CN110501400A - An enzyme biosensor for detecting inosinic acid, its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of biosensors and discloses an enzyme biosensor for detecting inosinic acid, its preparation method and application. The linear detection range is 0.313-210 μg/L, and the detection limit is 0.238 μg/L. An electrode, a counter electrode and a modified electrode obtained by solidifying a material recognition film sensitive to inosinic acid on the surface of the working electrode are composed of a composite solution a, a 5'-nucleotidase solution and a xanthine oxidase solution by volume The double-enzyme complex solution b and the bovine serum albumin solution composed of a ratio of 1:1 are composed of a volume ratio of 1:1:1, and the complex solution a is composed of MXene-Ti ₃ C ₂ Tx solution (T is selected from -OH, -O or -F ), chitosan solution, chloroauric acid solution and chloroplatinic acid solution in a volume ratio of 12:1.2:1:1. The enzyme biosensor of the invention is simple, fast, accurate and high in sensitivity, and can be used for quantitative detection of inosinic acid in food.

Description

An enzyme biosensor for detecting inosinic acid, its preparation method and application

technical field

The invention relates to the technical field of biosensors, in particular to an enzyme biosensor for detecting inosinic acid, its preparation method and application, which are used for the detection of inosinic acid (IMP) in food.

Background technique

Inosinic acid (IMP) is a single nucleotide and is a metabolic intermediate of ATP in organisms. Inosinic acid and its salts can produce umami, which is one of the important flavor substances in meat products. It can also act synergistically with sodium glutamate to double the umami, and is a commonly used flavoring agent. In addition, inosinic acid is also an important indicator internationally recognized to measure the freshness of meat. At present, methods for quantitative detection of inosinic acid include spectrophotometry, high performance liquid chromatography, etc., but these methods have problems such as high detection cost, long time consumption, and complicated operation. In comparison, electrochemical enzyme biosensors have It has high sensitivity and selectivity, can effectively amplify the signal, and the sample amount is small, the response is fast, the operation is simple, and it is more suitable for the detection of inosinic acid in food.

The key step in the preparation of enzyme electrodes is the immobilization of enzymes. In recent decades, researchers have been constantly looking for suitable enzyme carrier materials and immobilization methods. MXene-Ti ₃ C ₂ Tx (T=OH, O or F) material is a transition metal carbide with a graphene-like two-dimensional structure, high specific surface area, high conductivity, stability, and biocompatibility Well, it can promote the electron transfer between the enzyme active center and the electrode interface, and has a good application prospect in the biosensor enzyme carrier. Nano-Au and Pt particles can reduce the overpotential of the enzymatic hydrolysis product hydrogen peroxide, thereby improving the enzyme biosensor. Chitosan has good film-forming properties and is often used for enzyme immobilization.

Contents of the invention

In order to overcome the problems of high detection cost, long time-consuming and complicated operation of the existing methods for detecting inosinic acid, the primary purpose of the present invention is to provide an enzyme biosensor for detecting inosinic acid with good selectivity, sensitivity and stability .

Another object of the present invention is to provide a method for preparing the above-mentioned enzyme biosensor, and obtain an enzyme biosensor with stable performance through suitable enzyme carrier materials and immobilization methods.

Another object of the present invention is to provide the application of the above-mentioned enzyme biosensor in the quantitative detection of inosinic acid.

Above-mentioned purpose of the present invention is achieved through the following technical methods:

In the first aspect, the enzyme biosensor for detecting inosinic acid of the present invention is composed of a reference electrode, a counter electrode and a modified electrode, and the modified electrode is obtained by curing a material recognition film sensitive to inosinic acid on the surface of the working electrode; wherein:

The substance recognition membrane is composed of composite solution a, dual-enzyme composite solution b and 10 mg/mL bovine serum albumin solution in a volume ratio of 1:1:1; wherein, the composite solution a is composed of 0.5-1.25 mg/mL The MXene-Ti ₃ C ₂ Tx solution, the chitosan solution of 5mg/mL, the chloroauric acid solution of 5mmol/L and the chloroplatinic acid solution of 3mmol/L are composed by volume ratio 12:1.2:1:1, the described Dual-enzyme compound solution b is composed of 2mg/mL 5'-nucleotidase solution and 2mg/mL xanthine oxidase solution in a volume ratio of 1:1;

T in the MXene-Ti ₃ C ₂ Tx is selected from -OH, -O or -F;

The 5'-nucleotidase solution is obtained by dissolving 5'-nucleotidase in PBS solution with pH 6.0 and 0.01M, and the xanthine oxidase solution is obtained by dissolving xanthine oxidase in pH 6.0 and 0.01M PBS solution. PBS solution was obtained.

