Nitrite reductase electrode and method for preparing the same, electrochemical system

Abstract

The application relates to a nitrite reductase electrode and a preparation method thereof and an electrochemical system. The nitrite reductase electrode comprises a substrate layer and n electron transfer layers and n enzyme activity layers in the form of alternating layers above the substrate layer, n is in the range of 2-30, wherein the substrate layer comprises a carbon-based electrode loaded with a transition metal phosphide, and the enzyme activity layer comprises a nitrite reductase. The nitrite reductase electrode has low detection limit, high sensitivity, high enzyme loading rate, high electrocatalytic efficiency and good application prospect.

Description

Technical Field

[0001] This application relates to the field of electrochemical technology, and more particularly to a nitrite reductase electrode and its preparation method and electrochemical system. Background Technology

[0002] Nitrite, a common pollutant in industrial wastewater and contaminated groundwater, is also a potentially hazardous chemical. Excessive intake can cause methemoglobinemia, leading to tissue hypoxia, which can be fatal in severe cases. Long-term exposure may also increase the risk of certain types of cancer. Nitrite reductases (NiRs) are a class of key enzymes that can break down nitrite (NO2) into nitrogenous compounds. - Enzyme-catalyzed reactions are converted into nitric oxide (NO) or ammonia (NH3), playing a crucial role in the nitrogen cycle and nitrogen pollution control. While enzyme-catalyzed reactions are ubiquitous in nature, their limitations stem from the slow energy input and electron transfer processes within organisms, as well as the high cost and high susceptibility to inactivation of free enzymes. Enzyme-electrocatalysis, with immobilized enzymes at its core, combines the specificity of enzyme catalysis with the directional electron input of electrocatalysis, ensuring that the enzyme-electrocatalysis system simultaneously possesses the advantages of sensitivity, efficiency, economy, and stability.

[0003] Currently, enzyme immobilization techniques mainly include physicochemical processes such as adsorption, embedding, and covalent cross-linking. Adsorption is simple to operate and has mild immobilization conditions, but its stability is poor. Embedding is suitable for immobilizing various enzymes, but it may suffer from enzyme leakage and limitations in the diffusion of large substrate molecules. Covalent cross-linking, while providing good immobilization, requires harsh reaction conditions, often causing irreversible damage to enzyme activity, and has poor overall conductivity. Using an external electric field to induce (de)protonation of electrically neutral protein molecule functional groups makes it easier to form surface electrostatic potential traps and deposit them on the electrode surface. Electrodeposition technology directly loads enzymes onto conductive substrates through electrostatic attraction, significantly improving the sensitivity and conductivity of enzyme electrodes while preserving the enzyme's structure and catalytic stability. However, this method is prone to enzyme loss and lacks reproducibility in multiple cycles.

[0004] Therefore, there is a need for an improved nitrite reductase electrode, its preparation method, and an electrochemical system. Summary of the Invention

[0005] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of the claims.

[0006] This application provides a nitrite reductase electrode, comprising a base layer and alternating layers of n electron transport layers and n enzyme activity layers located above the base layer, wherein n is in the range of 2-30 (n is a positive integer), wherein the base layer comprises a carbon-based electrode loaded with transition metal phosphides, and the enzyme activity layers comprise nitrite reductase.

[0007] In one embodiment, n is in the range of 10-20.

[0008] In one embodiment, the loading rate of nitrite reductase on the carbon-based electrode is 10% to 70%.

[0009] In one embodiment, the loading of transition metal phosphide on the carbon-based electrode is 0.2-1.5 mg / cm³. 2 For example, 0.3-0.5 mg / cm³ 2 .

[0010] In one embodiment, the electron transport layer comprises reduced graphene oxide having a zeta potential in the range of -2 to -10 mV.

[0011] In one embodiment, the nitrite reductase is a cytochrome c nitrite reductase (C cNIR or NrfA) with a zeta potential in the range of 2 to 10 mV.

[0012] In this application, the loaded transition metal phosphide can be used to adsorb protons or dissociate water molecules to generate protons; the reduced graphene oxide forming the electron transport layer can be used to enhance electron transfer between the electrode and the enzyme and improve the compatibility between the enzyme and the metal layer; the nitrite reductase forming the enzyme active layer can play a major catalytic role as a catalytic site.

[0013] In one embodiment, the nitrite reductase electrode may further include a base layer and alternating layers of n electron transport layers and n-1 enzyme activity layers located above the base layer. That is, the outermost layer is reduced graphene oxide, which can be used to encapsulate the enzyme activity layers from the outside to form a stable composite structure and to protect the enzyme.

