CN102153772B - Phosphate radical sensitive membrane and preparation and using methods thereof - Google Patents
Phosphate radical sensitive membrane and preparation and using methods thereof Download PDFInfo
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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
Technical field
The invention belongs to electrochemica biological sensor and preparing technical field thereof; Particularly relate to a kind of phosphate radical sensitive membrane and preparation thereof, method of use; Promptly be the two enzyme phosphate radical sensitive membrane of maltose phosphorylase-P-FAD of matrix, detect phosphate radical with the nitrocellulose filter.
Background technology
Phosphate content is the important factor that influences fermenting process and products thereof quality, and phosphate content is the important indicator of food nutrition sanitary inspection quarantine in the food, and it is significant in fermentation and foodstuffs industry therefore fast, accurately to detect phosphate radical.The method that detects phosphate radical has multiple; Like colourimetry, turbidimetry, weighting method, ion chromatography etc.; But accuracy of detection, insufficient sensitivity height and the operating process of methods such as colourimetry, turbidimetry, weighting method are loaded down with trivial details, and ion chromatography needs expensive plant and instrument.In recent years, people begin one's study and detect phosphate radical with enzyme electrodes, have simple, quick, selectivity advantages of higher.The researchist has developed the multiple enzyme electrodes system that can be applied to the phosphate radical detection, and wherein more representational is pyruvate oxidation enzyme system and maltose phosphorylase system.
At document (1) Analytical Biochemistry; 2005; Among the 343:263-267; People such as Roger C.H.Kwan adopt Nafion solution that pyruvic oxidase is fixed on to have prepared the phosphate radical biosensor on the screen printing electrode, and this transmitter need add flavin adenine dinucleotide, diphosphothiamine and MgCl in test buffered soln
2As cofactor, pyruvic acid is as reaction substrate.Research shows that the phosphate radical linear response range is 7.5～625 μ mol/L under the best test condition, detects and is limited to 3.6 μ mol/L, and sensitivity is 523.89nA/ (mmol/L), and the time of response is 2 seconds, and 12 hours electric currents of ongoing operation keep 85%.The linear response range broad of this phosphate radical biosensor, response rapidly, the stable operation performance is good, but because test buffered soln constitutes complicatedly, so the kind of specimen is restricted, this transmitter mainly is applicable to the detection of human saliva.In addition, pyruvic oxidase and reaction cofactor all are expensive biochemical drugs, so cost is very high.
The employed enzyme price of maltose phosphorylase system is lower, on cost, has certain advantage.At document (2) Analytica ChimicaActa; 1995, among the 309:47-52, human LUTARALDEHYDEs such as N.Conrath are fixed in maltose phosphorylase, P-FAD, bovine serum albumin on the cuprophane film as linking agent; In citric acid solution, detecting phosphate radical does not attain the results expected; Have only add again mutarotase or/with acid phosphatase, be assembled into three enzymes, four enzyme biologic sensors, just realized detection to phosphate radical.Wherein the linear response range of four Enzyme sensors of performance the best is 0.1～1 μ mol/L.The phosphate radical biosensor sensitivity of document development is higher, reaches 4.4 μ A/ (mmol/L), but this system uses the kind of enzyme more, and base film is comparatively expensive, and the preparation process is more loaded down with trivial details, and cost is higher.
At document (3) Enzyme and Microbial Technology; 1997; Among the 21:413-420, human LUTARALDEHYDEs such as Stephan H ü wel are fixed on maltose phosphorylase, P-FAD, bovine serum albumin as linking agent and have constituted the phosphate radical biosensor on the platinum electrode, and this transmitter is at pH=6.5; Linear response range under 36 ℃ the best test condition is 0.5～10 μ mol/L; Detection is limited to 0.1 μ mol/L, and the time of response is 4 minutes, and enzymic activity is not lost in 6 hours.But because enzyme directly is fixed on the electrode, cause electrode to change, testing cost is high.
