CN102153772A - Phosphate radical sensitive membrane and preparation and using methods thereof - Google Patents

Abstract

The invention discloses a phosphate radical sensitive membrane and preparation and using methods thereof, and belongs to the technical fields of electrochemical biosensors and preparation thereof. The phosphate radical sensitive membrane consists of nitrocellulose membrane matrix, maltose phosphorylase, glucose oxidase and macromolecular substance glutaraldehyde of immobilized enzyme. The test buffer solution consists of maltose serving as a reaction substrate, 2-hydroxy pyridine serving as a glucose mutarotation catalyst, and citric acid-sodium citrate buffer solution. The phosphate radical biosensor realizes detection of phosphate radicals by using only two enzymes, is convenient for preparation and condition optimization, simultaneously has wide linear response range and good operating stability, and is particularly suitable to be used in the fields of fermentation industry, food inspection and the like.

Description

A kind of phosphate sensitive film and its preparation and use method

technical field

The invention belongs to the technical field of electrochemical biosensors and their preparation, in particular to a phosphate-sensitive membrane and its preparation and use methods, that is, maltose phosphorylase-glucose oxidase dual-enzyme phosphate based on a nitrocellulose membrane Sensitive membrane, detects phosphate.

Background technique

The phosphate content is an important factor affecting the fermentation process and product quality. The phosphate content in food is an important indicator of food nutrition and hygiene inspection and quarantine. Therefore, rapid and accurate detection of phosphate is of great significance in the fermentation and food industry. There are many methods for detecting phosphate, such as colorimetry, turbidimetry, gravimetric method, ion chromatography, etc., but the detection accuracy and sensitivity of colorimetric method, turbidimetric method, gravimetric method and other methods are not high enough and the operation process is cumbersome , while ion chromatography requires expensive equipment. In recent years, people have begun to study the detection of phosphate with enzyme electrodes, which has the advantages of simplicity, rapidity, and high selectivity. Researchers have developed a variety of enzyme electrode systems that can be applied to the detection of phosphate radicals, among which the pyruvate oxidase system and the maltose phosphorylase system are more representative.

In literature (1) Analytical Biochemistry, 2005, 343: 263-267, Roger CHKwan et al. used Nafion solution to immobilize pyruvate oxidase on a screen-printed electrode to prepare a phosphate biosensor. The sensor needs to be tested in a buffer solution Add flavin adenine dinucleotide, cocarboxylase and MgCl ₂ as cofactors, and pyruvate as reaction substrate. Studies have shown that under the best test conditions, the linear response range of phosphate is 7.5-625 μmol/L, the detection limit is 3.6 μmol/L, the sensitivity is 523.89nA/(mmol/L), the response time is 2 seconds, and the current operation lasts for 12 hours Keep 85%. The phosphate biosensor has a wide linear response range, rapid response, and good operational stability. However, due to the complex composition of the test buffer solution, the types of test samples are limited. The sensor is mainly suitable for the detection of human saliva. In addition, pyruvate oxidase and reaction cofactors are expensive biochemical drugs, so the cost is high.

The price of the enzyme used in the maltose phosphorylase system is relatively low, which has certain advantages in terms of cost. In document (2) Analytica ChimicaActa, 1995,309:47-52, people such as N.Conrath use glutaraldehyde as cross-linking agent that maltose phosphorylase, glucose oxidase, bovine serum albumin are immobilized on the copper spinning membrane, The detection of phosphate radicals in citric acid buffer solution did not achieve the desired effect. Only by adding mutarotase or/and acid phosphatase to assemble a three-enzyme and four-enzyme biosensor can the detection of phosphate radicals be realized. Among them, the four-enzyme sensor with the best performance has a linear response range of 0.1-1 μmol/L. The phosphate biosensor developed in this document has a high sensitivity of 4.4μA/(mmol/L), but the system uses many types of enzymes, the matrix membrane is relatively expensive, the preparation process is cumbersome, and the cost is high.

