenol sensor is characterized by comprising an electrode substrate, and a gold nanoparticle layer, basic protein and bilirubin oxidase which are sequentially assembled on the surface of the electrode substrate; the basic protein is bound to the surface of the gold nanoparticle in a covalent linkage mode through a carboxylic acid or amine compound with a sulfhydryl group, and the bilirubin oxidase is bound to the basic protein in a covalent linkage mode.

2. The novel unimolecular enzyme electrochemical phenol sensor according to claim 1, wherein the electrode substrate is made of gold, platinum, glassy carbon or graphite.

3. The novel unimolecular enzyme electrochemical phenol sensor according to claim 1, wherein the base protein is poly-heme cytochrome c, iron-sulfur protein, or bovine serum albumin.

4. A method for preparing a novel unimolecular enzyme electrochemical phenol sensor according to any one of claims 1 to 3, comprising the steps of: depositing gold nanoparticles on the surface of a conductive, inert solid analysis electrode; then modifying the self-assembled monolayer on the surface of the gold nanoparticle by means of carboxylic acid or amine compounds with sulfydryl; fixing the basic protein on the surface of the gold nanoparticle by virtue of a self-assembled monolayer in a covalent connection mode to obtain the monomolecular layer of the basic protein on the surface of the electrode; covalently linking bilirubin oxidase to basic protein to obtain the monomolecular enzyme electrochemical phenol sensor.

5. The method of claim 4, comprising the steps of:

(1) In a three-electrode electrochemical system, a conductive and inert solid analysis electrode is taken as a working electrode, a platinum wire is taken as a counter electrode, and silver/silver chloride is taken as a reference electrode, the electrodes are jointly immersed in an electrodeposition solution containing chloroauric acid, chloroauric acid radicals are reduced by a cyclic voltammetry method, and gold nanoparticles are deposited on the surface of the working electrode;

(2) Immersing the working electrode deposited with the gold nanoparticles in the carboxylation modification liquid for 6-48 h to obtain a surface carboxylation gold nanoparticle electrode;

(3) And (2) treating the nano gold particle electrode with surface carboxylation by using an EDC-NHS solution, soaking in a basic protein solution after rinsing with water to ensure that basic protein is covalently fixed on the surface of the gold particle, treating by using the EDC-NHS solution, rinsing with water, and soaking in a bilirubin oxidase solution to prepare the monomolecular enzyme electrochemical sensor.

6. The method of claim 5, wherein in step (1), the bath has the composition: 1mM chloroauric acid, 50mM sodium sulfate, 0.5M sulfuric acid, and water as a solvent; the potential range of the cyclic voltammetry is-1.4V- +0.6V, the scanning rate is 10-200 mV/s, and the number of scanning cycles is 5-20.

7. The method according to claim 5, wherein in the step (2), the composition of the carboxylated modification liquid is as follows: 9mM of beta-mercaptoethanol, 1mM of thioglycolic acid and water as a solvent.

8. The method according to claim 5, wherein in the step (3), the EDC-NHS solution has the following composition: 0.1M EDC,0.5M Sulfo-NHS,0.1M morpholine ethanesulfonic acid, solvent water.

9. The method of claim 5, wherein in step (3), the base protein is poly-heme cytochrome c, iron-sulfur protein, or bovine serum albumin; the concentration of the basic protein solution is 1 mu M, and the concentration of the bilirubin oxidase solution is 740U BOD/mL PBS buffer solution.

10. Use of a novel unimolecular enzyme electrochemical phenol sensor according to any one of claims 1 to 3 to detect phenol contaminants.

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