Preferably, the preparation method of the MXene-Ti ₃ C ₂ Tx is: slowly dissolve 1.6g LiF in 20mL 9mol/L HCl aqueous solution, stir for 5min, then add 1.0gTi ₃ AlC ₂ powder, and stir magnetically at 400rpm for 24h at room temperature ; Then centrifuge at 3500rpm for 5min, wash the obtained precipitate with ultrapure water; repeat the ultrasonic and washing operation 5 to 8 times, when the pH of the solution is 6, collect the precipitate, dissolve it in 100mL water, and store it under the protection of argon , sonicated in an ice bath at 4°C for 3h to delaminate the obtained Ti ₃ C ₂ T _x flakes; finally centrifuged at 8000rpm for 1h, collected the supernatant, and dried in vacuum at 60°C to obtain a black powder, namely MXene-Ti ₃ C ₂ T _x .

Preferably, the working electrode is a glassy carbon electrode, the reference electrode is an Ag/AgCl electrode, and the counter electrode is a platinum electrode.

Preferably, the linear detection range of the enzyme biosensor is 0.313-210 μg/L.

Preferably, the detection limit of the enzyme biosensor is 0.238 μg/L.

In a second aspect, the preparation method of the enzyme biosensor comprises the following steps:

(1) Carry out surface pretreatment to the working electrode;

(2) Mix the MXene-Ti ₃ C ₂ Tx solution, the chitosan solution, the chloroauric acid solution, and the chloroplatinic acid solution according to the ratio to obtain a composite solution a, and add it dropwise to step (1) surface pretreatment After the surface of the working electrode was dried at room temperature;

(3) Mix the 5'-nucleotidase solution and the xanthine oxidase solution according to the proportioning ratio to obtain a double-enzyme composite solution b, and add it dropwise to the electrode surface after step (2) treatment, and dry at room temperature;

(4) adding the bovine serum albumin solution dropwise to the electrode surface treated in step (3), and drying at room temperature to obtain a modified electrode;

(5) The modified electrode obtained in step (4) is combined with the reference electrode and the counter electrode to form a three-electrode system, to obtain final product;

Wherein, the volume ratio of the compound solution a, the double-enzyme compound solution b and the bovine serum albumin solution is 1:1:1.

Preferably, in step (1), the step of pre-treating the surface of the working electrode is: polishing the working electrode to a mirror surface on a polishing cloth with 0.3 μm and 0.05 μm alumina powder in sequence, then rinsing with ultrapure water, and then Ultrasonic treatment in ultrapure water for 1min, then placed in potassium ferricyanide solution for activation treatment, taken out, rinsed with ultrapure water, and blown dry with nitrogen; the potassium ferricyanide solution is composed of K ₃ [Fe(CN) ₆ ], K ₄ [Fe(CN) ₆ ] and KCl in a molar ratio of 1:1:100.

In the third aspect, the application of the above-mentioned biosensor in the quantitative detection of inosinic acid has a linear detection range of 0.313-210 μg/L and a detection limit of 0.238 μg/L.

The present invention selects a novel two-dimensional nanomaterial MXene-Ti ₃ C ₂ Tx (T=-OH, -O or -F), combines nano Au and Pt particles and utilizes the film-forming and embedding properties of chitosan to construct an electric The enzyme carrier of the chemical enzyme biosensor can increase the immobilization amount and stability of the enzyme catalyst on the surface of the electrode, and is helpful for the catalysis of the substrate. A double-enzyme compound solution composed of 5'-nucleotidase and xanthine oxidase was added dropwise, and then sealed with bovine serum albumin solution to form a modified electrode. Then, a three-electrode system was formed with reference electrode and counter electrode to prepare An enzyme biosensor for detecting inosinic acid was obtained, with a linear detection range of 0.313-210 μg/L and a detection limit of 0.238 μg/L.

Compared with prior art, the beneficial effect of the present invention is:

(1) The biosensor of the present invention has good electron transport property, can transfer the electrons generated by the reaction between the enzyme active center and the electrode surface well, and improve the reaction speed of the biosensor.

(2) The biosensor of the present invention has good selectivity, can accurately detect inosinic acid, has strong anti-interference ability, and has no current to interfering substances such as cysteine, methionine, inosine diphosphate and inosine triphosphate response.

(3) The biosensor of the present invention has good stability and reproducibility. It is continuously measured 10 times by square wave voltammetry, and the result only shows a relative standard deviation of 3.4%, which can still be reached after 2 weeks of storage at 4°C. 95% of the original response current.

(4) The biosensor of the present invention can be used for the detection of inosinic acid in food. The preparation process is simple and safe, and it can be detected at room temperature. It has a wide detection range, a low detection limit, and good application prospects.

Description of drawings

Fig. 1 is a schematic diagram of the overall structure of the enzyme biosensor in the present invention.

Fig. 2 is a flow chart of the preparation of the working electrode of the enzyme biosensor in the present invention.

Fig. 3 is the cyclic voltammetry curve diagram of enzyme biosensor working electrode in PBS solution (0.01M, pH=6.0) in embodiment 2; Wherein, a represents that the scan rate is 160mV/s, and b represents that the scan rate is 120mV/s , c means the scan rate is 80mV/s, d means the scan rate is 40mV/s.

