CN101726521B - Biosensor for rapidly detecting sterigmatocystin and assembling method thereof - Google Patents

Abstract

The invention discloses a biosensor for rapid detection of Aspergillus versicolor in a sample and an assembly method thereof. The biosensor includes a base electrode and a reaction layer assembled on the base electrode, and the reaction layer includes a polymer Membranes, electron transporters, chitosan gels and 6-methoxydifuranocoumarin oxidase. 6-Methoxybisfuranocoumarin oxidase was immobilized in the electron transporter-chitosan hybrid film, and then assembled on the substrate electrode modified by the polymer film to obtain a biosensor for the detection of versicolor . The biosensor of the invention has high sensitivity, fast response time and strong anti-interference ability, and the lower limit of detection of aspergillus versicolor reaches 3 ng/mL, can be used for actual sample detection, and its average recovery rate is 95.8%. The biosensor is simple to manufacture, convenient to use, good in stability and long in service life, and provides a new detection method for realizing the rapid detection of aspergillus versicolor in actual samples.

Description

Biosensor for rapid detection of versicolor and its assembly method

technical field

The invention belongs to the field of biosensors, and relates to a biosensor for rapid detection of versicolor and an assembly method thereof.

Background technique

Sterigmatocystin (ST) is a carcinogenic and teratogenic mycotoxin, and is the synthetic precursor of aflatoxin (Aflatoxin, AFT). Connection composition. ST is a Class 2B carcinogen classified by the International Agency for Research on Cancer. It is closely related to lung cancer, liver cancer, and gastric cancer. It is the main source of fungal pollution in grains such as grain, forage grass, corn, wheat, and peanuts. In our country, most of the areas with high incidence of malignant tumors are due to the pollution of ST in food and grains. However, people have little understanding of the acute and chronic toxicity of ST, so the determination of the content of ST is of great significance. At present, the methods for determining ST mainly include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and coupled mass spectrometry ( Tandem MS) et al (Turner NW, Subrahmanyam S, Piletsky SA. Analytical methods for determination of mycotoxins: A review. Anal Chim Acta 2009; 632: 168-180.). These methods have their own characteristics, but some require the use of expensive instruments, some are cumbersome to operate, and some sample pretreatment is troublesome; in addition, since the fluorescence intensity of ST is far less than that of aflatoxin, it is easier to use derivatization methods. Introducing large errors, sensitive and accurate detection usually requires the use of mass spectrometry detectors, making it difficult to achieve rapid detection of ST content in actual samples.

Enzyme biosensor is a better alternative method, which has the characteristics of fast detection speed and simple operation. The reported enzyme biosensor method, such as in the inventor's Chinese patent ZL 200410028018.9, provides a modified method using aflatoxin detoxification enzyme. The electrode preparation biosensor detects Aspergillus versicolor, the detection line is 10ng/mL, and the response time is 2min. However, this type of biosensor still needs to be improved to improve its sensitivity, stability and reproducibility.

Contents of the invention

The purpose of the present invention is to provide a biosensor that can be used for rapid detection of Aspergillus versicolor in actual samples in view of the deficiencies in the prior art. The biosensor has higher sensitivity, better stability and reproducibility, faster response time, and can be quickly used in biosensors and assembly methods for ST detection in actual samples.

A biosensor for rapid detection of Aspergillus versicolor according to the present invention includes a base electrode and a reaction layer assembled on the base electrode, and the reaction layer includes: a polymer film formed by electropolymerization on the base electrode The polymer film is modified on the base electrode; the electron transporter is an organic molecule or a polymer electron transporter; chitosan gel is made of a chitosan solution; and 6-methoxybisfuranocoumarin oxidase; wherein, the activated single-walled carbon nanotubes are uniformly dispersed in chitosan solution to form an electron transporter-chitosan gel mixed film; the 6-formazan Oxybisfuranocoumarin oxidase was embedded and immobilized on the electron transporter-chitosan gel hybrid membrane, and then assembled on the polymer membrane-modified substrate electrode to form a biosensor for the detection of versicolor .

The preferred polymer film of the present invention is poly-o-phenylenediamine, and o-phenylenediamine can be by electropolymerization, method such as organic synthesis makes polymer film on many conductive substrates, and it promptly is suitable for using in organic electrolyte, also can use In aqueous electrolyte solution, it has high Coulombic efficiency and stability.