At document (4) Analytica Chimica Acta; 2001; Among the 443:1-8, people such as Christine Mousty adopt glutaraldehyde as cross linker that maltose phosphorylase, mutarotase, P-FAD are fixed on the inorganic lithium saponite clay, have prepared the phosphate radical biosensor.Result of study shows, pH=6.5, and 40 ℃ best test condition lower linear responding range is 1～50 μ mol/L, and sensitivity is 10.3 μ A/ (mmol/L), and 8 hours electric currents of ongoing operation keep 90%, store 2 all after-current responses and can keep stable.The linear response range broad of phosphate radical biosensor, storge quality is good, and previous work has bigger lifting on performance relatively, but enzyme directly is fixed on the electrode, causes electrode to change, and testing cost is high.
Summary of the invention
The object of the present invention is to provide a kind of phosphate radical sensitive membrane and preparation thereof, method of use, promptly be the two enzyme phosphate radical sensitive membrane of maltose phosphorylase-P-FAD of matrix and detect phosphate radical with the nitrocellulose filter.Utilize performance advantages such as good chemicalstability of nitrocellulose filter and thermostability, bigger porosity, uniform pore size distribution and excellent biological compatibility; The activity that keeps maltose phosphorylase, P-FAD effectively; Improve sensitivity, linear response range and the stability etc. of biosensor, thereby improve the integrated performance index of biosensor.The present invention simultaneously is employed in the method catalyzed reaction intermediate product α-Pu Taotang mutarotation that adds 2 hydroxy pyrimidine in the test buffered soln, has played the effect of alternative mutarotase, has realized the detection of two enzyme systems for phosphate radical.
Phosphate radical sensitive membrane of the present invention is made up of the polymer substance LUTARALDEHYDE of nitrocellulose filter matrix, maltose phosphorylase, P-FAD 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 P-FAD is 7.0～32.0U/cm
2LUTARALDEHYDE is by the crosslinked introducing of glutaraldehyde water solution steam.
The preparation method of phosphate radical sensitive membrane of the present invention is: it is to handle in 6.5 the 0.05mol/L phosphate buffer solution to take out after 6～18 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-P-FAD 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 6～12 hours; Again enzyme membrane being immersed the pH value is in 6.5 the 0.05mol/L phosphate buffer solution; Combine unstable enzyme with flush away, process the phosphate radical sensitive membrane, be kept at temperature and be in 0～8 ℃ the refrigerator subsequent use.Wherein 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-P-FAD 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 P-FAD; What be used to produce the LUTARALDEHYDE steam is that massfraction is 50% glutaraldehyde water solution, and solvent is a redistilled water.
The method of phosphate radical is in the phosphate radical sensitive membrane specimen of the present invention: the phosphate radical sensitive membrane is enclosed within the platinum electrode top as working electrode, and platinum wire electrode is as counter electrode, and 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, utilizes 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 SANMALT-S 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.
Effect of the present invention can be found out from the electrochemica biological sensor that uses phosphate radical sensitive membrane of the present invention to make: adopt the response current of i-t method testing sensor to phosphate radical, the result is as shown in Figure 1,3 minutes time of response; Can be found out that by Fig. 2 the phosphate radical sensitive membrane is 0.5～6.0mmol/L to the linear response range of phosphate radical, response sensitivity is 4.24nA/ (mmol/L), and phosphate radical sensitive membrane of the present invention was carried out operate continuously 9 hours, and response signal keeps 98.3%.The contrast of phosphate radical biosensor of the present invention and data in literature is as shown in table 1.
Table 1. phosphate radical sensor performance relatively
The invention has the advantages that: aspect electrode design, the prepared two enzyme membranes of the present invention are easy to change, and have reduced the manufacturing cost and the maintenance cost of electrode; Aspect the matrix selection, the nitrocellulose filter that the present invention selects for use has excellent biological compatibility, can keep the activity of enzyme preferably, and cheap with respect to the cuprophane film that uses in the document (1); Aspect the preparation method, the two enzyme phosphate radical sensing membrane preparation methods of maltose phosphorylase-P-FAD of the present invention are simple and easy to do, and operation is less, and is consuming time shorter, and the preparation process cost is lower, is easy to apply; At aspect of performance; Only use two kinds of enzymes just to realize the detection of phosphate radical by the two prepared phosphate radical biosensors of enzyme phosphate radical sensitive membrane of maltose phosphorylase-P-FAD of the present invention; Be convenient to preparation and condition optimizing; Have simultaneously the linear response range of broad and operational stability preferably again, be particluarly suitable for using in the fields such as fermentation industry and food test.