Fig. 4 is the cyclic voltammetry curve figure of enzyme biosensor in the embodiment 3 in the PBS solution (0.01M, pH=6.0) that exists 3 μ mol/L inosinic acid and does not exist inosinic acid; Wherein, a represents that in the PBS solution There is inosinic acid at a concentration of 3 μmol/L, and b indicates the absence of inosinic acid in the PBS solution.

Fig. 5 is the differential pulse voltammetry graph of enzyme biosensor in embodiment 3 in the PBS solution (0.01M, pH=6.0) that exists 3 μ mol/L inosinic acid and does not exist inosinic acid; Wherein, a represents PBS solution There is no inosinic acid in the solution, b means there is 3 μmol/L inosinic acid in the PBS solution.

Fig. 6 is a graph showing the current-time response curves of the enzyme biosensor in Example 4 to different concentrations of inosinic acid under the conditions of a scan rate of 100 mV/s and a potential of -0.15 V in PBS solution.

7 is a standard curve of the response current of the enzyme biosensor in Example 4 to different concentrations of inosinic acid.

Fig. 8 is the current-time response curve of the enzyme biosensor in Example 5 to different interfering substances and inosinic acid in PBS solution.

Detailed ways

The present invention will be described in further detail below in conjunction with examples and accompanying drawings, but the embodiments of the present invention are not limited thereto.

The MXene-Ti ₃ C ₂ Tx material of the embodiment of the present invention can be a commercially available product or prepared by the following method: slowly dissolve 1.6g LiF in 20mL 9mol/L HCl aqueous solution, stir for 5min, then add 1.0gTi ₃ AlC ₂ powder, room temperature Stir magnetically at 400rpm for 24h under the same conditions; then centrifuge at 3500rpm for 5min, and wash the resulting precipitate with ultrapure water; repeat the ultrasonic and washing operations 5 to 8 times, when the pH of the solution is measured to be 6, collect the precipitate and dissolve it in 100mL In water, under the protection of argon, sonicate in an ice bath at 4°C for 3h to delaminate the obtained Ti ₃ C ₂ T _x flakes, and finally centrifuge at 8000rpm for 1h, collect the supernatant, and dry in vacuum at 60°C to obtain a black powder, which is MXene-Ti ₃ C ₂ T _x .

Example 1

The overall structure of the enzyme biosensor for detecting inosinic acid is shown in Figure 1. It consists of a reference electrode 2, a counter electrode 3 and a modified electrode 1. The modified electrode 1 consists of a working electrode 1-1 and a substance solidified on the surface of the working electrode. The recognition membrane 1-2 is composed of, wherein, the material recognition membrane 1-2 sensitive to inosinic acid is composed of MXene-Ti ₃ C ₂ Tx solution, chitosan solution, chloroauric acid solution, chloroplatinic acid solution, 5'-core The glucuronidase solution, the xanthine oxidase solution and the bovine serum albumin solution are prepared, and the above-mentioned enzyme biosensor is put into the test solution 4 to detect the content of inosinic acid in the test solution 4 .

The preparation process of the modified electrode 1 is shown in Figure 2, and the specific preparation method steps are:

(1) Working electrode pretreatment. The working electrode was polished to a mirror surface on a polishing cloth with alumina powder with a diameter of 0.3 μm and 0.05 μm in turn, then rinsed with ultrapure water, and then ultrasonically treated in ultrapure water for 1 min, after drying, the glassy carbon electrode Place in potassium ferricyanide solution (a mixed solution composed of K ₃ [Fe(CN) ₆ ], K ₄ [Fe(CN) ₆ ] and KCl in a molar ratio of 1:1:100), at -0.2～ The electrodes were activated by scanning 4 cycles with cyclic voltammetry at 0.6V, rinsed with ultrapure water, and dried with nitrogen to obtain pretreated glassy carbon electrodes.

(2) Add compound solution a dropwise. Add 5 μL a solution dropwise on the pretreated electrode surface; a solution is a mixed solution of MXene-Ti ₃ C ₂ Tx solution, chitosan solution, chloroauric acid solution, and chloroplatinic acid solution, in which MXene-Ti ₃ C ₂ The concentration of Tx solution is 0.5～1.25mg/mL, the concentration of chitosan solution is 5mg/mL, the concentration of chloroauric acid solution is 5mmol/L, the concentration of chloroplatinic acid solution is 3mmol/L, MXene-Ti ₃ C ₂ The volume ratio of Tx solution, chitosan solution, chloroauric acid solution, and chloroplatinic acid solution is 12:1.2:1:1;

(3) Add double-enzyme compound solution b dropwise. After the a solution dropped on the electrode surface is dry, add 5 μL b solution dropwise; b solution is a mixed solution of 5'-nucleotidase solution and xanthine oxidase solution, wherein the concentration of 5'-nucleotidase solution is 2 mg /mL, the concentration of xanthine oxidase solution is 2mg/mL, the volume ratio of 5'-nucleotidase solution and xanthine oxidase solution is 1:1;

(4) Add bovine serum albumin solution dropwise. After the solution b dropped on the surface of the electrode is dried, 5 μL of bovine serum albumin solution is added dropwise, and the modified electrode 1 is prepared after drying.

