CN114894871B - Preparation method and application of high-sensitivity nitrite reductase bioelectrode - Google Patents
Preparation method and application of high-sensitivity nitrite reductase bioelectrode Download PDFInfo
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- 108010025915 Nitrite Reductases Proteins 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
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- 238000004502 linear sweep voltammetry Methods 0.000 claims description 4
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- 238000002848 electrochemical method Methods 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
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- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 2
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P70/00—Climate change mitigation technologies in the production process for final industrial or consumer products
- Y02P70/50—Manufacturing or production processes characterised by the final manufactured product
Abstract
A preparation method and application of a high-sensitivity nitrite reductase bioelectrode relate to the technical field of environmental pollution monitoring and food safety detection, and comprise the steps of firstly loading a porous medium material on a conductive material to prepare a porous medium composite electrode, and then attaching nitrite reductase purified and extracted from microbial cells on the prepared composite electrode, so that the high-specificity nitrite reductase bioelectrode is constructed, and accurate qualitative and high-sensitivity quantitative detection of nitrite can be realized. The method for detecting the nitrite by the enzyme bioelectrode solves the problems of complex steps, large measurement error and the like in the traditional detection method, and has the advantages of quick detection, high sensitivity, strong anti-interference capability and good stability. The high-sensitivity nitrite reductase electrode constructed by the invention has great practical application value in the aspects of environmental monitoring and food safety evaluation of toxic nitrite.
Description
Technical Field
The invention relates to the technical field of environmental pollution monitoring and food safety detection, in particular to a preparation method and application of a high-sensitivity nitrite reductase bioelectrode.
Background
Nitrite is a typical toxic contaminant, widely found in fertilizers, industrial waste water, dyes, detergents and food additives. Nitrogen fertilizer is applied in large quantities in agricultural fields and nitrogen oxides are released into the atmosphere as a result of many domestic and industrial combustion processes, resulting in contaminated soil, surface water and groundwater supplies. Nitrite which is fed beyond the normal intake can cause irreversible oxidation of hemoglobin in blood to form cancerogenic nitrosamine, which has adverse effects on the ecosystem and human health. In view of serious ecotoxicology and health risks, the technical method of rapid qualitative detection and high-sensitivity quantitative analysis has important significance for nitrite environmental remediation and food safety evaluation.
To date, analytical methods for measuring nitrite content in an environment mainly include ion chromatography, capillary electrophoresis, chemiluminescence, spectrophotometry, fluorescence spectrometry, and the like. However, the above measurement method requires expensive processing equipment, and the measurement process is complicated and complicated, and the detection cost is high. These defects severely limit and prevent the actual detection of nitrite. Compared with the problems faced by the above treatment methods, the electrochemical method has the advantages of simple equipment, high sensitivity, low cost and the like, and is receiving more and more attention. However, the electrodes used in electrochemical analysis are all based on inorganic catalytic materials, are easily interfered by various physical and chemical factors, and are difficult to realize accurate qualitative and quantitative detection on complex environmental samples.
The enzyme is a high-efficiency specific biocatalyst, and can perform specific recognition and high-efficiency conversion on a substrate. Wherein the oxidoreductase, upon substrate catalysis, undergoes electron transfer. By utilizing the characteristic, the oxidation-reduction enzyme and the electrode can be coupled to prepare the enzyme bioelectrode with high specificity. In general, enzymatic bioelectrode detection techniques produce an electrical signal proportional to the amount of analyte by reacting with the analyte of interest. The electrode has the obvious advantages of good specificity, high stability, quick response, low cost, convenient operation, easy miniaturization and the like.
Nitrite reductase in microorganisms is capable of specifically recognizing and binding nitrite and reducing it by bioelectron. Therefore, the nitrite reductase is utilized to prepare a novel enzyme bioelectrode so as to realize the specific qualitative detection and high-sensitivity quantitative analysis of nitrite in complex samples, and the method has important application value for the control and treatment of nitrite pollutants.