The electron transporter described in the present invention can be an organic molecule or a polymer electron transporter. Organic molecular electron mediators mainly include ferrocene and its derivatives, organic dyes, quinones and their derivatives, ionic liquids, etc.; polymeric electron mediators mainly include carbon nanotubes, metal nanoparticles, variable-valence transition metal ions, and organic Redox polymers such as redox type etc. The preferred electron carrier in the present invention is activated single-walled carbon nanotubes, which can be activated by strong acid or strong oxidant. In the present invention, the single-walled carbon nanotubes are dispersed in the chitosan solution, and sol-gel (sol-gel) and its complexes, alum and some other compounds with film-forming ability can also be used to replace the chitosan.

A method for assembling a biosensor for rapid detection of versicolor according to the present invention comprises the following steps:

A. The o-phenylenediamine monomer is polymerized on the surface of the base electrode by an electrochemical method to obtain a base electrode modified by a poly-o-phenylenediamine polymer film;

B, chitosan solution is made chitosan gel;

C, after activating the single-walled carbon nanotubes, add them to the above-mentioned chitosan solution and mix evenly to obtain the single-walled carbon nanotubes-chitosan mixture, which is set aside;

D, prepare 6-methoxybisfuranocoumarin oxidase solution, standby;

E. Mix the 6-methoxybisfuranocoumarin oxidase solution with the single-walled carbon nanotube-chitosan mixture, and add it dropwise on the surface of the substrate electrode modified by the poly-o-phenylenediamine polymer film, at 4°C Assemble overnight to obtain the biosensor.

A preferred preparation method comprises the following steps:

a. Preparation of poly-o-phenylenediamine modified substrate electrode:

The cleaned base electrode is electrochemically pretreated in H ₂ SO ₄ before use, and scanned by cyclic voltammetry until the electrode signal is stable; the processed base electrode is inserted into a nitrogen-saturated environment containing 0.01-0.05 In 0.1-0.5mol/L H ₂ SO ₄ electropolymerization solution of L-o-phenylenediamine, 30-60 cycles of cyclic voltammetric electropolymerization at a sweep rate of 0.05-0.1V/s can form a layer of polymer on the surface of the base electrode. O-phenylenediamine film;

B, the preparation of chitosan solution:

Dissolve the chitosan powder with acetic acid to make the concentration between 0.1%-1.5%, filter out the insoluble impurities after fully dissolving, adjust the pH value of the chitosan solution to 5.5, and store it at 4°C for later use;

c, the preparation of single-walled carbon nanotube-chitosan mixed solution:

Activate single-walled carbon nanotubes with strong acid or strong oxidant, wash until neutral and dry, dissolve in the chitosan solution prepared in step b, and disperse ultrasonically to obtain evenly dispersed 0.1mg/mL-2.5mg/mL Single-walled carbon nanotube-chitosan solution;

d, the preparation of 6-methoxybisfuranocoumarin oxidase:

The genetically engineered Escherichia coli cloned with methoxybisfuranocoumarin oxidase was induced to express, the bacteria were collected, the cells were disrupted by ultrasonication, and then purified with an affinity chromatography column, and the fusion protein was digested to obtain a single 6-methoxy Difuranocoumarin oxidase; chromatographically purify the digested product, collect the enzyme activity peak components in the chromatography process, and concentrate to obtain 6-methoxydifuranocoumarin oxidase; the protein concentration is 2mg/mL Enzyme solution, spare;

Preparation of e, 6-methoxybisfuranocoumarin oxidase-modified biosensor:

Take the single-walled carbon nanotube-chitosan mixture prepared in step c and add it dropwise on the base electrode modified by poly-o-phenylenediamine in step a, and dry it at room temperature to form a film; then add dropwise the The prepared enzyme solution was assembled overnight at 4° C. to obtain a 6-methoxybisfuranocoumarin oxidase-modified biosensor.

The biosensor of the present invention mainly utilizes 6-methoxybisfuranocoumarin oxidase to detect versicolor (ST) as a molecular recognition element. Under the catalysis of coumarin oxidase, a redox reaction occurs, and with the transfer of electrons, a detectable electrical signal is generated. The biosensor of the present invention has high specificity, high selectivity and high sensitivity for the detection of versicolor, and can fully meet the detection of the versicolor content in actual samples.