The above-mentioned modified electrode 1, the reference electrode 2 and the counter electrode 3 constitute a three-electrode system to obtain an enzyme biosensor for detecting inosinic acid.

Example 2

The enzyme biosensor for detecting inosinic acid, its preparation steps are as follows:

(1) A glassy carbon electrode with a diameter of 3 mm was polished to a mirror surface on a polishing cloth with alumina powders with a diameter of 0.3 μm and 0.05 μm in sequence, then rinsed with ultrapure water, and then ultrasonically treated in ultrapure water for 1 min, and left to dry After drying, place the glassy carbon electrode in a potassium ferricyanide solution (a mixture of K ₃ [Fe(CN) ₆ ], K ₄ [Fe(CN) ₆ ] and KCl in a molar ratio of 1:1:100 Solution), at -0.2 ~ 0.6V, use cyclic voltammetry to scan for 4 circles to activate the electrode, take it out, wash it with ultrapure water, and dry it with nitrogen to obtain a pretreated glassy carbon electrode.

(2) Disperse 125mg of MXene-Ti ₃ C ₂ Tx material in 100mL of ultrapure water to obtain a MXene-Ti ₃ C ₂ Tx solution with a concentration of 1.25mg/mL; use 1wt% acetic acid solution to prepare a shell with a concentration of 5mg/mL Polysaccharide solution; 98.50mg tetrachloroauric acid trihydrate was dissolved in 50mL ultrapure water to obtain a chloroauric acid solution with a concentration of 5mmol/L; 77.70mg hexachloroplatinic acid hexahydrate was dissolved in 50mL ultrapure water, Obtain the chloroplatinic acid solution that concentration is 3mmol/L; Dissolve 20mg5'-nucleotidase in 10mL PBS solution (pH 6.0,0.01M), obtain the 5'-nucleotidase solution that concentration is 2mg/mL; Dissolve 20mg of xanthine oxidase in 10mL of PBS solution (pH 6.0, 0.01M) to obtain a xanthine oxidase solution with a concentration of 2mg/mL, and dissolve 500mg of bovine serum albumin in 50mL of ultrapure water to obtain a concentration of 10mg/mL bovine serum albumin solution.

(3) Mix MXene-Ti ₃ C ₂ Tx solution, chitosan solution, chloroauric acid solution and chloroplatinic acid solution according to the volume ratio of 12:1.2:1:1 to obtain composite solution a, take 5 μL composite solution a is added dropwise to the surface of the glassy carbon electrode whose surface has been pretreated in step (1), and dried at room temperature.

(4) Mix the 5'-nucleotidase solution and the xanthine oxidase solution according to the volume ratio of 1:1 to obtain the double-enzyme compound solution b, take 5 μL of the double-enzyme compound solution b and add it dropwise to step (3) The glassy carbon electrode surface was dried at room temperature.

(5) 5 μL of bovine serum albumin solution was added dropwise to the surface of the glassy carbon electrode in step (4), and dried at room temperature to obtain a modified electrode.

(6) The modified electrode was combined with the Ag/AgCl electrode (KCl concentration: 3mol/L) as the reference electrode and the platinum wire as the counter electrode to form a three-electrode system to obtain an enzyme biosensor for detecting inosinic acid.

Cyclic voltammetry is used to test the enzyme biosensor prepared in Example 2. The specific steps are: at room temperature, the enzyme biosensor for detecting inosinic acid is immersed in a PBS buffer solution with a pH of 6.0 and 0.01M, and the enzyme biosensor is tested by cyclic voltammetry. For electrochemical test, the scanning rate is 40, 80, 120, 160mV/s, and the scanning potential range is -0.6～0.6V.

Fig. 3 is the cyclic voltammetry curve diagram of the enzyme biosensor working electrode detecting inosinic acid in PBS solution of pH 6.0, 0.01M, wherein, a indicates that the scan rate is 160mV/s, b indicates that the scan rate is 120mV/s, c indicates that the scan rate is 80mV/s, d indicates that the scan rate is 40mV/s; the cathode and anode peak currents increase linearly with the increase of the scan rate, and the peak current is proportional to the scan rate, indicating that 5'-inosinase/ The electron transfer between the xanthine enzyme and the working electrode can be performed rapidly on the substance recognition membrane.