Disclosure of Invention
Aiming at the defects of complex operation, high cost, insufficient anti-interference capability and the like of the current nitrite detection analysis method, the invention separates and purifies nitrite reductase from microbial cells based on the advantages of high specificity and high sensitivity of enzyme, and fixes the nitrite reductase on the surface of a high-adsorptivity composite electrode to prepare a novel nitrite reductase bioelectrode so as to realize high-efficiency specific qualitative and quantitative detection of nitrite pollutants in complex samples.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a preparation method of a high-sensitivity nitrite reductase bioelectrode comprises the steps of firstly loading a porous medium material on a conductive material to prepare a porous medium composite electrode, and then attaching nitrite reductase purified and extracted from microbial cells to the prepared composite electrode, so that the high-specificity nitrite reductase bioelectrode is constructed, and accurate qualitative and high-sensitivity quantitative detection of nitrite can be realized.
The invention prepares a novel nitrite reductase bioelectrode based on the oxidation-reduction characteristic of nitrite reductase capable of losing electrons by utilizing the high specificity identification and high sensitivity reduction of nitrite, and rapidly and stably detects nitrite reduction signals by an electrochemical method by coupling a porous medium composite electrode, thereby realizing high-accuracy qualitative analysis and high-sensitivity quantitative detection of nitrite.
As a preferable technical scheme of the preparation method of the high-sensitivity nitrite reductase bioelectrode, the preparation method comprises the following steps:
(1) Extracting nitrite reductase in purified microbial cells, and freezing and preserving the nitrite reductase at a low temperature in a dark place;
(2) Adding Indium Tin Oxide (ITO) powder into the acetic acid-ethanol solution, and uniformly mixing by ultrasonic to prepare an ITO solution;
(3) Dropping ITO solution on the high-conductivity electrode to form a uniform film, naturally air-drying, performing high-temperature anaerobic treatment, and cooling to obtain the porous medium composite electrode;
(4) And (3) taking the nitrite reductase frozen in the step (1), and dripping the nitrite reductase onto the porous medium composite electrode after thawing, so as to obtain the high-sensitivity nitrite reductase bioelectrode.
In the production method of the present invention, the nitrite reductase in the step (1) may be derived from a bacterium having nitrite-reducing ability, preferably by purification from bacterium Shewanella oneidensis MR-1 or bacterium Paracoccus denitrificans.
In the preparation method, the volume ratio of acetic acid to absolute ethyl alcohol in the acetic acid-ethyl alcohol solution in the step (2) is 1:2-3, and the mass of Indium Tin Oxide (ITO) powder accounts for 15-25% of the mass of the acetic acid-ethyl alcohol solution.
In the preparation method, the porous medium composite material is prepared by utilizing ITO powder and a high-conductivity electrode material. The porous medium attached to the conductive electrode is favorable for adsorbing nitrite reductase, and improves the local concentration of the substrate, thereby improving the detection sensitivity. Among them, the high conductivity electrode is preferably ITO conductive glass or conductive carbon paper. Meanwhile, after the ITO solution is dripped on the surface to form a film uniformly, naturally air-drying, then performing anaerobic treatment at 400-500 ℃ for 10-30 minutes, and finally naturally cooling to room temperature.
As another object of the invention, the invention also provides an application of the prepared nitrite reductase bioelectrode in accurate qualitative and high-sensitivity quantitative detection of nitrite, which comprises the following specific steps: the electrochemical workstation is utilized, a three-electrode system is adopted, a nitrite reductase electrode is used as a working electrode, ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, and after a sample solution containing nitrite is added into an electrolyte, a proper electrochemical scanning method is selected for detecting a reduction signal of the nitrite, so that accurate qualitative and high-sensitivity quantitative detection of the nitrite is realized. This detection technique is based on the reduction signal of nitrite rather than using the oxidation signal.
As a preferred technical solution for this specific application, a PBS electrolyte was used with a ph=6.5 at 10mm, wherein the concentrations of the components were: 8g/L of sodium chloride, 0.2g/L of potassium chloride, 3.58g/L of disodium hydrogen phosphate dodecahydrate and 0.27g/L of sodium dihydrogen phosphate. The electrolyte system is split into a 50mL system by a serum bottle, and is stored in a refrigerator at 4 ℃ after being aerated with nitrogen for 30 minutes so as to ensure the electrocatalytic activity of the enzyme. The volume of the electrolyte used for single detection is 50mL, and the volume of the nitrite solution is 0.5-1.5% of the volume of the electrolyte.
