A single-molecule enzyme electrochemical phenol sensor and its preparation method and application

Abstract

The present invention discloses a single-molecule enzyme electrochemical phenol sensor, its preparation method, and its application. Specifically, the present invention utilizes bilirubin oxidase as a phenol receptor, using the Faradaic current generated when it oxidizes phenolic compounds as the basis for sensing. An electron transfer channel is constructed between the electrode and the bilirubin oxidase, and the bilirubin oxidase is efficiently assembled on the electrode surface using gold nanoparticles and a base protein, thereby constructing a single-molecule enzyme electrochemical phenol 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, enabling real-time online qualitative and quantitative detection of phenolic pollutants.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical detection, and in particular to a single-molecule enzyme electrochemical phenol sensor and a preparation method and application thereof. Background Art

[0002] Phenolic compounds are a significant class of toxic organic pollutants in the ecological environment. Phenolic compounds in the environment come from a wide range of sources, primarily industrial and domestic wastewater discharges, and the degradation of organophosphorus pesticides. They pose a serious threat to ecological security and human health, necessitating the urgent need to strengthen monitoring, early warning, and control of phenolic pollutants.

[0003] Traditionally, analytical detection methods for phenolic compounds have primarily relied on chromatography and mass spectrometry, making them difficult to implement for low-cost, high-throughput, real-time online monitoring. Bioelectrochemical sensors offer a promising solution for real-time online detection of specific pollutants. However, current bioelectrochemical sensors, which typically convert pollutant concentrations into a detectable current through protein coating and electron-mediated conductivity, suffer from low sensitivity, rapid enzyme inactivation, poor anti-interference capabilities, and limited quantitative reliability. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a single-molecule enzyme electrochemical phenol sensor and its preparation method and application.

[0005] In order to construct a bioelectrochemical phenol sensor with technical and economic value, the present invention selects bilirubin oxidase (BOD, the same below, derived from the genus Myrotheca) as the electrochemical receptor of phenol, uses nanogold and basic protein to design and realize a unique single-molecule interface between BOD and the electrode substrate, further improves the activity and stability of the enzyme electrode, and constructs a single-molecule enzyme electrochemical phenol sensor based on the "nanogold-basic protein-BOD" system.

[0006] The technical concept of the present invention is to deposit gold nanoparticles on the surface of a conductive, inert solid analytical electrode using an electrochemical method; then, using a carboxylic acid or amine compound with a thiol group, modify the gold nanoparticle surface with a self-assembled monolayer; covalently attach a base protein to the gold nanoparticle surface using the self-assembled monolayer to form a monolayer of the base protein on the electrode surface; and covalently attach bilirubin oxidase to the base protein to create a single-molecule enzyme electrochemical phenol sensor. By introducing the base protein between the electrode surface and the enzyme, the activity and stability of the enzyme electrode are significantly improved. At an appropriate potential, the oxidation of phenolic compounds generates a specific current signal, which serves as a basis for the qualitative and quantitative determination of phenolic compounds in water samples.

[0007] Therefore, the first object of the present invention is to provide a single-molecule enzyme electrochemical phenol sensor, which includes an electrode substrate, and a gold nanoparticle layer, a basic protein and a bilirubin oxidase assembled in sequence on the surface of the electrode substrate, wherein the basic protein is covalently bound to the surface of the gold nanoparticles through a carboxylic acid or amine compound with a thiol group, and the bilirubin oxidase is covalently bound to the basic protein.

[0008] Preferably, the electrode substrate is made of gold, platinum, glassy carbon or graphite.

[0009] Preferably, the base protein is a conductive protein or a non-conductive protein.

[0010] Preferably, the conductive protein is multi-heme cytochrome c or iron-sulfur protein; and the obtained non-conductive protein is bovine serum albumin.

[0011] A second object of the present invention is to provide a method for preparing the above-mentioned single-molecule enzyme electrochemical phenol sensor, comprising the following steps: depositing gold nanoparticles on the surface of a conductive, inert solid analytical electrode; then modifying a self-assembled monolayer on the surface of the gold nanoparticles with a carboxylic acid or amine compound having a thiol group; covalently immobilizing a basic protein on the surface of the gold nanoparticles with the self-assembled monolayer to obtain a monomolecular layer of the basic protein on the electrode surface; and covalently linking bilirubin oxidase to the basic protein to obtain the single-molecule enzyme electrochemical phenol sensor.