[0014] In this application, the cytochrome c nitrite reductase can be formed from cytochrome c nitrite reductase extracted from commercially available Shewanella bacteria, and its zeta potential is approximately 2–10 mV.

[0015] In one embodiment, the transition metal phosphide is selected from the group consisting of copper phosphide, cobalt phosphide, nickel phosphide, and iron phosphide.

[0016] In one embodiment, the carbon-based electrode is selected from one of glassy carbon electrode, graphite electrode, carbon paper, carbon cloth, and carbon felt.

[0017] In one embodiment, the carbon felt includes one of polyacrylonitrile-based carbon felt, viscose-based carbon felt, pitch-based carbon felt, and wood fiber-based carbon felt.

[0018] On the other hand, this application provides a method for preparing the above-mentioned nitrite reductase electrode, comprising applying an alternating electric field for 2-30 cycles to sequentially electrodeposit reduced graphene oxide and nitrite reductase in a mixed solution onto a carbon-based electrode loaded with transition metal phosphides, thereby forming the nitrite reductase electrode immobilized with nitrite reductase.

[0019] In one embodiment, the mixed solution is formed by stirring and mixing 200-800 μL of nitrite reductase solution with 10 mL of phosphate buffer solution, and then adding 5 mL of reduced graphene oxide dispersion and stirring.

[0020] In one embodiment, the nitrite reductase solution is prepared by dissolving 0.1–1.0 mg / mL of nitrite reductase in a buffer solution (such as HEPES buffer) with a pH range of 6.5–7.

[0021] In one embodiment, the nitrite reductase solution and the phosphate buffer solution are stirred and mixed at 2-8°C.

[0022] In one embodiment, the reduced graphene oxide dispersion is obtained by mixing graphene oxide powder in 0.1-1.0 mg / mL ascorbic acid and ammonia water, and then hydrothermally treating it at 90-120°C for 1-5 hours to obtain a reduced graphene oxide dispersion with a concentration of 0.5-1.0 mg / mL. The pH of the reduced graphene oxide dispersion is adjusted to 3-9, preferably 3-6.

[0023] In one embodiment, the transition metal phosphide can be synthesized by first synthesizing a metal hydroxide precursor via a hydrothermal method, followed by pyrolysis gas-solid phase phosphating. The preparation of a carbon-based electrode loaded with transition metal phosphides can include placing a pretreated (e.g., acid-washed) carbon-based electrode, such as a carbon felt, in a solution of transition metal nitrates (e.g., cobalt nitrate) and urea for hydrothermal reaction to obtain a carbon-based electrode loaded with a metal hydroxide (e.g., cobalt hydroxide) precursor, followed by phosphating with sodium hypophosphite as a phosphorus source to obtain a carbon-based electrode loaded with metal phosphides.

[0024] In one embodiment, the total volume of the mixed solution is 20-40 mL.

[0025] In one embodiment, the total stirring time can be 20 to 60 minutes.

[0026] In one embodiment, applying an alternating electric field includes using the mixed solution as the electrolyte, a carbon-based electrode loaded with transition metal phosphides as the working electrode, a platinum sheet as the counter electrode, and silver-silver chloride as the reference electrode, alternating positive and negative potentials for a total of 2 to 30 cycles to sequentially deposit reduced graphene oxide and nitrite reductase onto the working electrode to form the initial electrode, and then freeze-drying the initial electrode to obtain the nitrite reductase electrode.

[0027] In one embodiment, the conditions of the alternating electric field include a positive potential of +0.7 to +1.0V and a single duration of 10 to 20 seconds; and a negative potential of -0.5 to -0.8V and a single duration of 10 to 15 seconds.

[0028] In one embodiment, the last applied alternating electric field is a positive potential. In this application, the last application of a positive potential forms a reduced graphene oxide layer as the outermost electron transport layer.

[0029] In one embodiment, after forming the initial electrode, the process may include immersing the initial electrode in pure water for 10 to 30 minutes to remove residual phosphate ions in the electrode gaps and on the surface; then freeze-drying the initial electrode to obtain the nitrite reductase electrode.

[0030] In another aspect, this application provides an electrochemical system for detecting nitrite content in water, including the aforementioned nitrite reductase electrode as a cathode.

[0031] In another aspect, this application provides a biosensor including the aforementioned nitrite reductase electrode.