As a preferred embodiment of this particular application, the electrochemical methods employed in the present invention are linear sweep voltammetry and cyclic voltammetry. And applying a linearly-changing voltage to the electrode to detect the reduction signal of the nitrite, thereby realizing qualitative analysis and quantitative detection of the nitrite in the environmental sample. Wherein, cyclic voltammetry (-0.7-0V, 10 mV/s) is selected for qualitative detection of nitrite; quantitative detection of nitrite with different concentrations is carried out by using a linear sweep voltammetry (0 to-0.7V, 10 mV/s).
Compared with the prior art, the invention has the beneficial effects that:
1. aiming at the defects of a nitrite high-sensitivity detection method in the environment and food, the invention loads the nitrite reductase amplified and purified on the surface of an electrode based on the specificity and high sensitivity of nitrite reductase of microorganisms, constructs a novel high-specificity nitrite reductase bioelectrode, and realizes qualitative and quantitative detection of nitrite by using an electrochemical method.
2. The method for detecting the nitrite by the enzyme bioelectrode solves the problems of complex steps, large measurement error and the like in the traditional detection method, and has the advantages of quick detection, high sensitivity, strong anti-interference capability and good stability. The high-sensitivity nitrite reductase electrode constructed by the invention has great practical application value in the aspects of environmental monitoring and food safety evaluation of toxic nitrite.
Drawings
FIG. 1 shows the qualitative detection of nitrite by the nitrite reductase electrode prepared in example 1.
FIG. 2 shows quantitative detection of nitrite at different concentrations by the nitrite reductase electrode prepared in example 2.
Detailed Description
Example 1
This example prepared a nitrite reductase bioelectrode from Shewanella oneidensis MR-1 for rapid detection of nitrite content. Shewanella oneidensis MR-1 wild-type strain was deposited at the American type Collection (ATCC) and strain number was ATCC700550. This strain can be purchased directly from the center. The preparation method and detection method of the enzyme bioelectrode comprise the following specific steps:
(1) The nitrite reductase in Shewanella oneidensis MR-1 is extracted and purified, and the specific steps are as follows:
(1) inoculating: the target bacterial liquid (Shewanella oneidensis MR-1/PBBP 1-nrfas-his) was inoculated at 1:100 into 400mL of LB containing GM 10ug/mL, and cultured at 30℃for 24 hours at 200 rpm.
(2) Collecting bacteria: 400mL of the bacterial liquid was centrifuged at 200 g for 10 minutes at 4℃and the supernatant was discarded.
(3) Protein collection: precipitation with PBS buffer (137mM NaCl,2.7mM KCl,10mM Na) 2 HPO 4 ，2mM KH 2 PO 4 0.1mM PMSF) was additionally added, and the suspension was resuspended, spun at 4℃for 10 minutes at 8000g, and the supernatant was discarded, and after repeating twice, resuspended with 100mL PBS buffer, and broken by high-pressure homogenization.
(4) And (3) precipitating and freezing at 4 ℃ for standby.
(5) After washing the nickel column with the ultra-pure water filtered by suction, the protein sample is added, and after letting it flow down slowly, imidazole is added to elute with different concentration gradients (10 mM,50mM,100mM,250mM,500 mM), and the sample is taken up for each elution.
(6) Washing with 1M imidazole after using nickel column, washing with ultrapure water, and sealing and preserving with 20% ethanol.
(7) SDS-PAGE gel (protein buffer (50 mM Tris-HCl,200mM NaCl,1mM MgCl,1mM PMSF,10mM beta-mercaptoethanol)), and after the concentration of imidazole eluted by the nitrite reductase solution was determined, a nitrite reductase solution was obtained, and the enzyme solution concentration was measured using a Siemens Fei BCA protein detection kit and was 0.918mg/mL.
(8) Packaging into centrifuge tube packed with tinfoil paper, and storing in a refrigerator at-80deg.C in dark place.
(2) An acetic acid-ethanol solution (7.5 mL of absolute ethanol, 3mL of acetic acid) was prepared, to which 1.8g of ITO powder was added, and sonicated for 20 minutes until no powder remained, to obtain an ITO solution.