[0012] Preferably, the method for preparing the single-molecule enzyme electrochemical phenol sensor comprises the following steps:

[0013] (1) In a three-electrode electrochemical system, a conductive, inert solid analytical electrode is used as the working electrode, a platinum wire is used as the counter electrode, and silver / silver chloride is used as the reference electrode. The electrodes are immersed in an electrodeposition solution containing chloroauric acid, and chloroaurate is reduced by cyclic voltammetry to deposit gold nanoparticles on the surface of the working electrode.

[0014] (2) immersing the working electrode deposited with gold nanoparticles in a carboxylation modification solution for 6 to 48 hours to obtain a surface carboxylated gold nanoparticle electrode;

[0015] (3) The surface carboxylated gold nanoparticle electrode was treated with EDC-NHS solution, rinsed with water, and immersed in a basic protein solution to covalently fix the basic protein to the surface of the gold nanoparticle. The electrode was then treated with EDC-NHS solution, rinsed with water, and immersed in a bilirubin oxidase solution to prepare a single-molecule enzyme electrochemical sensor.

[0016] Preferably, in step (1), the conductive, inert solid analytical electrode is made of gold, platinum, glassy carbon or graphite.

[0017] 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 to +0.6V, the scan rate is 10 to 200mV / s, and the number of scan cycles is 5 to 20.

[0018] Preferably, in step (2), the composition of the carboxyl modification solution is: 9 mM β-mercaptoethanol, 1 mM thioglycolic acid, and the solvent is water.

[0019] 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.

[0020] Preferably, in step (3), the basic protein is heme cytochrome c, iron-sulfur protein or bovine serum albumin; the concentration of the basic protein solution is 1 μM, and the concentration of the bilirubin oxidase solution is 740 U BOD / mL PBS buffer.

[0021] The third object of the present invention is to provide the use of the above-mentioned single-molecule enzyme electrochemical phenol sensor in detecting phenol pollutants.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention utilizes bilirubin oxidase as a phenol receptor, using the Faradaic current generated when it oxidizes phenolic compounds as the basis for sensing. An electron transfer channel is established between the electrode and the bilirubin oxidase. Using gold nanoparticles and a base protein, the bilirubin oxidase is efficiently assembled on the electrode surface, thereby constructing a single-molecule enzyme electrochemical phenol sensor. Compared with traditional enzyme electrochemical sensors, the present invention offers the technical advantages of high detection sensitivity, high quantitative accuracy, and high enzyme activity stability for the detection of phenolic pollutants. It can achieve real-time, online qualitative and quantitative detection of phenolic pollutants, and has high economic and technical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the "nanogold-CctA-BOD" electrode.

[0025] Figure 2 Cyclic voltammograms of the "nano-gold-BOD" and "nano-gold-CctA-BOD" systems sensing 2,4-dichlorophenol.

[0026] Figure 3 The fitted linear plot of 2,4-dichlorophenol concentration and sensor test current.

[0027] Figure 4Figure 3 shows the response signal changes of the sensor lacking CctA ("nanogold-BOD") and the sensor with added CctA ("nanogold-CctA-BOD") to 2,4-dichlorophenol during multiple scans.

[0028] Figure 5 The response signal changes of the sensor with BSA added ("nanogold-BSA-BOD") to 2,4-dichlorophenol during multiple scans. DETAILED DESCRIPTION

[0029] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0030] Example 1

[0031] 1. Preparation of single-molecule enzyme electrochemical phenol sensor ("nano-gold-CctA-BOD" electrode)

[0032] (1) Preparation of gold nanoparticle-modified electrodes

[0033] In this case, a gold disk electrode was used as the substrate. Before use, it was wet-polished with 1μm and then 50nm alumina powder. Once the electrode surface was smooth and shiny, it was ultrasonically cleaned three times with ethanol and then with pure water. A conventional three-electrode electrolysis system was assembled, with the polished and cleaned gold disk electrode as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride reference electrode immersed in an electroplating solution containing chloroauric acid (1mM chloroauric acid, 50mM sodium sulfate, 0.5M sulfuric acid, and water as the solvent). The system was then connected to an electrochemical workstation. Cyclic voltammetry was performed under vigorous stirring (potential range -1.4V to +0.6V, scan rate 50mV / s, 10 scan cycles) to reduce the chloroaurate ion and deposit gold nanoparticles on the electrode surface. The results showed that the electrode surface changed from a smooth, golden-yellow color to a frosted, purple-red color, demonstrating the formation of a dense nano-gold layer.

[0034] (2) Carboxylation of gold nanoparticles

[0035] The working electrode deposited with gold nanoparticles obtained in step (1) was rinsed with pure water, air-dried, and then immersed in a carboxylation modification solution (9 mM β-mercaptoethanol, 1 mM thioglycolic acid, and water as solvent) for 18 h. After being taken out, it was rinsed with water again to obtain a surface carboxylated gold nanoparticle electrode.