[0032] This application combines nitrite reductase with reduced graphene oxide, a two-dimensional flexible nanomaterial, and utilizes the principle of electrostatic self-assembly to form a near-neutral nanoassembly of nitrite reductase and reduced graphene oxide in solution, which have opposite charges. Then, under the action of an alternating electric field, the assemblies migrate to the surface of a metal phosphide with a stronger local field through surface dipoles and electrophoretic forces, thereby forming a stable enzyme electrode. Through the periodic reversal of the electric field, the reduced graphene oxide and nitrite reductase form a stable conformation on the carbon-based electrode surface, and the coverage of the assembly on the electrode surface is effectively controlled, thereby maximizing the electron-donating capacity of the electrode and the catalytic activity of the enzyme.

[0033] The nitrite reductase electrode of this application overcomes the problems of low efficiency and poor selectivity in enzyme electrodes with enzymes as active sites during nitrite detection and conversion. It provides an important supplement to the detection and conversion technology of nitrite nitrogen pollution in surface water and groundwater.

[0034] Existing unmodified carbon-based conductive substrates, due to their smooth surface and uniform electric field distribution, are difficult to synthesize enzyme electrodes through alternating electric field deposition, resulting in enzyme loading of less than 5%. In contrast, this application introduces metal phosphides onto the surface of a conductive carbon-based electrode to enhance its local micro-electric field, thereby inducing the loading of reduced graphene oxide and nitrite reductase assemblies onto the electrode surface.

[0035] This application is simple to operate and highly reproducible, and has significant advantages in synthesizing enzyme electrodes and controlling the loading and catalytic activity of nitrite reductase.

[0036] The nitrite reductase electrode of this application not only achieves high sensitivity and high selectivity in the detection of nitrite, but also promotes the reduction of nitrite through electrochemical methods, providing a new approach for nitrogen cycling and nitrogen pollution remediation.

[0037] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and drawings. Attached Figure Description

[0038] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0039] Figure 1 Cyclic voltammetry curves of the nitrite reductase electrode provided in Example 1 of this application at different nitrite concentrations;

[0040] Figure 2 Cyclic voltammetry curves of a cobalt phosphide / carbon felt electrode without nitrite reductase loading provided for Comparative Example 1 of this application at different nitrite concentrations;

[0041] Figure 3 The catalytic conversion effect of the nitrite reductase electrode provided in this application on nitrite with different initial concentrations; and,

[0042] Figure 4 This paper compares the catalytic nitrite conversion effects of the nitrite reductase electrode provided in this application with those of different control electrodes. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application are described in detail below. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be arbitrarily combined with each other.

[0044] Unless otherwise specified, all materials used in the following examples and comparative examples are commercially available.

[0045] Example 1

[0046] The preparation method of the nitrite reductase electrode may include the following steps:

[0047] Cell disruption and extraction were performed on Shewanella strain (commercially available, available from the China General Microbiological Culture Collection Center (CGMCC), strain accession number CGMCC 19242), followed by protein purification to obtain cytochrome c nitrite reductase with a molecular weight of 45-55 kDa, which was dissolved in HEPES buffer and stored. The concentration of nitrite reductase was determined to be 0.9 mg / mL by BCA protein quantification method.

[0048] 50 mg of graphene oxide powder was mixed in a 0.1 mg / mL solution of ascorbic acid and ammonia, and hydrothermally treated at 100 °C for 2 h to obtain a reduced graphene oxide dispersion with a concentration of 0.5 mg / mL. The pH of the reduced graphene oxide dispersion was adjusted to 4.

[0049] Take 2.0*2.5cm 2 Commercially available polyacrylonitrile carbon felt was pretreated with 68% nitric acid to remove surface impurities. The pretreated carbon felt was then hydrothermally reacted at 110°C for 5 hours in a solution of 100 mg / L cobalt nitrate and 50 mg / L urea to obtain carbon felt supported on a cobalt hydroxide precursor. Subsequently, using sodium hypophosphite as the phosphorus source, it was phosphated at 300°C for 2 hours to obtain cobalt phosphide-supported carbon felt with a cobalt phosphide loading of 0.4 mg / cm³. 2 .

[0050] 400 μL of nitrite reductase solution with a protein concentration of 0.9 mg / mL was added to 10 mL of phosphate buffer solution and stirred at 4 °C until homogeneous. Then, 5 mL of reduced graphene oxide dispersion with pH 4 was added and stirred at room temperature for 30 minutes to obtain a mixed solution of nitrite reductase-reduced graphene oxide.