(3) Placing ITO conductive glass (1 cm x 3 cm) in a culture dish, wherein the smooth surface is glass, the rough surface is electrode, the electrode surface is upward, 75 mu L of ITO solution is dripped into the ITO conductive glass (10 mu L is dripped for a few times each time, the ITO solution is slowly dripped into a 1cm x 2cm area on one side of the conductive glass as much as possible, the ITO conductive glass is slowly diffused, and leads are loaded in the rest area), forming a uniform membranous shape, naturally airing, then placing the membranous shape on a high-temperature resistant vessel, carrying out anaerobic treatment for 20 minutes at 450 ℃, and naturally cooling to obtain the porous medium ITO electrode.
(4) 10mM PBS electrolyte (8 g/L of sodium chloride, 0.2g/L of potassium chloride, 3.58g/L of disodium hydrogen phosphate dodecahydrate and 0.27g/L of sodium dihydrogen phosphate) with pH=6.5 is prepared, and 100mL serum bottles are packaged (50 mL system) and stored in a refrigerator at 4 ℃ after being aerated with nitrogen for 30 minutes to ensure the electrocatalytic activity of the enzyme.
(5) Taking out nitrite reductase under the condition of light shading, thawing on ice, transferring 10 mu L of nitrite reductase, dripping the nitrite reductase onto a porous medium ITO electrode, and washing off excessive enzyme by using ultrapure water after 1 minute to obtain the enzyme electrode.
(6) By means of an electrochemical workstation, a three-electrode system is adopted in an anaerobic reactor, an enzyme electrode is used as a working electrode, ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, 0.5mL of 500 mu M nitrite solution is sucked by an injector, air is discharged and then is extended below the liquid level, the solution is slowly added into electrolyte, stirring is carried out for 5 seconds at 1000rpm, and cyclic voltammetry (-0.7-0V, 10 mV/s) is selected for qualitative detection of nitrite after the liquid level is stable.
As can be seen from FIG. 1, the enzyme electrode prepared in this example has a distinct reduction electric signal at-0.49V in the presence of nitrite, indicating that it can detect the presence of nitrite with high specificity.
Example 2
This example prepares a nitrite reductase bioelectrode from Paracoccus denitrificans for rapid detection of nitrite content. Paracoccus denitrificans wild-type strain was deposited at the American type Collection (ATCC) and strain number was ATCC19367. This strain can be purchased directly from the center. The preparation method and detection method of the enzyme bioelectrode comprise the following specific steps:
(1) The nitrite reductase in Paracoccus denitrificans was extracted and purified in the step (1) of example 1, and the resulting mixture was packed into a centrifuge tube packed with tinfoil paper, and stored in a refrigerator at-80℃in a dark place.
(2) An acetic acid-ethanol solution (7.5 mL of absolute ethanol, 3mL of acetic acid) was prepared, to which 1.8g of ITO powder was added, and sonicated for 20 minutes until no powder remained, to obtain an ITO solution.
(3) Placing conductive carbon paper (1 cm x 3 cm) in a culture dish, dropwise adding 75 mu L of ITO solution (10 mu L is dropwise added each time, a small amount of ITO solution is dropwise added for many times, the ITO solution is slowly dripped on a 1cm x 2cm area at one side of the conductive carbon paper as much as possible, the rest area is slowly diffused, and leads are loaded) to form a uniform membranous shape, naturally airing, then placing the membranous shape on a high-temperature resistant vessel, carrying out anaerobic treatment for 20 minutes at 450 ℃, and naturally cooling to obtain the porous medium carbon paper electrode.
(4) 10mM PBS electrolyte (8 g/L of sodium chloride, 0.2g/L of potassium chloride, 3.58g/L of disodium hydrogen phosphate dodecahydrate and 0.27g/L of sodium dihydrogen phosphate) with pH=6.5 is prepared, and 100mL serum bottles are packaged (50 mL system) and stored in a refrigerator at 4 ℃ after being aerated with nitrogen for 30 minutes to ensure the electrocatalytic activity of the enzyme.
(5) Under the condition of avoiding light, taking out nitrite reductase, placing the nitrite reductase on ice for thawing, transferring 10 mu L (the concentration of enzyme solution is 0.918 mg/mL), dripping the nitrite reductase on a porous medium carbon paper electrode, and washing off excessive enzyme by using ultrapure water after 1 minute to obtain the enzyme electrode.