Description of drawings
Fig. 1. the phosphate radical sensitive membrane is to the i-t response curve of the phosphate radical of 1mmol/L.X-coordinate-time, unit: second (s); Ordinate zou-response current, unit: receive peace (nA).
Fig. 2. the relation curve of phosphate radical sensitive membrane response current and phosphate concentration.X-coordinate-phosphate concentration, unit: mmole/liter (mmol/L); Ordinate zou-response current, unit: receive peace (nA).
Embodiment:
With mean pore size is 0.45 μ m, and thickness is that the nitrocellulose filter of 100 μ m is immersed in 0.05mol/L, and the pH value is to handle 6 hours in 6.5 the phosphate buffer solution; Take out then, dry up with high pure nitrogen, and be bonded on the O shape rubber ring; 20 μ L are contained the two enzyme mixing solutionss of 0.30U/ μ L maltose phosphorylase and 0.25U/ μ L P-FAD to drop on the nitrocellulose filter of handling; In 4 ℃ refrigerator dry 2 hours, in refrigerator, using massfraction was crosslinked 12 hours of 50% glutaraldehyde water solution steam, at last enzyme membrane is immersed 0.05mol/L; The pH value is in 6.5 the phosphoric acid buffer; Combine not firm enzyme with flush away, promptly forming with the nitrocellulose filter is the two enzyme phosphate radical sensitive membrane of maltose phosphorylase-P-FAD of matrix, is kept in 4 ℃ the refrigerator subsequent use.
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 as reference electrode, test system be contain 10mmol/L SANMALT-S, 400mmol/L 2 hydroxy pyrimidine pH=6.5 concentration is the citric acid-sodium citrate damping fluid of 0.1mol/L, adopts the CHI660B electrochemical workstation that this phosphate radical biosensor is carried out electrochemical Characterization; Adopt the response current of timing current methods testing sensor to phosphate radical; Record i-t curve, the result is as shown in Figure 1,3 minutes time of response; Found out that by Fig. 2 transmitter is 0.5～6.0mmol/L to the linearity range of phosphate radical response, response sensitivity is 4.24nA/ (mmol/L); The operate continuously of phosphate radical sensitive membrane uses 9 hours its response signals to keep 98.3%.
With mean pore size is 1.20 μ m, and thickness is that the nitrocellulose filter of 150 μ m is immersed in 0.05mol/L, and the pH value is to handle 12 hours in 6.5 the phosphate buffer solution; Take out then, dry up with high pure nitrogen, and be bonded on the O shape rubber ring; 20 μ L are contained the two enzyme mixing solutionss of 0.15U/ μ L maltose phosphorylase and 0.125U/ μ L P-FAD to drop on the nitrocellulose filter of handling; In 0 ℃ refrigerator dry 0.5 hour, in refrigerator, using massfraction was crosslinked 18 hours of 50% glutaraldehyde water solution steam, at last enzyme membrane is immersed 0.05mol/L; The pH value is in 6.5 the phosphoric acid buffer; Combine not firm enzyme with flush away, promptly forming with the nitrocellulose filter is the two enzyme phosphate radical sensitive membrane of maltose phosphorylase-P-FAD of matrix, is kept in 0 ℃ the refrigerator subsequent use.
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 as reference electrode; Test system be contain 1mmol/L SANMALT-S, 100mmol/L 2 hydroxy pyrimidine pH=6.5 concentration is the citric acid-sodium citrate damping fluid of 0.1mol/L, adopts the CHI660B electrochemical workstation that this phosphate radical biosensor is carried out electrochemical Characterization, adopts the response current of timing current methods testing sensor to phosphate radical; Record i-t curve, 3 minutes time of response; Transmitter is 0.3～4.0mmol/L to the linearity range of phosphate radical response, and response sensitivity is 3.19nA/ (mmol/L); The operate continuously of phosphate radical sensitive membrane uses 9 hours its response signals not see obvious decline.