[0051] Using the mixed solution as the electrolyte, a carbon felt electrode loaded with cobalt phosphide as the working electrode, a platinum sheet as the counter electrode, and silver-silver chloride as the reference electrode, deposition was carried out on an electrochemical workstation under an alternating electric field. Under positive current, the positive potential used was +0.7V with a single duration of 15s, and the negative potential used was -0.7V with a single duration of 10s. A total of 20 cycles of alternating positive and negative potentials were performed to obtain an electrode immobilized with nitrite reductase.

[0052] The initial electrode was removed and immersed in pure water for 20 minutes to remove residual phosphate buffer from the electrode pores and surface. After freeze-drying the electrode, it was stored at 4°C. By testing the change in protein concentration in the mixed solution before and after deposition, the actual loading rate (enzyme loading rate) on the electrode was found to be 68%.

[0053] The nitrite reductase-reduced graphene oxide-cobalt phosphide electrode synthesized in Example 1 was applied to the electrochemical detection of low-concentration nitrite. Cyclic voltammetry experiments were conducted within a voltage range of +0.2V to -1.0V at a scan rate of 50mV / s. The electrolyte consisted of 0.01M PBS and different concentrations of nitrite. The results showed... Figure 1 middle.

[0054] from Figure 1 As can be seen, the nitrite reductase electrode in this embodiment exhibits a significant reduction peak at a potential of -0.67V, and a significant increase in current occurs when the nitrite concentration is 140μM. Furthermore, the peak current density increases with the increase of nitrite concentration.

[0055] Comparative Example 1

[0056] The preparation method of the nitrite reductase electrode may include the following steps:

[0057] Cell disruption and extraction were performed on commercially available Sheva strains, followed by protein purification to obtain cytochrome c nitrite reductase with a molecular weight of 45-55 kDa. The concentration of nitrite reductase was determined to be 0.9 mg / mL by BCA protein quantification method.

[0058] 50 mg of commercially available graphene oxide powder was mixed in a 0.1 mg / mL solution of ascorbic acid and ammonia, and hydrothermally treated at 100 °C for 2 h to obtain a reduced graphene oxide dispersion with a concentration of 0.5 mg / mL. The pH of the reduced graphene oxide dispersion was adjusted to 4.

[0059] Take 2.0*2.5cm 2 Commercially available polyacrylonitrile carbon felt is pretreated with 68% nitric acid to remove surface impurities, resulting in pretreated carbon felt.

[0060] 400 μL of nitrite reductase solution with a protein concentration of 0.9 mg / mL was added to 10 mL of phosphate buffer solution and stirred at 4 °C until homogeneous. Then, 5 mL of reduced graphene oxide dispersion was added and stirred at room temperature for 30 minutes to obtain a mixed solution of nitrite reductase-reduced graphene oxide.

[0061] Using a mixed solution as the electrolyte, a polyacrylonitrile carbon felt electrode as the working electrode, a platinum sheet as the counter electrode, and silver-silver chloride as the reference electrode, deposition was carried out on an electrochemical workstation under an alternating electric field. Under positive current, the positive potential used was +0.7V with a single duration of 15s, and the negative potential used was -0.7V with a single duration of 10s. A total of 20 cycles of alternating positive and negative potentials were performed to obtain the initial electrode for immobilized nitrite reductase.

[0062] The initial electrode was removed and immersed in pure water for 20 minutes to remove residual phosphate buffer from the electrode pores and surface. After freeze-drying the electrode, it was stored at 4°C. By testing the change in protein concentration in the mixed solution before and after deposition, the actual loading rate (enzyme loading rate) on the electrode was found to be only 3.4%.

[0063] The nitrite reductase-reduced graphene oxide-carbon felt electrode synthesized in Comparative Example 1 was applied to the electrochemical detection of low-concentration nitrite. Cyclic voltammetry experiments were conducted within a voltage range of +0.2V to -1.0V at a scan rate of 50mV / s. The electrolyte consisted of 0.01M PBS and different concentrations of nitrite. The results showed... Figure 2 middle.

[0064] Depend on Figure 2 It can be seen that when the concentration of nitrite added was 420 μM, no significant reduction peak or increase in peak current appeared on the cyclic voltammetry curve of this comparative example, indicating that its sensitivity to low concentrations of nitrite is low.

[0065] Example 2

[0066] The preparation method of the nitrite reductase electrode may include the following steps:

[0067] Cell disruption and extraction were performed on commercially available Sheva strains, followed by protein purification to obtain cytochrome c nitrite reductase with a molecular weight of 45-55 kDa. The concentration of nitrite reductase was determined to be 0.9 mg / mL by BCA protein quantification method.