In the literature (3) Enzyme and Microbial Technology, 1997, 21: 413-420, Stephan Hüwel et al. used glutaraldehyde as a cross-linking agent to immobilize maltose phosphorylase, glucose oxidase, and bovine serum albumin on platinum electrodes to form A phosphate biosensor was developed. The sensor has a linear response range of 0.5-10 μmol/L, a detection limit of 0.1 μmol/L, and a response time of 4 minutes under the optimal test conditions of pH=6.5 and 36°C. Activity is not lost. However, since the enzyme is directly immobilized on the electrode, the electrode is not suitable for replacement and the test cost is high.

In literature (4) Analytica Chimica Acta, 2001, 443: 1-8, Christine Mousty et al. used glutaraldehyde as a cross-linking agent to immobilize maltose phosphorylase, mutarotase, and glucose oxidase on inorganic laponite clay Above, a phosphate biosensor was prepared. The research results show that the linear response range is 1-50 μmol/L under the optimal test conditions of pH=6.5 and 40°C, the sensitivity is 10.3 μA/(mmol/L), the current keeps 90% after 8 hours of continuous operation, and after 2 weeks of storage The current response is able to remain stable. The phosphate biosensor has a wide linear response range and good storage performance. Compared with previous work, the performance has been greatly improved. However, the enzyme is directly fixed on the electrode, which makes the electrode not suitable for replacement and high testing costs.

Contents of the invention

The object of the present invention is to provide a phosphate-sensitive membrane and its preparation and use method, that is, a maltose phosphorylase-glucose oxidase dual-enzyme phosphate-sensitive membrane with a nitrocellulose membrane as a substrate and a phosphate-sensitive membrane. Utilize the advantages of good chemical and thermal stability, large porosity, relatively uniform pore size distribution and good biocompatibility of nitrocellulose membrane to effectively maintain the activity of maltose phosphorylase and glucose oxidase , improve the sensitivity, linear response range and stability of biosensors, etc., thereby improving the comprehensive performance indicators of biosensors. At the same time, the present invention adopts the method of adding 2-hydroxypyridine in the test buffer solution to catalyze the mutarotation of the reaction intermediate product α-glucose, which plays the role of replacing mutarotase and realizes the detection of phosphate by the two-enzyme system.

The phosphate radical sensitive membrane of the invention is composed of nitrocellulose membrane matrix, maltose phosphorylase, glucose oxidase and high molecular substance glutaraldehyde immobilizing the enzyme. Wherein, the nitrocellulose membrane is a nitrocellulose microporous membrane with an average pore size of 0.20-1.20 μm and a thickness of 50-150 μm; the loading capacity of maltose phosphorylase is 9.0-28.0 activity units (U)/cm ² ; The loading capacity of glucose oxidase is 7.0-32.0 U/cm ² ; glutaraldehyde is introduced by vapor cross-linking of glutaraldehyde aqueous solution.

The preparation method of the phosphate radical sensitive membrane of the present invention is: immerse the nitrocellulose membrane in the 0.05mol/L phosphate buffer solution that the pH value is 6.5, take out after processing for 6～18 hours, dry with high-purity nitrogen; The cellulose membrane is stuck on the O-shaped rubber ring, and the maltose phosphorylase-glucose oxidase dual enzyme mixed solution is dropped on the nitrocellulose membrane, and dried in a refrigerator at a temperature of 0-8°C for 0.5-2 hours; then Use glutaraldehyde aqueous vapor to cross-link at 0-8°C for 6-12 hours, and then immerse the enzyme membrane in a 0.05mol/L phosphate buffer solution with a pH value of 6.5 to wash away the weakly bound enzymes. Form a phosphate-sensitive film and store in a refrigerator at a temperature of 0-8°C for later use. Wherein the solvent of the 0.05mol/L phosphate buffer solution with a pH value of 6.5 is twice distilled water; the solvent of the maltose phosphorylase-glucose oxidase dual enzyme mixed solution is a 0.05mol/L phosphate buffer solution with a pH=6.5, and the solute is 0.1 ~0.5U/μL maltose phosphorylase and 0.1~0.5U/μL glucose oxidase; glutaraldehyde aqueous solution with a mass fraction of 50% is used to generate glutaraldehyde vapor, and the solvent is double distilled water.

