CN114894871B - Preparation method and application of high-sensitivity nitrite reductase bioelectrode - Google Patents

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

Preparation method and application of a highly sensitive nitrite reductase bioelectrode

Technical field

The invention relates to the technical fields of environmental pollution monitoring and food safety detection, and specifically to a preparation method and application of a highly sensitive nitrite reductase bioelectrode.

Background technique

Nitrite is a typical toxic pollutant that is widely present in chemical fertilizers, industrial wastewater, dyes, detergents and food additives. Contamination of soil, surface water and groundwater supplies is caused by the heavy application of nitrogen fertilizers on farmland and the release of nitrogen oxides into the atmosphere from many domestic and industrial burning processes. Eating more nitrite than normal intake will cause irreversible oxidation of hemoglobin in the blood, forming carcinogenic nitrosamines, which will have adverse effects on the ecosystem and human health. In view of its serious ecotoxicological and health risks, technical methods for rapid qualitative detection and highly sensitive quantitative analysis are of great significance for the environmental remediation of nitrite and food safety assessment.

So far, the analysis and determination methods for nitrite content in the environment mainly include ion chromatography, capillary electrophoresis, chemiluminescence, spectrophotometry and fluorescence spectroscopy. However, most of the above determination methods require expensive processing equipment, the determination process is complex and cumbersome, and the detection cost is high. These deficiencies severely limit and hinder the practical detection of nitrite. Compared with the problems faced by the above treatment methods, electrochemical methods have the advantages of simple equipment, high sensitivity, and low cost, and have attracted more and more attention. However, the electrodes commonly used in electrochemical analysis are based on inorganic catalytic materials, which are susceptible to interference from a variety of physical and chemical factors, making it difficult to achieve accurate qualitative and quantitative detection of complex environmental samples.

Enzymes are efficient and specific biocatalysts that can specifically recognize and efficiently transform substrates. Among them, oxidoreductase will transfer electrons when catalyzing the substrate. Using this characteristic, oxidoreductases and electrodes can be coupled to prepare highly specific enzyme bioelectrodes. Typically, enzymatic bioelectrode detection techniques react with the analyte involved to produce an electrical signal proportional to the amount of analyte. This type of electrode has obvious advantages such as good specificity, high stability, rapid response, low cost, easy operation and easy miniaturization.

Nitrite reductase in microorganisms can specifically recognize and bind nitrite and reduce it using biological electrons. Therefore, using nitrite reductase to prepare a new type of enzymatic bioelectrode to achieve specific qualitative detection and highly sensitive quantitative analysis of nitrite in complex samples has important application value for the control and treatment of nitrite pollutants.

Contents of the invention

In view of the shortcomings of current nitrite detection and analysis methods, such as cumbersome operation, high cost and insufficient anti-interference ability, the present invention is based on the advantages of high specificity and high sensitivity of enzymes, and isolates and purifies nitrite reductase from microbial cells, and Fixed on the surface of a highly adsorbent composite electrode, a new type of nitrite reductase bioelectrode was prepared to achieve efficient, specific, qualitative and quantitative detection of nitrite contaminants in complex samples.

In order to achieve the above objects, the technical solutions adopted by the present invention are:

A method for preparing a highly sensitive nitrite reductase bioelectrode. First, a porous medium material is loaded on a conductive material to form a porous medium composite electrode, and then the nitrite reductase purified and extracted from microbial cells is attached to the prepared On the composite electrode, a highly specific nitrite reductase bioelectrode can be constructed, which can achieve accurate qualitative and highly sensitive quantitative detection of nitrite.

The present invention is based on the redox characteristics of nitrite reductase that can gain and lose electrons, and utilizes its high specific recognition and high sensitivity reduction of nitrite to prepare a new type of nitrite reductase bioelectrode by coupling porous media composite electrodes. The chemical method quickly and stably detects the nitrite reduction signal, thereby achieving high-precision qualitative analysis and highly sensitive quantitative detection of nitrite.

As the preferred technical solution for the preparation method of high-sensitivity nitrite reductase bioelectrode of the present invention, the specific preparation method steps are as follows:

(1) Extract and purify nitrite reductase from microbial cells, and freeze and store it at low temperature away from light;

(2) Add indium tin oxide (ITO) powder to the acetic acid-ethanol solution and mix with ultrasonic to prepare an ITO solution;

(3) Drop the ITO solution on the highly conductive electrode to form a uniform film, dry it naturally and treat it in an oxygen-free manner at high temperature. After cooling, a porous medium composite electrode is obtained;

(4) Take the frozen nitrite reductase in step (1), thaw it and add it dropwise to the porous medium composite electrode, thereby obtaining a highly sensitive nitrite reductase bioelectrode.