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

(1) Utilize the biosensor described in the present invention to realize the fast detection to actual sample, only need carry out simple pretreatment to actual sample, just can real-time online monitor, and its response time is fast, less than 10s, more sensitive to ST response ;

(2) The biosensor of the present invention has higher sensitivity, the lower limit of ST detection in the PBS detection system of 0.1mol/L pH7.0 is 3ng/mL, and the linear range is wide, being 10ng/mL-310ng/mL;

(3) The biosensor of the present invention has good stability and reproducibility; strong anti-interference ability, can quickly realize the detection of single or batch samples; the recovery rate of detecting actual samples is 87.6-105.5%, and the detection speed Fast, stable and long service life;

(4) The biosensor of the present invention is time-saving and labor-saving to use, has a fast detection speed, is simple and convenient to operate, and can be popularized and used without training.

Description of drawings

Figure 1 shows the cyclic voltammetry curves of the MDCO/CS-SWCNTs/POPD/Au modified electrode in PBS containing different concentrations of ST; among them, the change of the curve from a to c in the figure corresponds to the concentration of ST being 0ng/mL and 10ng respectively /mL, 20ng/mL.

Figure 2 is the response of the chronoamperometry sensor to ST; the inset in the figure is the relationship between the response current and ST concentration.

Detailed ways

Example 1: Biosensor for Rapid Detection of Aspergillus versicolor

The biosensor for rapid detection of Aspergillus versicolor in actual samples according to the present invention includes a gold electrode (Au) as a base electrode and a reaction layer assembled on the gold electrode, and the reaction layer includes a polymer film, Electron transporter, chitosan gel and 6-methoxydifuranocoumarin oxidase. First, a layer of polymer film is assembled on the gold electrode by electrochemical method, and then the electron transporter is evenly dispersed in chitosan, and then 6-methoxybisfuranocoumarin oxidase is embedded in the electron transporter. In the mixture of body-chitosan, the formed electron transporter-6-methoxybisfuranocoumarin oxidase-chitosan hybrid was assembled on the gold electrode modified by the polymer film to form a 6- Methoxybisfuranocoumarin oxidase-modified biosensor.

In this example, poly-ortho-phenylenediamine was selected as the polymer film assembled on the gold electrode, and the electron mediator used was activated single-walled carbon nanotubes. Single-walled carbon nanotubes were uniformly dispersed in chitosan to form a single-walled carbon nanotube-chitosan suspension, and 6-methoxybisfuranocoumarin oxidase was immobilized in the mixed film by embedding method, and assembled into Sensitive enzyme biosensor for Aspergillus versicolor.

Example 2: Preparation of a biosensor for detecting Aspergillus versicolor

(1) Preparation of materials

(1) Working electrode: a gold electrode with an inner diameter of 2mm was selected from Shanghai Chenhua Instrument Company.

(2) Ortho-phenylenediamine (OPD): chemically pure, purchased from Shanghai Chemical Reagent Co., Ltd.

(3) Single-walled carbon nanotubes (SWCNTs): diameter 2nm, length 5-15μm, purity 95%, purchased from Shenzhen Nanoport.

(4) Chitosan (CS): 85-90% degree of deacetylation, average molecular weight 1×10 ⁶ g/mol, purchased from Zhejiang Aoxing Biotechnology Co., Ltd.

(2) Preparation of poly-o-phenylenediamine-modified gold electrodes

Soak the gold electrode in a solution consisting of 7 volumes of 98% H ₂ SO ₄ and 3 volumes of 30% H ₂ O ₂ for 30 minutes, rinse it with double distilled water, and then wash it with particle sizes of 1.0 μm, 0.3 μm and 0.05μm alumina powder is polished to the mirror surface, cleaned and dried for later use. The gold electrode was electrochemically pretreated in H ₂ SO ₄ before use, and cyclic voltammetry was used to scan until the electrode signal was stable. The treated gold electrode was inserted into a nitrogen-saturated 0.1-0.5mol/L H2SO4 electropolymerization solution containing 0.01-0.05mol/L o-phenylenediamine, and cyclic voltammetric electropolymerization was carried out at a sweep rate of 0.05-0.1V/s for 30 -60 cycles, a layer of poly-o-phenylenediamine film can be formed on the surface of the gold electrode, denoted as POPD/Au.

(3) Preparation of chitosan solution

An appropriate amount of chitosan powder is dissolved in acetic acid solution to form a 0.1%-1.5% chitosan solution. After fully dissolving, adjust the pH value of the chitosan solution to 5.5 and store it at 4°C for future use.