With mean pore size is 0.20 μ m, and thickness is that the nitrocellulose filter of 50 μ m is immersed in 0.05mol/L, and the pH value is to handle 9 hours in 6.5 the phosphate buffer solution; Take out then, dry up with high pure nitrogen, and be bonded on the O shape rubber ring; 20 μ L are contained the two enzyme mixing solutionss of 0.45U/ μ L maltose phosphorylase and 0.5U/ μ L P-FAD to drop on the nitrocellulose filter of handling; In 8 ℃ refrigerator dry 1 hour, in refrigerator, using massfraction was crosslinked 6 hours of 50% glutaraldehyde water solution steam, at last enzyme membrane is immersed 0.05mol/L; The pH value is in 6.5 the phosphoric acid buffer; Combine not firm enzyme with flush away, promptly forming with the nitrocellulose filter is the two enzyme phosphate radical sensitive membrane of maltose phosphorylase-P-FAD of matrix, is kept in 8 ℃ the refrigerator subsequent use.
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 as reference electrode; Test system be contain 40mmol/L SANMALT-S, 500mmol/L 2 hydroxy pyrimidine pH=6.5 concentration is the citric acid-sodium citrate damping fluid of 0.1mol/L, adopts the CHI660B electrochemical workstation that this phosphate radical biosensor is carried out electrochemical Characterization, adopts the response current of timing current methods testing sensor to phosphate radical; Record i-t curve, 4 minutes time of response; Transmitter is 0.7～8.0mmol/L to the linearity range of phosphate radical response, and response sensitivity is 5.32nA/ (mmol); The operate continuously of phosphate radical sensitive membrane uses 9 hours its response signals not see obvious decline.
Claims (4)
1. phosphate radical sensitive membrane, it is characterized in that: this phosphate radical sensitive membrane is made up of the LUTARALDEHYDE of nitrocellulose filter matrix, maltose phosphorylase, P-FAD 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 9.0～28.0U/cm
2, the charge capacity of P-FAD is 7.0～32.0U/cm
2, wherein U is the unit of activity of enzyme; LUTARALDEHYDE 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, process 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-P-FAD 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 being immersed the pH value is in 6.5 the 0.05mol/L phosphate buffer solution; Combine unstable enzyme with flush away, process the phosphate radical sensitive membrane, be kept at temperature and be in 0～8 ℃ the refrigerator subsequent use;
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 9.0～28.0U/cm
2The charge capacity of P-FAD is 7.0～32.0U/cm
2
3. according to the described method of claim 2, it is characterized in that: 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-P-FAD 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 P-FAD; What be used to produce the LUTARALDEHYDE steam is that massfraction is 50% glutaraldehyde water solution, and solvent is a redistilled water.
4. the method for use of the said 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, and 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; Is being 0.6V with potential setting with respect to the Ag/AgCl electrode; Adopt timing current methods test phosphate radical sensitive membrane to the response current i of the phosphate anion change curve of t in time; Brief note according to the standard working curve of i-t curve plotting response current and phosphate concentration relation, utilizes this standard working curve to carry out the detection by quantitative of phosphate concentration in the testing sample for the i-t curve; Wherein test buffered soln and be and in the 0.1mol/L of pH=6.5 citric acid-sodium citrate buffer, add reaction substrate SANMALT-S 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5288613A (en) * | 1988-02-17 | 1994-02-22 | Her Majesty The Queen In Right Of Canada, As Represented By The National Research Council Of Canada | Enzyme-based biosensor system for monitoring the freshness of fish |
| JP2007003280A (en) * | 2005-06-22 | 2007-01-11 | Techno Medica Co Ltd | Electrode structure and enzyme sensor including it for measuring phosphoric acid in body fluids |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5288613A (en) * | 1988-02-17 | 1994-02-22 | Her Majesty The Queen In Right Of Canada, As Represented By The National Research Council Of Canada | Enzyme-based biosensor system for monitoring the freshness of fish |
| JP2007003280A (en) * | 2005-06-22 | 2007-01-11 | Techno Medica Co Ltd | Electrode structure and enzyme sensor including it for measuring phosphoric acid in body fluids |
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