In the preparation method of the present invention, the nitrite reductase in step (1) can be derived from bacteria with nitrite reducing ability, and is preferably purified and extracted from the bacterium Shewanella oneidensis MR-1 or the bacterium Paracoccus denitrificans.

In the preparation method of the present invention, the volume ratio of acetic acid and absolute ethanol in the acetic acid-ethanol solution in 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-ethanol solution. %.

In the preparation method of the present invention, a porous medium composite material prepared by ITO powder and high conductive electrode material is used. The porous medium attached to the conductive electrode facilitates the adsorption of nitrite reductase and increases the local concentration of the substrate, thereby improving the sensitivity of the detection. Among them, the highly conductive electrode is preferably ITO conductive glass or conductive carbon paper. At the same time, drop the ITO solution on the surface to form a uniform film, then air-dry it naturally, then treat it without oxygen at 400-500°C for 10-30 minutes, and finally cool it to room temperature naturally.

As another object of the present invention, the present invention also proposes an application of the prepared nitrite reductase bioelectrode in accurate qualitative and highly sensitive quantitative detection of nitrite, specifically as follows: using an electrochemical workstation, using three The electrode system uses the nitrite reductase electrode as the working electrode, Ag/AgCl as the reference electrode, and platinum wire as the counter electrode. After adding the nitrite-containing sample solution to the electrolyte, select an appropriate electrochemical scanning method for detection. The reduction signal of nitrite enables accurate qualitative and highly sensitive quantitative detection of nitrite. This detection technology is based on the reduction signal of nitrite rather than the oxidation signal.

As the preferred technical solution for this specific application, the electrolyte system used is 10mM, pH=6.5 PBS electrolyte, in which the concentration of each component is: sodium chloride 8g/L, potassium chloride 0.2g/L, twelve Hydrated disodium hydrogen phosphate 3.58g/L, sodium dihydrogen phosphate 0.27g/L. The electrolyte system was divided into 50 mL systems through serum bottles, exposed to nitrogen for 30 minutes, and then stored in a 4°C refrigerator to ensure the electrocatalytic activity of the enzyme. The volume of electrolyte used for a single test is 50 mL, and the volume of nitrite solution is 0.5-1.5% of the volume of the electrolyte.

As the preferred technical solution for this specific application, the electrochemical methods used in the present invention are linear scan voltammetry and cyclic voltammetry. A linearly changing voltage is applied to the electrode to detect the reduction signal of nitrite, thereby achieving qualitative analysis and quantitative detection of nitrite in environmental samples. Among them, cyclic voltammetry (-0.7~0V, 10mV/s) was selected for qualitative detection of nitrite; linear scanning voltammetry (0~-0.7V, 10mV/s) was used for quantitative determination of nitrite at different concentrations. detection.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. The present invention aims at the lack of high-sensitivity detection methods for nitrite in the environment and food. Based on the specificity and high sensitivity of microbial nitrite reductase, the amplified and purified nitrite reductase is loaded onto the electrode surface to construct a A new type of highly specific nitrite reductase bioelectrode uses electrochemical methods to achieve qualitative and quantitative detection of nitrite.

2. The enzymatic bioelectrode method for detecting nitrite proposed by the present invention solves the problems of traditional detection methods such as complex steps and large measurement errors, and has the advantages of rapid detection, high sensitivity, strong anti-interference ability and good stability. The highly sensitive nitrite reductase electrode constructed by the invention has great practical application value in environmental monitoring of toxic nitrite and food safety assessment.

Description of the drawings

Figure 1 shows the qualitative detection of nitrite by the nitrite reductase electrode prepared in Example 1.

Figure 2 shows the quantitative detection of nitrite at different concentrations by the nitrite reductase electrode prepared in Example 2.

Detailed ways

Example 1

In this example, a nitrite reductase bioelectrode from Shewanella oneidensis MR-1 was prepared for rapid detection of nitrite content. The wild-type strain of Shewanella oneidensis MR-1 is deposited in the American Type Collection (ATCC), and the strain number is ATCC700550. This strain can be purchased directly from the center. The specific steps for preparing enzyme bioelectrodes and detecting them are as follows:

(1) Extract and purify nitrite reductase from Shewanella oneidensis MR-1. The specific steps are:

①. Inoculation of bacteria: Inoculate the target bacterial solution (Shewanella oneidensis MR-1/PBBP1-nrfASO-his) into 400mL of LB containing GM 10ug/mL at a ratio of 1:100, and culture at 30°C and 200rpm for 24 hours.

②. Collect bacteria: add 8000g of 400mL bacterial liquid, centrifuge at 4℃ for 10 minutes, and discard the supernatant.

③. Collect protein: Resuspend the pellet in PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ , 2mM KH ₂ PO ₄ , add additional 0.1mM PMSF), 8000g, 10 minutes, centrifuge at 4°C, discard Clear, repeat twice, resuspend in 100 mL PBS buffer, and homogenize under high pressure.