[0036] (3) Acquisition of expression of multi-heme cytochrome c

[0037] In this case, the polyheme cytochrome c (CctA) from Shewanella oneidensis MR-1 was used as the base protein in the electrode. To extract this protein, the DNA of CctA (GenBank: AAN55755.1) was recombined into the plasmid pBAD202 / D-TOPO (purchased from ThermoFisher, and maintained as a circular plasmid in Escherichia coli by laboratory subculture) and a 6×His tag was added to the N-terminus. The recombinant strain was then retransfected into the bacteria. The recombinant strain was activated in LB medium until mid-logarithmic growth phase, and expression was induced by adding L-arabinose (1 mM). After 10 hours of induction, the bacteria were harvested and lysed by ultrasonication. The supernatant was collected by ultracentrifugation and passed through a nickel affinity resin. The resin was washed as usual, and the CctA protein was eluted from the resin using imidazole. Buffer replacement was performed using an ultrafiltration cup to dissolve the protein in PBS buffer (20 mM NaH2PO4, 80 mM Na2HPO4, pH 7.4, solvent: water) to obtain a CctA protein solution with a concentration of 1 μM.

[0038] (4) CctA covalently assembled onto the surface of gold nanoparticles

[0039] The surface carboxylated gold nanoparticle electrode obtained in step (2) was immersed in an EDC-NHS solution (containing 0.1 M MEDC, 0.5 M Sulfo-NHS, 0.1 M morpholineethanesulfonic acid, pH 6.0, and water) for 1 hour, then removed and rinsed with pure water and air-dried. The electrode was then transferred and immersed in a 1 μM CctA protein solution for 1 hour to covalently immobilize the conductive protein CctA on the surface of the gold nanoparticle.

[0040] (5) BOD covalently assembled onto the CctA surface

[0041] The electrode surface obtained in step (4) was gently rinsed with pure water, and then immersed in EDC-NHS solution for 1 hour, taken out and rinsed with pure water. Transfer and soak in BOD solution (740U BOD / mL PBS buffer) for 1 hour, and then gently rinse the electrode to obtain the "nano-gold-CctA-BOD" electrode ( Figure 1 ).

[0042] The preparation steps of the "nano-gold-BOD" electrode are similar to those of the "nano-gold-CctA-BOD" electrode. Specifically, the electrode with deposited gold nanoparticles obtained in step (1) is rinsed with pure water, immersed in EDC-NHS solution for 1 hour, removed and rinsed with pure water. The electrode is then transferred and immersed in BOD solution (740U BOD / mL PBS buffer) for 1 hour, and then gently rinsed to obtain the "nano-gold-BOD" electrode.

[0043] 2. Electrochemical Sensing of 2,4-Dichlorophenol

[0044] Electrochemical sensing tests were conducted using 2,4-dichlorophenol as a representative analyte in a three-electrode electrolysis system controlled by an electrochemical workstation. The experiments were conducted using the "nano-gold-BOD" or "nano-gold-CctA-BOD" electrode prepared above as the working electrode, a platinum wire electrode as the counter electrode, and a saturated silver / silver chloride electrode as the reference electrode. The electrolyte used was PBS supplemented with 50 mM NaCl as the supporting electrolyte, and 2 mM 2,4-dichlorophenol was added. The redox reaction of 2,4-dichlorophenol was performed using cyclic voltammetry (potential range -0.6 V to +0.8 V, scan rate 10 mV / s, and scan cycle number 2).

[0045] Cyclic voltammogram of reduction-oxidation transformation of 2,4-dichlorophenol ( Figure 2 ), a clear oxidation peak was observed in the high potential region (+0.575V), which serves as a characteristic signal of BOD oxidation. This result proves that the "nano-gold-CctA-BOD" electrode designed in this invention has the ability to sense 2,4-dichlorophenol.

[0046] 3. Standard curve of 2,4-dichlorophenol concentration

[0047] In the system of step 2, cyclic voltammetry was performed on 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. The peak current value at +0.575 V was recorded for each cyclic voltammetry, and a linear regression equation of concentration-peak current was established, as shown in the following example: 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.