(4) Preparation of single-walled carbon nanotube-chitosan mixture

Single-walled carbon nanotubes are purified before use, activated with strong acid or strong oxidant, washed to neutral and dried, then dissolved in the chitosan solution prepared in step 3, ultrasonically dispersed, and uniformly dispersed 0.1mg/mL-2.5mg/mL single-walled carbon nanotube-chitosan solution.

(5) Preparation of 6-methoxydifuranocoumarin oxidase

The ORF (open reading frame) of the 6-methoxydifuranocoumarin oxidase (MDCO) gene was cloned into the pMAL-C2X vector (purchased from NEB, USA) containing the fusion tag MBP (maltose binding protein) to construct a prokaryotic The recombinant expression plasmid pMAL-C2X-MDCO was introduced into Escherichia coli Rosetta (DE3) (purchased from Tianjin Bomeike Biotechnology Co., Ltd.), and the recombinant methoxybisfuranocoumarin oxidase gene engineering Escherichia coli (Rosetta (DE3)-C2X-MDCO), induced by IPTG (isopropylthioβ-D-galactoside), achieved high expression of MBP_MDCO fusion protein. Bacterial cells were collected, cells were disrupted by ultrasonication, and purified with an affinity chromatography column filled with amylose medium (amylose was purchased from Beijing NEB), and the elution peaks were collected. The fusion protein was cleaved with Factor Xa to obtain a single MDCO (6-methoxydifuranocoumarin oxidase). The digested product was further purified by PhenylSepharose 6 Fast Flow hydrophobic column chromatography (Phenyl Sepharose 6 Fast Flow hydrophobic column chromatography is a product of Pharmacia, Sweden, phenyl Sepharose hydrophobic chromatography medium 6Fast Flow model), using a continuous linear method with decreasing salt concentration. Gradient elution, the elution A solution is 0.05mol/L PBS, pH 6.5, the elution B solution is 0.05mol/L PBS, 2mol/L (NH ₄ )2SO4, pH 6.5, and the enzyme activity peak group during the chromatographic process is collected After concentration, 6-methoxydifuranocoumarin oxidase (MDCO) was obtained. Prepare an enzyme solution with a protein concentration of 2 mg/mL for later use.

(6) Preparation of 6-methoxybisfuranocoumarin oxidase-modified biosensor

Take an appropriate amount of the single-walled carbon nanotube-chitosan mixture prepared in step 4 and add it dropwise on the gold electrode modified by poly-o-phenylenediamine in step 2, and dry to form a film; then add an appropriate amount of step 5 The enzyme solution prepared in , assembled overnight at 4°C, the biosensor modified with 6-methoxybisfuranocoumarin oxidase was obtained, which was denoted as MDCO/CS-SWCNTs/POPD/Au, and the electrode was pre-heated at 0.1M before use. Soak in PBS with pH 7.0 for 2 min, and store in a dry place at 4°C when not in use.

In this example, Aspergillus versicolor (ST) was selected as the test substrate.

Example 3: Electrochemical response of MDCO/CS-SWCNTs/POPD/Au modified electrode to versicolor

The MDCO/CS-SWCNTs/POPD/Au modified electrode prepared in Example 2 was used as a working electrode, together with a common counter electrode and an optional reference electrode to form a three-electrode system.

In this embodiment, an Ag/AgCl electrode (saturated KCl) is selected as the reference electrode, a Pt wire electrode is used as the counter electrode to form a three-electrode system, and 0.1mol/L PBS with a pH of 7.0 is used as the electrolytic support solution.

(1), cyclic voltammetry detection of ST

Using CHI660C electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.), using a three-electrode system (reference electrode: Ag/AgCl electrode, counter electrode: Pt wire electrode, working electrode: MDCO/CS-SWCNTs/POPD/Au modified electrode), Select the cyclic voltammetry scan mode and set the electrochemical parameters as follows:

Working potential: -0.8V-+0.2V

Scan rate: 100mV/s

Rest time: 2s

After setting the parameters, different concentrations of ST were added dropwise to 0.1mol/L PBS with pH 7.0, mixed well and detected by cyclic voltammetry. As shown in Figure 1, in the electrolyte solution without ST, MDCO/CS-SWCNTs/POPD/Au exhibited a pair of aligned and reversible redox peaks, enabling the direct electrochemical reaction of MDCO on the modified electrode (curve a) . With the increase of ST concentration (such as curve b=10ng/mL ST, c=20ng/mL ST), the reduction peak current of the MDCO modified electrode increases, while the oxidation peak current decreases, and the oxidation peak and reduction peak potential remain basically the same. Change, indicating that MDCO/CS-SWCNTs/POPD/Au has a good electrocatalytic reduction effect on ST.