④. Precipitate and freeze at 4°C for later use.

⑤. After flushing the nickel column with filtered ultrapure water, add the protein sample and let it flow down slowly. Then add imidazole with different concentration gradients (10mM, 50mM, 100mM, 250mM, 500mM) for elution. Need to take samples.

⑥. After using the nickel column, rinse it with 1M imidazole, then rinse it with ultrapure water, and finally add 20% ethanol and seal it for storage.

⑦. Run SDS-PAGE gel (protein buffer (50mM Tris-HCl, 200mM NaCl, 1mM MgCl, 1mMPMSF, 10mM β-mercaptoethanol)), determine the concentration of imidazole eluted from the nitrite reductase solution, and obtain nitrite reductase. Solution, use Thermo Fisher BCA Protein Assay Kit to determine the concentration of the enzyme solution, which is 0.918mg/mL.

⑧. Dispense into centrifuge tubes wrapped with tin foil and store in a -80°C refrigerator away from light.

(2) Prepare an acetic acid-ethanol solution (7.5 mL absolute ethanol, 3 mL acetic acid), add 1.8 g of ITO powder to it, and sonicate for 20 minutes until no powder remains to obtain an ITO solution.

(3) Place the ITO conductive glass (1cm*3cm) in a petri dish. The smooth side is the glass and the rough side is the electrode. Turn the electrode side up and add 75 μL of ITO solution dropwise (10 μL each time). , a small amount multiple times, try to slowly drop it on a 1cm*2cm area on one side of the conductive glass, spread it slowly, and load the remaining area with leads) to form a uniform film. After natural air drying, place it on a high-temperature-resistant vessel, 450 ℃ anaerobic treatment for 20 minutes, and after natural cooling, a porous media ITO electrode was obtained.

(4) Prepare 10mM, pH=6.5 PBS electrolyte (sodium chloride 8g/L, potassium chloride 0.2g/L, disodium hydrogen phosphate dodecahydrate 3.58g/L, sodium dihydrogen phosphate 0.27g/L) , aliquot into 100mL serum bottles (50mL system), expose to nitrogen for 30 minutes and store in a 4°C refrigerator to ensure the electrocatalytic activity of the enzyme.

(5) Under dark conditions, take out the nitrite reductase and place it on ice for thawing. Then pipet 10 μL and drop it onto the porous media ITO electrode. After 1 minute, wash away the excess enzyme with ultrapure water to obtain the enzyme electrode.

(6) With the help of an electrochemical workstation, a three-electrode system is used in an anaerobic reactor, with the enzyme electrode as the working electrode, Ag/AgCl as the reference electrode, and the platinum wire as the counter electrode. Use a syringe to absorb 0.5 mL of 500 μM nitrite. Solution, after exhausting the air, extend it below the liquid level and slowly add it to the electrolyte, stir at 1000rpm for 5 seconds, and select cyclic voltammetry (-0.7~0V, 10mV/s) for qualitative detection of nitrite after the liquid level stabilizes.

It can be seen from Figure 1 that the enzyme electrode prepared in this example has an obvious reduction electrical 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. The wild-type strain of Paracoccus denitrificans is deposited in the American Type Collection (ATCC), and the strain number is ATCC19367. This strain can be purchased directly from the center. The specific steps for preparing enzyme bioelectrodes and detecting them are as follows:

(1) Extract and purify the nitrite reductase in Paracoccus denitrificans according to step (1) of Example 1, aliquot into centrifuge tubes wrapped with tin foil, and then store in a -80°C refrigerator in the dark.

(2) Prepare an acetic acid-ethanol solution (7.5 mL absolute ethanol, 3 mL acetic acid), add 1.8 g of ITO powder to it, and sonicate for 20 minutes until no powder remains to obtain an ITO solution.

(3) Place the conductive carbon paper (1cm*3cm) in a petri dish, and add 75 μL of ITO solution dropwise (10 μL each time, a small amount multiple times, as slowly as possible in a 1cm*2cm area on one side of the conductive carbon paper) , slowly spread, and the remaining area is loaded with leads) to form a uniform film. After natural air drying, place it on a high-temperature-resistant vessel and treat it without oxygen at 450°C for 20 minutes. After natural cooling, a porous medium carbon paper electrode is obtained.

(4) Prepare 10mM, pH=6.5 PBS electrolyte (sodium chloride 8g/L, potassium chloride 0.2g/L, disodium hydrogen phosphate dodecahydrate 3.58g/L, sodium dihydrogen phosphate 0.27g/L) , aliquot into 100mL serum bottles (50mL system), expose to nitrogen for 30 minutes and store in a 4°C refrigerator to ensure the electrocatalytic activity of the enzyme.