The method for testing the phosphate in the sample by the phosphate sensitive film of the present invention is as follows: the phosphate sensitive film is placed on the top of the platinum electrode as a working electrode, the platinum wire electrode is used as a counter electrode, and the Ag/AgCl electrode is used as a reference electrode to form a three-electrode system; Place the three-electrode system in a test buffer solution with a pH value of 6.5, set the potential at 0.6V (vs.Ag/AgCl), and use the chronoamperometry method to test the response current of the phosphate sensitive membrane to phosphate ions, record i-t curve, draw the standard working curve of the relationship between the response current and the concentration of phosphate according to the i-t curve, and use this standard working curve to quantitatively detect the concentration of phosphate in the sample to be tested. Wherein the test buffer solution is obtained by adding reaction substrate maltose and glucose mutarotational catalyst 2-hydroxypyridine in 0.1mol/L citric acid-sodium citrate buffer solution with pH=6.5, and the concentration of maltose is 1～40mmol/L, 2 The concentration of -hydroxypyridine is 100-500mmol/L.

Effect of the present invention can find out from the electrochemical biosensor that uses phosphate radical sensitive film of the present invention to make: adopt i-t method test sensor to the response electric current of phosphate radical, result as shown in Figure 1, response time 3 minutes; By Figure 2 can It can be seen that the linear response range of the phosphate sensitive film to phosphate is 0.5～6.0mmol/L, and the response sensitivity is 4.24nA/(mmol/L). The phosphate sensitive film of the present invention is continuously operated for 9 hours, and the response signal remains 98.3%. The comparison between the phosphate radical biosensor of the present invention and literature data is shown in Table 1.

Table 1. Phosphate sensor performance comparison

The advantages of the present invention are: in terms of electrode design, the double-enzyme membrane prepared by the present invention is easy to replace, which reduces the manufacturing cost and maintenance cost of the electrode; in terms of substrate selection, the nitrocellulose membrane selected by the present invention has good biophase Capacitance, can keep the activity of enzyme better, and with respect to the copper spun membrane used in document (1) cheap; The method is simple and easy to implement, with fewer procedures, less time-consuming, lower preparation process cost, and easy popularization and application; in terms of performance, the phosphate-based biological preparation prepared by the maltose phosphorylase-glucose oxidase dual-enzyme phosphate-sensitive membrane of the present invention The sensor realizes the detection of phosphate using only two enzymes, which is convenient for preparation and condition optimization, and has a wide linear response range and good operational stability, and is especially suitable for use in the fields of fermentation industry and food inspection.

Description of drawings

Figure 1. The i-t response curve of the phosphate-sensitive membrane to 1 mmol/L phosphate. Abscissa - time, unit: second (s); ordinate - response current, unit: nanoampere (nA).

Figure 2. The relationship between the response current of the phosphate-sensitive membrane and the concentration of phosphate. Abscissa-phosphate concentration, unit: mmol/L (mmol/L); ordinate-response current, unit: nanoampere (nA).

Detailed ways:

Example 1

Immerse the nitrocellulose membrane with an average pore size of 0.45 μm and a thickness of 100 μm in a 0.05mol/L, pH value of 6.5 phosphate buffer solution for 6 hours, then take it out, dry it with high-purity nitrogen, and stick it on the O-shaped membrane. On the rubber ring, drop 20 μL of a double-enzyme mixture solution containing 0.30 U/μL maltose phosphorylase and 0.25 U/μL glucose oxidase on the treated nitrocellulose membrane, dry it in a refrigerator at 4 °C for 2 hours, and place it in the refrigerator. 50% glutaraldehyde aqueous vapor cross-linking for 12 hours, and finally immerse the enzyme membrane in 0.05mol/L phosphate buffer solution with a pH value of 6.5 to wash away the loosely bound enzyme, that is, to form The maltose phosphorylase-glucose oxidase dual-enzyme phosphate-sensitive membrane based on the nitrocellulose membrane was stored in a refrigerator at 4°C for use.