(2) Detect the current-time curve of ST

CHI660C electrochemical workstation selects the current-time detection mode, adopts a three-electrode system (reference electrode: Ag/AgCl electrode, counter electrode: Pt wire electrode, working electrode: MDCO/CS-SWCNTs/POPD/Au modified electrode), set the electrode The chemical parameters are as follows:

Detection potential: -0.4V

Rest time: 2s

Stirring speed: 50r/min

After the background current is stable, add ST dropwise to 10mL 0.1mol/L PBS with pH7.0 for a medium time, which can be ST of different concentrations or ST of equal concentration. The change was 10 ng/mL. It can be seen from Figure 2 that as the ST concentration gradient changes, the response current continues to increase, and the enzyme biosensor responds quickly and sensitively, with an average response time of less than 10 s. The response current of the enzyme electrode has a linear relationship with the ST concentration in the range of 10-310ng/mL (Figure 2 illustration), and the linear regression equation is: I(μA)=0.0389c(ng/ml)+2.5825(r=0.997), The detection limit was 3ng/mL.

Embodiment four: Stability and reproducibility of enzyme electrode

A MDCO/CS-SWCNTs/POPD/Au modified electrode was placed in a pH 7.0 PBS solution containing 20 ng/mL ST, and the cyclic voltammetry was scanned for 100 cycles, and the peak shape remained almost unchanged, indicating that the enzyme biosensor of the present invention Has very good stability. The average relative standard deviation (RSD) of the current response was 3.9% when 20ng/mL ST was measured 11 times by this biosensor, which indicated that the electrode had good detection repeatability. In addition, seven enzyme-modified electrodes were prepared at the same time, and the RSD of the response current to 20ng/mL ST was 4.4% under the same conditions. It shows that the modified electrode has good fabrication reproducibility. When the electrode is not in use, it is stored above the pH 7.0 PBS solution at 4°C. During intermittent use, the response signal is 95.5% of the initial value after 4 days, and can still maintain 91% after 30 days. The biosensor of the present invention can be used for a long time .

Example 5: Anti-interference ability of MDCO/CS-SWCNTs/POPD/Au modified electrode

The anti-interference ability of MDCO/CS-SWCNTs/POPD/Au modified electrode was studied by chronoamperometry, the test condition parameters were set as shown in Example 3 (two), in 10mL containing 50ng/mL ST 0.1mol/L pH7 .0 PBS, drop the following possible interferents at different concentrations to analyze their anti-interference ability.

Types of interfering substances added dropwise:

PO ₄ ^3- , NO ^3- , citrate, fructose, glucose at 1000 times ST concentration;

EDTA, NH ⁴⁺ , oxalic acid, α-lactose, ascorbic acid 500 times the concentration of ST;

100 times the ST concentration of glycine, L-serine, uric acid, methanol, oleic acid;

10 times the ST concentration of Fe ³⁺ , p-nitrophenol, acetic acid.

Test effect: when the control relative error does not exceed ±5% of the current response value of 50ng/mL ST, 1000 times of PO ₄ ^3- , NO ^3- , citrate, fructose, glucose; 500 times of EDTA, NH ⁴⁺ , oxalic acid, α-lactose, ascorbic acid; 100 times of glycine, L-serine, uric acid, methanol, oleic acid; 10 times of Fe ³⁺ , p-nitrophenol, acetic acid, etc. will not interfere with the determination.

Embodiment six: Actual sample determination and recovery rate experiment

The enzyme biosensor of the present invention is used for actual sample determination and detection of its recovery rate. The actual sample can be a series of agricultural and sideline products and food crops such as corn, peanut, rice, wheat, etc., and can also be feed, beverage, A series of products that may contain ST, such as soy sauce, olive oil, and peanut oil.

In this example, corn samples were selected for detection.

Treatment of corn samples: Take 1g of high-quality corn flour, add different concentrations of ST, shake and extract in 5ml 80% methanol solution for 45min, centrifuge (5000r/min, 10min), and use 0.1mol/L PBS with pH7.0 for the supernatant According to 1:5 (V/V) dilution and detection. Other samples can be detected after simple pretreatment according to the similar method.

Cyclic voltammetry detection: test condition parameter setting is as shown in (one) of embodiment three, carries out cyclic voltammetry detection to the corn sample that respectively contains 10ng/nL, 50ng/nL, 100ng/nL, 150ng/nL ST, each The concentration of corn samples were measured 5 times. The test results are shown in Table 1.