Example 3

The enzyme biosensor for detecting inosinic acid, its preparation steps are as follows:

(1) A glassy carbon electrode with a diameter of 3 mm was polished to a mirror surface on a polishing cloth with alumina powders with a diameter of 0.3 μm and 0.05 μm in sequence, then rinsed with ultrapure water, and then ultrasonically treated in ultrapure water for 1 min, and left to dry After drying, place the glassy carbon electrode in a potassium ferricyanide solution (a mixture of K ₃ [Fe(CN) ₆ ], K ₄ [Fe(CN) ₆ ] and KCl in a molar ratio of 1:1:100 Solution), at -0.2 ~ 0.6V, use cyclic voltammetry to scan for 4 circles to activate the electrode, take it out, wash it with ultrapure water, and dry it with nitrogen to obtain a pretreated glassy carbon electrode.

(2) Disperse 125mg of MXene-Ti ₃ C ₂ Tx material in 100mL of ultrapure water to obtain a MXene-Ti ₃ C ₂ Tx solution with a concentration of 1.25mg/mL; use 1wt% acetic acid solution to prepare a shell with a concentration of 5mg/mL Polysaccharide solution; 98.50mg tetrachloroauric acid trihydrate was dissolved in 50mL ultrapure water to obtain a chloroauric acid solution with a concentration of 5mmol/L; 77.70mg hexachloroplatinic acid hexahydrate was dissolved in 50mL ultrapure water, Obtain the chloroplatinic acid solution that concentration is 3mmol/L; Dissolve 20mg5'-nucleotidase in 10mL PBS solution (pH 6.0,0.01M), obtain the 5'-nucleotidase solution that concentration is 2mg/mL; Dissolve 20mg of xanthine oxidase in 10mL of PBS solution (pH 6.0, 0.01M) to obtain a xanthine oxidase solution with a concentration of 2mg/mL, and dissolve 500mg of bovine serum albumin in 50mL of ultrapure water to obtain a concentration of 10mg/mL bovine serum albumin solution.

(3) Mix MXene-Ti ₃ C ₂ Tx solution, chitosan solution, chloroauric acid solution and chloroplatinic acid solution according to the volume ratio of 12:1.2:1:1 to obtain composite solution a, take 5 μL composite solution a is added dropwise to the surface of the glassy carbon electrode whose surface has been pretreated in step (1), and dried at room temperature.

(4) Mix the 5'-nucleotidase solution and the xanthine oxidase solution according to the volume ratio of 1:1 to obtain the double-enzyme compound solution b, take 5 μL of the double-enzyme compound solution b and add it dropwise to step (3) The glassy carbon electrode surface was dried at room temperature.

(5) 5 μL of bovine serum albumin solution was added dropwise to the surface of the glassy carbon electrode in step (4), and dried at room temperature to obtain a modified electrode.

(6) The modified electrode was combined with the Ag/AgCl electrode (KCl concentration: 3mol/L) as the reference electrode and the platinum wire as the counter electrode to form a three-electrode system to obtain an enzyme biosensor for detecting inosinic acid.

Cyclic voltammetry is used to test the enzyme biosensor prepared in Example 3. The specific steps are: at room temperature, the enzyme biosensor for detecting inosinic acid is immersed in a PBS buffer solution of pH 6.0 and 0.01M, and injected into the PBS buffer solution. 3μmol/L inosinic acid, the electrochemical test was carried out by cyclic voltammetry, the scan rate was 100mV/s, and the scan potential range was -0.2～0.6V.

Figure 4 is a cyclic voltammetry curve of the enzyme biosensor working electrode for detecting inosinic acid in PBS solution with pH 6.0 and 0.01M, wherein a indicates that there is 3 μmol/L inosinic acid in the PBS solution, and b indicates the PBS solution There is no inosinic acid in the medium, when 3 μmol/L inosinic acid is injected, the oxidation current and reduction current increase significantly, indicating that the 5'-inosinase and xanthinase immobilized on the electrode have high biocatalytic properties and can Sensitive current response to inosinic acid in solution, rapid electron transfer on the electrode surface.

The enzyme biosensor prepared in Example 3 was tested by differential pulse voltammetry. The specific steps were as follows: at room temperature, the enzyme biosensor for detecting inosinic acid was immersed in a PBS buffer solution of pH 6.0 and 0.01M, and in the PBS buffer solution, 3μmol/L inosinic acid was injected into the medium, and the electrochemical test was carried out by differential pulse voltammetry, and the scanning potential range was -0.2～0.6V.

Fig. 5 is the differential pulse voltammetry curve diagram of the working electrode of the enzyme biosensor for detecting inosinic acid in the PBS solution of pH 6.0 and 0.01 M, wherein, a indicates that there is no inosinic acid in the PBS solution, and b indicates that in the PBS solution Inosinic acid was present at 3 μmol/L. Differential pulse voltammetry applies a step potential to the electrodes. Compared with cyclic voltammetry, it has higher sensitivity and resolution, and has good detection ability. Compared with a and b in the figure, the current peak value of the b curve with inosinic acid in the PBS solution is higher, and the current peak is around -0.15V, indicating that it is observed near the relatively low -0.15V (relative to Ag/AgCl) potential The maximum response current of the enzyme biosensor to inosinic acid, so -0.15V can be used as a fixed voltage for further electrochemical characterization by current-time method, thereby effectively reducing background interference factors and lowering the detection limit.