Put the phosphate sensitive film on the top of the platinum electrode as the working electrode, the platinum wire electrode as the counter electrode, and the Ag/AgCl electrode as the reference electrode. The test system contains 10mmol/L maltose, 400mmol/L 2-hydroxypyridine pH=6.5 concentration 0.1mol/L citric acid-sodium citrate buffer solution, CHI660B electrochemical workstation was used to electrochemically characterize the phosphate biosensor, and the chronoamperometry method was used to test the response current of the sensor to phosphate, and the i-t curve was recorded. The results are as follows: As shown in Figure 1, the response time is 3 minutes; it can be seen from Figure 2 that the linear range of the sensor's response to phosphate is 0.5-6.0mmol/L, and the response sensitivity is 4.24nA/(mmol/L); the phosphate-sensitive membrane operates continuously Its response signal remains 98.3% after 9 hours of use.

Example 2

Immerse the nitrocellulose membrane with an average pore size of 1.20 μm and a thickness of 150 μm in a 0.05 mol/L, pH value of 6.5 phosphate buffer solution for 12 hours, then take it out, dry it with high-purity nitrogen, and stick it on the O-shaped membrane. On the rubber ring, drop 20 μL of a dual-enzyme mixture solution containing 0.15 U/μL maltose phosphorylase and 0.125 U/μL glucose oxidase on the treated nitrocellulose membrane, dry it in a refrigerator at 0 °C for 0.5 hours, and place it in the refrigerator 50% glutaraldehyde aqueous vapor cross-linking for 18 hours, and finally immerse the enzyme membrane in 0.05mol/L phosphate buffer solution with a pH value of 6.5 to wash away the loosely bound enzyme, that is, to form The maltose phosphorylase-glucose oxidase dual-enzyme phosphate-sensitive membrane based on the nitrocellulose membrane was stored in a refrigerator at 0°C for use.

Put the phosphate sensitive film on the top of the platinum electrode as the working electrode, the platinum wire electrode as the counter electrode, and the Ag/AgCl electrode as the reference electrode. The test system contains 1mmol/L maltose, 100mmol/L 2-hydroxypyridine pH=6.5 concentration 0.1mol/L citric acid-sodium citrate buffer solution, CHI660B electrochemical workstation was used to perform electrochemical characterization of the phosphate biosensor, and the chronoamperometry method was used to test the response current of the sensor to phosphate, and the i-t curve and response time were recorded. 3 minutes; the linear range of the sensor's response to phosphate is 0.3-4.0mmol/L, and the response sensitivity is 3.19nA/(mmol/L); the response signal of the phosphate-sensitive membrane has not significantly decreased after 9 hours of continuous operation.

Example 3

Immerse the nitrocellulose membrane with an average pore size of 0.20 μm and a thickness of 50 μm in a 0.05mol/L, pH value of 6.5 phosphate buffer solution for 9 hours, then take it out, dry it with high-purity nitrogen, and stick it on the O-shaped membrane. On the rubber ring, drop 20 μL of a double-enzyme mixture solution containing 0.45 U/μL maltose phosphorylase and 0.5 U/μL glucose oxidase on the treated nitrocellulose membrane, and dry it in a refrigerator at 8°C for 1 hour. 50% glutaraldehyde aqueous vapor cross-linking for 6 hours, and finally immerse the enzyme membrane in 0.05mol/L phosphate buffer solution with a pH value of 6.5 to wash away the loosely bound enzyme, that is, to form the following The maltose phosphorylase-glucose oxidase dual-enzyme phosphate-sensitive membrane based on the nitrocellulose membrane was stored in a refrigerator at 8°C for use.