Table 1: Determination results of ST content in corn samples

As shown in the table above, the relative standard deviation of the measured samples was between 2.8% and 4.1%, the recovery rate of standard addition was between 87.6% and 105.5%, and the average recovery rate was 95.8%.

Claims (1)

1. A biosensor for rapid detection of aspergillus versicolor, comprising a substrate electrode and a reaction layer assembled on the substrate electrode, characterized in that, the reaction layer comprises: a polymer film consisting of poly-o-phenylenediamine electropolymerized on a base electrode; the polymer film is decorated on said base electrode; The electron transporter is a polymer electron transporter; chitosan gel, made from a solution of chitosan; and 6-methoxydifuranocoumarin oxidase; Wherein, the activated single-walled carbon nanotubes are evenly dispersed in the chitosan solution to form an electron transporter-chitosan gel mixed film; the 6-methoxybisfuranocoumarin oxidase is embedded and fixed on Electron transporter-chitosan gel mixed membrane, and then assembled on the substrate electrode modified by polymer membrane to form a biosensor for detection of Aspergillus versicolor. 2 . The biosensor according to claim 1 , wherein the electron mediator is a single-walled carbon nanotube activated by a strong acid or a strong oxidant. 3. The assembly method of biosensor according to claim 1, is characterized in that, comprises the following steps: A. The o-phenylenediamine monomer is polymerized on the surface of the base electrode by an electrochemical method to obtain a base electrode modified by a poly-o-phenylenediamine polymer film; B, chitosan solution is made chitosan gel; C, after activating the single-walled carbon nanotubes, add them to the above-mentioned chitosan solution and mix evenly to obtain the single-walled carbon nanotubes-chitosan mixture, which is set aside; D, prepare 6-methoxybisfuranocoumarin oxidase solution, standby; E. Mix the 6-methoxybisfuranocoumarin oxidase solution with the single-walled carbon nanotube-chitosan mixture, and add it dropwise on the surface of the substrate electrode modified by the poly-o-phenylenediamine polymer film, at 4°C Assemble overnight to obtain the biosensor. 4. The assembly method of biosensor according to claim 3, is characterized in that, comprises the following steps: a. Preparation of poly-o-phenylenediamine modified substrate electrode: The cleaned base electrode is electrochemically pretreated in H ₂ SO ₄ before use, and scanned by cyclic voltammetry until the electrode signal is stable; the processed base electrode is inserted into a nitrogen-saturated environment containing 0.01-0.05 In 0.1-0.5mol/L H ₂ SO ₄ electropolymerization solution of L-o-phenylenediamine, 30-60 cycles of cyclic voltammetric electropolymerization at a sweep rate of 0.05-0.1V/s can form a layer of polymer on the surface of the base electrode. O-phenylenediamine film; B, the preparation of chitosan solution: Dissolve the chitosan powder with acetic acid to make the concentration between 0.1%-1.5%. After fully dissolving, filter out the insoluble impurities, adjust the pH value of the chitosan solution to 5.5, and store it at 4°C for later use; c, the preparation of single-walled carbon nanotube-chitosan mixed solution: Activate single-walled carbon nanotubes with strong acid or strong oxidant, wash until neutral and dry, dissolve in the chitosan solution prepared in step b, and disperse ultrasonically to obtain evenly dispersed 0.1mg/mL-2.5mg/mL Single-walled carbon nanotube-chitosan solution; d, the preparation of 6-methoxybisfuranocoumarin oxidase: The genetically engineered Escherichia coli cloned with methoxybisfuranocoumarin oxidase was induced to express, the bacteria were collected, the cells were disrupted by ultrasonication, and then purified with an affinity chromatography column, and the fusion protein was digested to obtain a single 6-methoxy Difuranocoumarin oxidase; chromatographically purify the digested product, collect the enzyme activity peak components in the chromatography process, and concentrate to obtain 6-methoxydifuranocoumarin oxidase; the protein concentration is 2mg/mL Enzyme solution, spare; Preparation of e, 6-methoxybisfuranocoumarin oxidase-modified biosensor: Take the single-walled carbon nanotube-chitosan mixture prepared in step c and add it dropwise on the base electrode modified by poly-o-phenylenediamine in step a, and dry it at room temperature to form a film; then add dropwise the The prepared enzyme solution was assembled overnight at 4° C. to obtain a 6-methoxybisfuranocoumarin oxidase-modified biosensor.

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