Example 4

The enzyme biosensor for detecting inosinic acid, its preparation steps are as follows:

(1) A glassy carbon electrode with a diameter of 3 mm was polished to a mirror surface on a polishing cloth with alumina powders with a diameter of 0.3 μm and 0.05 μm in sequence, then rinsed with ultrapure water, and then ultrasonically treated in ultrapure water for 1 min, and left to dry After drying, place the glassy carbon electrode in a potassium ferricyanide solution (a mixture of K ₃ [Fe(CN) ₆ ], K ₄ [Fe(CN) ₆ ] and KCl in a molar ratio of 1:1:100 Solution), at -0.2 ~ 0.6V, use cyclic voltammetry to scan for 4 circles to activate the electrode, take it out, wash it with ultrapure water, and dry it with nitrogen to obtain a pretreated glassy carbon electrode.

(2) Disperse 50 mg of MXene-Ti ₃ C ₂ Tx material in 100 mL of ultrapure water to obtain a MXene-Ti ₃ C ₂ Tx solution with a concentration of 0.5 mg/mL; use 1 wt% acetic acid solution to prepare a shell with a concentration of 5 mg/mL Polysaccharide solution; 98.50mg tetrachloroauric acid trihydrate was dissolved in 50mL ultrapure water to obtain a chloroauric acid solution with a concentration of 5mmol/L; 77.70mg hexachloroplatinic acid hexahydrate was dissolved in 50mL ultrapure water, Obtain the chloroplatinic acid solution that concentration is 3mmol/L; Dissolve 20mg5'-nucleotidase in 10mL PBS solution (pH 6.0,0.01M), obtain the 5'-nucleotidase solution that concentration is 2mg/mL; Dissolve 20mg of xanthine oxidase in 10mL of PBS solution (pH 6.0, 0.01M) to obtain a xanthine oxidase solution with a concentration of 2mg/mL, and dissolve 500mg of bovine serum albumin in 50mL of ultrapure water to obtain a concentration of 10mg/mL bovine serum albumin solution.

(3) Mix MXene-Ti ₃ C ₂ Tx solution, chitosan solution, chloroauric acid solution and chloroplatinic acid solution according to the volume ratio of 12:1.2:1:1 to obtain composite solution a, take 5 μL composite solution a is added dropwise to the surface of the glassy carbon electrode whose surface has been pretreated in step (1), and dried at room temperature.

(4) Mix the 5'-nucleotidase solution and the xanthine oxidase solution according to the volume ratio of 1:1 to obtain the double-enzyme compound solution b, take 5 μL of the double-enzyme compound solution b and add it dropwise to step (3) The glassy carbon electrode surface was dried at room temperature.

(5) 5 μL of bovine serum albumin solution was added dropwise to the surface of the glassy carbon electrode in step (4), and dried at room temperature to obtain a modified electrode.

(6) The modified electrode was combined with the Ag/AgCl electrode (KCl concentration: 3mol/L) as the reference electrode and the platinum wire as the counter electrode to form a three-electrode system to obtain an enzyme biosensor for detecting inosinic acid.

The enzyme biosensor prepared in Example 4 was tested by the current-time method. The specific steps were as follows: at room temperature, the enzyme biosensor for detecting inosinic acid was immersed in 10 mL of PBS buffer solution with pH 6.0 and 0.05 M, and buffered in PBS every 30 seconds. 3 μL of inosinic acid with a concentration of 3 μmol/L was added to the solution, and stirred continuously with a magnetic stirrer, and the electrochemical test was carried out by the current-time method, with a fixed potential of -0.15 V and a scan rate of 100 mV/s.

Fig. 6 is the time-current curve of the enzyme biosensor prepared in Example 4 in response to different concentrations of inosinic acid at a fixed potential of -0.15V and a scan rate of 100mV/s. It can be seen that after adding 3 μL inosinic acid (3 μmol/L) every 30 s, the image presents a gradient curve, and the response time of the modified working electrode to inosinic acid is less than 5 s (reaching 98% of the steady-state current), indicating that the electrode surface The substance recognition membranes maintained good biocatalytic activity within the concentration range of inosinic acid tested.

What Fig. 7 shows is the standard curve of the response current of the enzyme biosensor that detects inosinic acid in embodiment 4 to different concentrations of inosinic acid; The current has a linear relationship with the concentration of inosinic acid, the linear regression equation is: I(μA)=0.9488+2036.4C(μmol/L), the correlation coefficient R2 is ^0.9973 , and the detection limit is 0.238μg/L. Increase the concentration of inosinic acid added dropwise, repeat the above experiment, it can be obtained: when the concentration of inosinic acid is within 0.313 ~ 210μg/L, the response current has a linear relationship with the concentration of inosinic acid, and the detection limit is 0.238μg/L . Therefore, the enzyme biosensor of the present invention can be used for quantitative detection of inosinic acid.