[0048] 4. Effect of cytochrome c on sensor stability

[0049] In an electrolyte containing 2 mM 2,4-dichlorophenol, multiple sensing tests were carried out in a three-electrode system using "nano-gold-BOD" and "nano-gold-CctA-BOD" electrodes as working electrodes, and the changes in the characteristic oxidation peak current signal of 2,4-dichlorophenol were used to reflect the changes in sensor activity (such as Figure 4 The peak current signal of the oxidation peak of the sensor "nanogold-BOD" lacking the auxiliary protein CctA gradually decreased and became inactive during multiple rounds of testing. The sensor "nanogold-CctA-BOD" with the addition of the auxiliary protein CctA significantly improved the activity and stability of BOD.

[0050] Through practical tests, the single-molecule enzyme electrochemical phenol sensor for phenol pollutants in the present invention has the advantages of high sensitivity and high enzyme activity stability, is suitable for online industrial detection and environmental monitoring of phenol pollutants, and has high economic and technical value.

[0051] Example 2

[0052] 1. Preparation of single-molecule enzyme electrochemical phenol sensor ("nanogold-BSA-BOD" electrode)

[0053] The preparation steps of the "nanogold-bovine serum albumin (BSA)-BOD" electrode in this case are the same as those in Example 1, except that the basic protein in the "nanogold-BSA-BOD" electrode is the non-conductive protein BSA.

[0054] 2. Electrochemical Sensing of 2,4-Dichlorophenol

[0055] The stability of the "nano-gold-BSA-BOD" electrode was tested in an electrolyte containing 2 mM 2,4-dichlorophenol. Multiple sensing tests were performed in a three-electrode system. The changes in the characteristic oxidation peak current signal of 2,4-dichlorophenol were used to reflect the changes in sensor activity. The specific steps were the same as in Example 1. The results are shown in FIG. Figure 5 As shown. Figure 5 It can be seen that the sensor "nanogold-BSA-BOD" with the addition of basic protein BSA also significantly improved the activity and stability of BOD.

[0056] Through the above-mentioned example tests, the single-molecule enzyme electrochemical phenol sensor for phenol pollutants in the present invention can greatly improve the activity and stability of the enzyme electrode, effectively extend the service life of the enzyme electrode, make the enzyme electrode have better reproducibility, and have the ability of long-term monitoring, so that the enzyme electrode has higher economic and technical value.

[0057] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A single-molecule enzyme electrochemical phenol sensor, characterized in that: The sensor comprises an electrode substrate, and a gold nanoparticle layer, a base protein, and a bilirubin oxidase sequentially assembled on the surface of the electrode substrate; the base protein is covalently bound to the surface of the gold nanoparticles via a carboxylic acid or amine compound with a thiol group, and the bilirubin oxidase is covalently bound to the base protein; the base protein is heme cytochrome c, iron-sulfur protein, or bovine serum albumin; the electrode substrate is made of gold, platinum, glassy carbon, or graphite; The preparation method of the single-molecule enzyme electrochemical phenol sensor comprises the following steps: (1) In a three-electrode electrochemical system, a conductive, inert solid analytical electrode is used as the working electrode, a platinum wire is used as the counter electrode, and silver / silver chloride is used as the reference electrode. The electrodes are immersed in an electrodeposition solution containing chloroauric acid, and chloroaurate is reduced by cyclic voltammetry to deposit gold nanoparticles on the surface of the working electrode. (2) immersing the working electrode deposited with gold nanoparticles in a carboxylation modification solution for 6 to 48 hours to obtain a surface carboxylated gold nanoparticle electrode; (3) The surface carboxylated gold nanoparticle electrode was treated with EDC-NHS solution, rinsed with water, and immersed in a basic protein solution to covalently fix the basic protein to the surface of the gold nanoparticle. The electrode was then treated with EDC-NHS solution, rinsed with water, and immersed in a bilirubin oxidase solution to prepare a single-molecule enzyme electrochemical sensor.

2. The single-molecule enzyme electrochemical phenol sensor according to claim 1, characterized in that: 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 to +0.6V, the scan rate is 10 to 200mV / s, and the number of scan cycles is 5 to 20.

3. The single-molecule enzyme electrochemical phenol sensor according to claim 1, characterized in that: In step (2), the composition of the carboxyl modification solution is: 9 mM β-mercaptoethanol, 1 mM thioglycolic acid, and the solvent is water.

4. The single-molecule enzyme electrochemical phenol sensor according to claim 1, characterized in that: 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.

5. The single-molecule enzyme electrochemical phenol sensor according to claim 1, characterized in that: In step (3), the basic protein is heme cytochrome c, iron-sulfur protein or bovine serum albumin; the concentration of the basic protein solution is 1 μM, and the concentration of the bilirubin oxidase solution is 740 U BOD / mL PBS buffer.

6. Use of the single-molecule enzyme electrochemical phenol sensor according to claim 1 in detecting phenol pollutants.

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