Put the phosphate sensitive film on the top of the platinum electrode as the working electrode, the platinum wire electrode as the counter electrode, and the Ag/AgCl electrode as the reference electrode. The test system contains 40mmol/L maltose, 500mmol/L 2-hydroxypyridine pH=6.5 concentration 0.1mol/L citric acid-sodium citrate buffer solution, CHI660B electrochemical workstation was used to perform electrochemical characterization of the phosphate biosensor, and the chronoamperometry method was used to test the response current of the sensor to phosphate, and the i-t curve and response time were recorded. 4 minutes; the linear range of the sensor's response to phosphate is 0.7-8.0mmol/L, and the response sensitivity is 5.32nA/(mmol); the response signal of the phosphate-sensitive membrane has not significantly decreased after 9 hours of continuous operation.

Claims (4)

1. phosphate radical sensitive membrane, it is characterized in that: this phosphate radical sensitive membrane is made of the polymer substance glutaraldehyde of nitrocellulose filter matrix, maltose phosphorylase, glucose oxidase and immobilized enzyme; Wherein, nitrocellulose filter is the nitrocellulose millipore filtration, and its mean pore size is 0.20～1.20 μ m, and thickness is 50～150 μ m; The charge capacity of maltose phosphorylase is 9.0～28.0 unit of activity (U)/cm ²The charge capacity of glucose oxidase is 7.0～32.0U/cm ²Glutaraldehyde is by the crosslinked introducing of glutaraldehyde water solution steam.

2. a method for preparing the described phosphate radical sensitive membrane of claim 1 is characterized in that, processing step is: it is to handle in 6.5 the 0.05mol/L phosphate buffer solution to take out after 6～12 hours that nitrocellulose filter is immersed the pH value, dries up with high pure nitrogen; Nitrocellulose filter after handling being bonded on the O shape rubber ring, the two enzyme mixing solutionss of maltose phosphorylase-glucose oxidase are dropped on this nitrocellulose filter, is in 0～8 ℃ the refrigerator dry 0.5～2 hour in temperature; Use the glutaraldehyde water solution steam subsequently under 0～8 ℃ condition crosslinked 6～18 hours, again enzyme membrane is immersed the pH value and be in 6.5 the 0.05mol/L phosphate buffer solution, in conjunction with unstable enzyme, make the phosphate radical sensitive membrane with flush away, be kept at temperature and be in 0～8 ℃ the refrigerator standby.

Described nitrocellulose filter is the nitrocellulose millipore filtration, and its mean pore size is 0.20～1.20 μ m, and thickness is 50～150 μ m; The charge capacity of maltose phosphorylase is 9.0～28.0 unit of activity (U)/cm ²The charge capacity of glucose oxidase is 7.0～32.0U/cm ²

3. it is characterized in that in accordance with the method for claim 2: the pH value is that the solvent of 6.5 0.05mol/L phosphate buffer solution is a redistilled water; The solvent of the two enzyme mixing solutionss of maltose phosphorylase-glucose oxidase is the 0.05mol/L phosphate buffer solution of pH=6.5, and solute is 0.1～0.5U/ μ L maltose phosphorylase and 0.1～0.5U/ μ L glucose oxidase; What be used to produce the glutaraldehyde steam is that massfraction is 50% glutaraldehyde water solution, and solvent is a redistilled water.

4. the using method of the described phosphate radical sensitive membrane of claim 1, it is characterized by: the detection by quantitative that is used for the sample phosphate radical, testing method is: the phosphate radical sensitive membrane is enclosed within the platinum electrode top as working electrode, platinum wire electrode is as counter electrode, the Ag/AgCl electrode is formed three-electrode system as reference electrode; It is 6.5 test buffered soln that this three-electrode system is placed the pH value, with potential setting at 0.6V (vs.Ag/AgCl), adopt the response current of timing current methods test phosphate radical sensitive membrane to phosphate anion, record i-t curve, according to the standard working curve of i-t curve plotting response current and phosphate concentration relation, utilize this standard working curve to carry out the detection by quantitative of phosphate concentration in the testing sample; Wherein test buffered soln and be and in the 0.1mol/L of pH=6.5 citric acid-sodium citrate buffer, add reaction substrate maltose and glucose mutarotation catalyzer 2 hydroxy pyrimidine obtains, maltose concentration is 1～40mmol/L, and the concentration of 2 hydroxy pyrimidine is 100～500mmol/L.

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