Testing on actual samples:

Select 4 different meat samples (chicken, pork, beef, mutton), take 5g each, add 20mL 5% perchloric acid to 5g meat samples, use a homogenizer to homogenize at 10000rpm for 3min, and then use 5% perchloric acid Dilute to 25 mL, centrifuge the homogenate at 15,000 rpm for 10 minutes at 4°C, collect the supernatant, adjust the pH to 5.5 with KOH, and centrifuge at 15,000 rpm for 10 minutes at 4°C, collect the supernatant for inosinic acid content analysis. The detection results of the enzyme biosensor prepared in Example 4 were compared with the detection results of liquid chromatography, and the specific detection results are shown in Table 1 below.

Table 1. Comparison of detection results of inosinic acid content in actual samples (unit: mg/g)

The results in Table 1 show that the detection results of the enzyme biosensor prepared in Example 4 are not much different from those of liquid chromatography, indicating that the constructed enzyme biosensor can be applied to the analysis and detection of inosinic acid content in actual samples.

Example 5

The enzyme biosensor for detecting inosinic acid, its preparation steps are as follows:

(1) A glassy carbon electrode with a diameter of 3 mm was polished to a mirror surface on a polishing cloth with alumina powders with a diameter of 0.3 μm and 0.05 μm in sequence, then rinsed with ultrapure water, and then ultrasonically treated in ultrapure water for 1 min, and left to dry After drying, the glassy carbon electrode was placed in potassium ferricyanide solution (a mixed solution composed of K ₃ [Fe(CN) ₆ ], K ₄ [Fe(CN) ₆ ] and KCl in a molar ratio of 1:1:100 ), at -0.2 ~ 0.6V, use cyclic voltammetry to scan for 4 circles to activate the electrode, take it out, wash it with ultrapure water, and dry it with nitrogen to get a pretreated glassy carbon electrode.

(2) Disperse 50 mg of MXene-Ti ₃ C ₂ Tx material in 100 mL of ultrapure water to obtain a MXene-Ti ₃ C ₂ Tx solution with a concentration of 0.5 mg/mL; use 1 wt% acetic acid solution to prepare a shell with a concentration of 5 mg/mL Polysaccharide solution; 98.50mg tetrachloroauric acid trihydrate was dissolved in 50mL ultrapure water to obtain a chloroauric acid solution with a concentration of 5mmol/L; 77.70mg hexachloroplatinic acid hexahydrate was dissolved in 50mL ultrapure water, Obtain the chloroplatinic acid solution that concentration is 3mmol/L; Dissolve 20mg5'-nucleotidase in 10mL PBS solution (pH 6.0,0.01M), obtain the 5'-nucleotidase solution that concentration is 2mg/mL; Dissolve 20mg of xanthine oxidase in 10mL of PBS solution (pH 6.0, 0.01M) to obtain a xanthine oxidase solution with a concentration of 2mg/mL, and dissolve 500mg of bovine serum albumin in 50mL of ultrapure water to obtain a concentration of 10mg/mL bovine serum albumin solution.

(3) Mix MXene-Ti ₃ C ₂ Tx solution, chitosan solution, chloroauric acid solution and chloroplatinic acid solution according to the volume ratio of 12:1.2:1:1 to obtain composite solution a, take 5 μL composite solution a is added dropwise to the surface of the glassy carbon electrode whose surface has been pretreated in step (1), and dried at room temperature.

(4) Mix the 5'-nucleotidase solution and the xanthine oxidase solution according to the volume ratio of 1:1 to obtain the double-enzyme compound solution b, take 5 μL of the double-enzyme compound solution b and add it dropwise to step (3) The glassy carbon electrode surface was dried at room temperature.

(5) 5 μL of bovine serum albumin solution was added dropwise to the surface of the glassy carbon electrode in step (4), and dried at room temperature to obtain a modified electrode.

(6) The modified electrode was combined with the Ag/AgCl electrode (KCl concentration: 3mol/L) as the reference electrode and the platinum wire as the counter electrode to form a three-electrode system to obtain an enzyme biosensor for detecting inosinic acid.

The enzyme biosensor prepared in Example 5 is tested by the current-time method. The specific steps are: at room temperature, the enzyme biosensor for detecting inosinic acid is immersed in the PBS buffer solution of pH 6.0 and 0.05M, and then poured into the PBS buffer solution successively. Add inosine triphosphate (1g/L), cysteine (1g/L), inosinic acid (0.1g/L), inosine diphosphate (1g/L), inosinic acid (0.1g/L) , methionine (1g/L), inosinic acid (0.1g/L), and continuously stirred with a magnetic stirrer, the electrochemical test was carried out by the current-time method, the fixed potential was -0.15V, and the scan rate was 100mV/s.