(6) By means of an electrochemical workstation, a three-electrode system is adopted in an anaerobic reactor, an enzyme electrode is used as a working electrode, ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, 0.5mL of nitrite solutions with different concentrations (1-50 mu M) are respectively sucked by using a syringe, air is discharged and then is slowly added into electrolyte after reaching the lower part of the liquid level, stirring is carried out at 1000rpm for 5 seconds, and after the liquid level is stable, the nitrite is quantitatively detected by using a linear scanning voltammetry (0 to-0.7V, 10 mV/s).
As can be seen from FIG. 2, the enzyme electrode prepared in this example was able to quantitatively detect nitrite at different concentrations.
The foregoing is merely illustrative and explanatory of the principles of the invention, as various modifications and additions may be made to the specific embodiments described, or similar thereto, by those skilled in the art, without departing from the principles of the invention or beyond the scope of the appended claims.
Claims (8)
1. A preparation method of a high-sensitivity nitrite reductase bioelectrode is characterized in that a porous medium material is firstly loaded on a conductive material to prepare a porous medium composite electrode, and then nitrite reductase purified and extracted from microbial cells is attached to the prepared composite electrode, so that the high-specificity nitrite reductase bioelectrode is constructed, and accurate qualitative and high-sensitivity quantitative detection of nitrite can be realized; wherein the nitrite reductase attached to the composite electrode is derived from bacteriaShewanella oneidensisMR-1 or bacteriaParacoccus denitrificansPurifying and extracting;
the preparation method comprises the following steps:
(1) Extracting nitrite reductase in purified microbial cells, and freezing and preserving the nitrite reductase at a low temperature in a dark place;
(2) Adding Indium Tin Oxide (ITO) powder into the acetic acid-ethanol solution, and uniformly mixing by ultrasonic to prepare an ITO solution;
(3) Dropping ITO solution on the high-conductivity electrode to form a uniform film, naturally air-drying, performing high-temperature anaerobic treatment, and cooling to obtain the porous medium composite electrode;
(4) And (3) taking the nitrite reductase frozen in the step (1), and dripping the nitrite reductase onto the porous medium composite electrode after thawing, so as to obtain the high-sensitivity nitrite reductase bioelectrode.
2. The method according to claim 1, wherein the volume ratio of acetic acid to absolute ethanol in the acetic acid-ethanol solution in the step (2) is 1:2-3, and the mass of Indium Tin Oxide (ITO) powder is 15-25% of the mass of the acetic acid-ethanol solution.
3. The preparation method of claim 1, wherein in the step (3), the high conductivity electrode is selected from ITO conductive glass or conductive carbon paper, and after the ITO solution is dropped on the surface to form a film uniformly, the film is naturally air-dried, and then the film is subjected to anaerobic treatment at 400-500 ℃ for 10-30 minutes, and finally naturally cooled to room temperature.
4. The use of a nitrite reductase bioelectrode prepared by the method as claimed in any one of claims 1 to 3 in the accurate qualitative and high-sensitivity quantitative detection of nitrite, wherein an electrochemical workstation is used, a three-electrode system is adopted, a nitrite reductase electrode is used as a working electrode, ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, and a sample solution containing nitrite is added into an electrolyte, and then a proper electrochemical scanning method is selected to detect the reduction signal of nitrite, thereby realizing the accurate qualitative and high-sensitivity quantitative detection of nitrite.
5. The use according to claim 4, wherein the electrolyte system used is 10mm, ph=6.5 in PBS electrolyte, wherein the concentration of the components is: sodium chloride 8g/L, potassium chloride 0.2g/L, disodium hydrogen phosphate dodecahydrate 3.58g/L, sodium dihydrogen phosphate 0.27g/L.
6. The use according to claim 5, wherein the electrolyte system is dispensed into a 50mL system by serum bottles and stored in a refrigerator at 4 ℃ after 30 minutes of nitrogen exposure to ensure electrocatalytic activity of the enzyme.
7. The use according to claim 6, wherein the volume of electrolyte used for the single test is 50mL and the volume of nitrite solution is 0.5-1.5% of the volume of electrolyte.
8. The use according to claim 7, wherein the qualitative detection of nitrite is performed by cyclic voltammetry with cyclic voltammetry parameters of-0.7 to 0v,10 mV/s; quantitative detection of nitrite with different concentrations is carried out by using a linear sweep voltammetry, wherein the parameters of the linear sweep voltammetry are 0 to-0.7V and 10 mV/s.
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