(5) Under dark conditions, take out the nitrite reductase and place it on ice for thawing. Then pipet 10 μL (enzyme solution concentration is 0.918 mg/mL) and drop it onto the porous media carbon paper electrode. After 1 minute, use ultrasonic Pure water washes away excess enzyme to obtain the enzyme electrode.

(6) With the help of an electrochemical workstation, a three-electrode system is used in the anaerobic reactor, with the enzyme electrode as the working electrode, Ag/AgCl as the reference electrode, and the platinum wire as the counter electrode. Use a syringe to draw 0.5 mL of different concentrations ( 1-50μM) nitrite solution, exhaust the air and slowly add it to the electrolyte below the liquid level. Stir at 1000rpm for 5 seconds. After the liquid level stabilizes, use linear scanning voltammetry (0~-0.7V, 10mV/s ) for quantitative detection of nitrite.

It can be seen from Figure 2 that the enzyme electrode prepared in this embodiment can quantitatively detect nitrite at different concentrations.

The above contents are only examples and explanations of the concept of the invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or substitute them in similar ways, as long as they do not deviate from the concept of the invention. or beyond the scope defined by the claims, shall belong to the protection scope of the present invention.

Claims (8)

1. A method for preparing a highly sensitive nitrite reductase bioelectrode, which is characterized by first loading porous media materials on conductive materials to form porous media composite electrodes, and then reducing the nitrite extracted from microbial cells. The enzyme is attached to the prepared composite electrode, thereby constructing a highly specific nitrite reductase bioelectrode, which can achieve accurate qualitative and highly sensitive quantitative detection of nitrite; among them, the nitrite reductase attached to the composite electrode It is purified and extracted from the bacterium Shewanella oneidensis MR-1 or the bacterium Paracoccus denitrificans ; The specific preparation method steps are as follows: (1) Extract and purify nitrite reductase from microbial cells, and freeze and store it at low temperature away from light; (2) Add indium tin oxide (ITO) powder to the acetic acid-ethanol solution and mix ultrasonically to prepare an ITO solution; (3) Add the ITO solution dropwise on the highly conductive electrode to form a uniform film, dry it naturally and treat it at high temperature without oxygen, and then cool it to obtain a porous medium composite electrode; (4) Take the frozen nitrite reductase in step (1), thaw it and add it dropwise to the porous medium composite electrode, thereby obtaining a highly sensitive nitrite reductase bioelectrode. 2. The preparation method as claimed in claim 1, characterized in that in step (2), the volume ratio of acetic acid and absolute ethanol in the acetic acid-ethanol solution is 1:2-3, and the mass of indium tin oxide (ITO) powder accounts for 15-25% of the mass of acetic acid-ethanol solution. 3. The preparation method according to claim 1, characterized in that in step (3), ITO conductive glass or conductive carbon paper is selected as the high conductivity electrode, and the ITO solution is dripped on the surface to form a uniform film, and then dried naturally, and then dried at 400 Oxygen-free treatment at -500℃ for 10-30 minutes, and finally cooled to room temperature naturally. 4. The application of the nitrite reductase bioelectrode prepared by the method of any one of claims 1 to 3 in the accurate qualitative and highly sensitive quantitative detection of nitrite, characterized in that an electrochemical workstation is used and three electrodes are used The system uses the nitrite reductase electrode as the working electrode, Ag/AgCl as the reference electrode, and platinum wire as the counter electrode. After adding a sample solution containing nitrite to the electrolyte, select an appropriate electrochemical scanning method to detect nitrite. The reduction signal of nitrate enables accurate qualitative and highly sensitive quantitative detection of nitrite. 5. Application as claimed in claim 4, characterized in that the electrolyte system used is 10 mM, PBS electrolyte with pH=6.5, wherein the concentration of each component is: sodium chloride 8 g/L, chloride Potassium 0.2 g/L, sodium hydrogen phosphate dodecahydrate 3.58 g/L, sodium dihydrogen phosphate 0.27 g/L. 6. The application according to claim 5, characterized in that the electrolyte system is divided into 50 mL systems through serum bottles, and is stored in a 4°C refrigerator after being exposed to nitrogen for 30 minutes to ensure the electrocatalytic activity of the enzyme. 7. The application as claimed in claim 6, characterized in that the volume of the electrolyte used for a single detection is 50 mL, and the volume of the nitrite solution is 0.5-1.5% of the volume of the electrolyte. 8. The application as claimed in claim 7, characterized in that cyclic voltammetry is selected to carry out qualitative detection of nitrite, and the cyclic voltammetry parameters are -0.7 ~ 0 V, 10 mV/s; linear sweep voltammetry is used The method was used to quantitatively detect nitrite at different concentrations. The linear scan voltammetry parameters were 0 ~ -0.7 V and 10 mV/s.