Fig. 8 is the time-current curve of the enzyme biosensor prepared in Example 5 in response to different interfering substances and inosinic acid at a fixed potential of -0.15V and a scan rate of 100mV/s. It can be seen that the current signal becomes larger when inosinic acid is added to PBS buffer, but there is no current response when interfering substances are added, indicating that the prepared enzyme biosensor has good specificity and anti-interference.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (9)

1. detecting the enzyme biologic sensor of inosinicacid, which is characterized in that its linear detection range is 0.313~210 μ g/L, by joining It is formed than electrode, to electrode and modified electrode, and the modified electrode is solidified by working electrode surface to inosine acid-sensitive Material identification film obtains；Wherein:

The material identification film by composite solution a, double enzyme composite solution b and 10mg/mL bovine serum albumin solution according to body Product is than being that 1:1:1 is formed；

The composite solution a by 0.5~1.25mg/mL MXene-Ti₃C₂Tx solution, the chitosan solution of 5mg/mL, 5mmol/ 12:1.2:1:1 is formed the platinum acid chloride solution of the chlorauric acid solution of L and 3mmol/L by volume；

Double enzyme composite solution b press body by the 5'-NT solution of 2mg/mL and the xanthine oxidase solution of 2mg/mL Product is formed than 1:1；

The MXene-Ti₃C₂T is selected from-OH ,-O or-F in Tx.

2. the enzyme biologic sensor of detection inosinicacid as described in claim 1, which is characterized in that

The 5'-NT solution is dissolved in pH 6.0 by 5'-NT, the PBS solution of 0.01M obtains；

The xanthine oxidase solution is dissolved in pH 6.0 by xanthine oxidase, the PBS solution of 0.01M obtains.

3. the enzyme biologic sensor of detection inosinicacid as described in claim 1, which is characterized in that the MXene-Ti₃C₂Tx's The preparation method comprises the following steps: 1.6gLiF to be slowly dissolved in the HCL aqueous solution of 20mL9mol/L, 5min is stirred, 1.0gTi is then added₃AlC₂ Powder, under room temperature for 24 hours with 400rpm magnetic agitation；Then 5min is centrifuged with 3500rpm, by gained sediment ultrapure water Washing；Ultrasound and washing operation 5~8 times are repeated, when measuring pH value of solution is 6, sediment is collected, is dissolved in 100mL water, In Under argon gas protection, the ultrasound 3h in 4 DEG C of ice baths makes the Ti generated₃C₂T_xThin slice delamination；It is centrifuged 1h with 8000rpm, collects supernatant Liquid, 60 DEG C of vacuum drying obtain black powder, as MXene-Ti₃C₂T_x。

4. the enzyme biologic sensor of detection inosinicacid as described in claim 1, which is characterized in that the working electrode is glass carbon Electrode, the reference electrode are Ag/AgCl electrode, and described is platinum electrode to electrode.

5. the enzyme biologic sensor of detection inosinicacid as described in claim 1, which is characterized in that the enzyme biologic sensor Detection is limited to 0.238 μ g/L.

6. the preparation method of any one of Claims 1 to 5 enzyme biologic sensor, which comprises the following steps:

(1) surface preparation is carried out to working electrode；

(2) the composite solution a is added drop-wise to the working electrode surface after step (1) surface preparation, room temperature is dried；

(3) double enzyme composite solution b are added drop-wise to step (2) treated electrode surface, room temperature is dried；

(4) bovine serum albumin solution is added drop-wise to step (3) treated electrode surface, room temperature is dried, and electricity must be modified Pole；

(5) by modified electrode obtained by step (4) and the reference electrode and it is described to electrode composition three-electrode system to get；Its In: the dripping quantity volume ratio of the composite solution a, double enzyme composite solution b and bovine serum albumin solution are 1:1:1.

7. the preparation method of enzyme biologic sensor as claimed in claim 6, which is characterized in that in step (1), the working electrode The step of surface preparation are as follows: working electrode successively uses to 0.3 μm and 0.05 μm of alumina powder polishes on polishing cloth At mirror surface, then with ultrapure water, it is then ultrasonically treated 1min in ultrapure water, is subsequently placed in potassium ferricyanide solution at activation Reason is taken out, with ultrapure water, is dried with nitrogen；Wherein, the potassium ferricyanide solution is by K₃[Fe(CN)₆]、K₄[Fe(CN)₆] It is in molar ratio the mixed solution of 1:1:100 composition with KCl.

8. application of any one of Claims 1 to 5 biosensor in inosinicacid quantitative detection, which is characterized in that institute The linear detection range for stating inosinicacid is 0.313~210 μ g/L.

9. application as claimed in claim 8, which is characterized in that the detection of the inosinicacid is limited to 0.238 μ g/L.