[0068] 50 mg of graphene oxide powder was mixed in a 0.1 mg / mL solution of ascorbic acid and ammonia, and hydrothermally treated at 100 °C for 2 h to obtain a reduced graphene oxide dispersion with a concentration of 0.5 mg / mL. The pH of the reduced graphene oxide dispersion was adjusted to 3.

[0069] Take 2.0*2.5cm 2Commercially available polyacrylonitrile carbon felt was pretreated with 68% nitric acid to remove surface impurities. The pretreated carbon felt was then hydrothermally reacted at 110°C for 5 hours in a solution of 100 mg / L cobalt nitrate and 50 mg / L urea to obtain carbon felt supported on a cobalt hydroxide precursor. Subsequently, using sodium hypophosphite as the phosphorus source, it was phosphated at 300°C for 2 hours to obtain cobalt phosphide-supported carbon felt with a cobalt phosphide loading of 0.4 mg / cm³. 2 .

[0070] 400 μL of nitrite reductase solution with a protein concentration of 0.9 mg / mL was added to 10 mL of acidic phosphate buffer solution and stirred at 4 °C until homogeneous. Then, 5 mL of acidic reduced graphene oxide dispersion with pH adjusted to 3 was added and stirred at room temperature for 30 minutes to obtain a mixed solution of nitrite reductase-reduced graphene oxide.

[0071] Using a mixed solution as the electrolyte, a carbon felt electrode loaded with cobalt phosphide as the working electrode, a platinum sheet as the counter electrode, and silver-silver chloride as the reference electrode, deposition was carried out on an electrochemical workstation under an alternating electric field. Under positive current, the positive potential used was +0.7V with a single duration of 15s, and the negative potential used was -0.7V with a single duration of 10s. A total of 20 cycles of alternating positive and negative potentials were performed to obtain an electrode immobilized with nitrite reductase.

[0072] The initial electrode was removed and immersed in pure water for 20 minutes to remove residual phosphate buffer from the electrode pores and surface. After freeze-drying the electrode, it was stored at 4°C. By testing the change in protein concentration in the mixed solution before and after deposition, the actual loading rate (enzyme loading rate) on the electrode was found to be 42%.

[0073] Example 3

[0074] The preparation method of the nitrite reductase electrode may include the following steps:

[0075] Cell disruption and extraction were performed on commercially available Sheva strains, followed by protein purification to obtain cytochrome c nitrite reductase with a molecular weight of 45-55 kDa. The concentration of nitrite reductase was determined to be 0.9 mg / mL by BCA protein quantification method.

[0076] 50 mg of graphene oxide powder was mixed in a 0.1 mg / mL solution of ascorbic acid and ammonia, and hydrothermally treated at 100 °C for 2 h to obtain a reduced graphene oxide dispersion with a concentration of 0.5 mg / mL. The pH of the reduced graphene oxide dispersion was adjusted to 4.

[0077] Take 2.0*2.5cm 2Commercially available polyacrylonitrile carbon felt was pretreated with 68% nitric acid to remove surface impurities. The pretreated carbon felt was then placed in a solution of 100 mg / L ferric nitrate and 50 mg / L urea and reacted hydrothermally at 110°C for 5 hours to obtain carbon felt supported by ferric hydroxide precursor. Subsequently, using sodium hypophosphite as the phosphorus source, it was phosphated at 300°C for 2 hours to obtain carbon felt supported by ferric phosphide, with a ferric phosphide loading of 0.3 mg / cm³. 2 .

[0078] 400 μL of nitrite reductase solution with a protein concentration of 0.9 mg / mL was added to 10 mL of acidic phosphate buffer solution and stirred at 4 °C until homogeneous. Then, 5 mL of acidic reduced graphene oxide dispersion with pH adjusted to 4 was added and stirred at room temperature for 30 minutes to obtain a mixed solution of nitrite reductase-reduced graphene oxide.

[0079] Using a mixed solution as the electrolyte, a carbon felt electrode loaded with iron phosphide as the working electrode, a platinum sheet as the counter electrode, and silver-silver chloride as the reference electrode, deposition was carried out on an electrochemical workstation under an alternating electric field. Under positive current, the positive potential used was +0.7V with a single duration of 15s, and the negative potential used was -0.7V with a single duration of 10s. A total of 20 cycles of alternating positive and negative potentials were performed to obtain an electrode immobilized with nitrite reductase.

[0080] The initial electrode was removed and immersed in pure water for 20 minutes to remove residual phosphate buffer from the electrode pores and surface. After freeze-drying the electrode, it was stored at 4°C. By testing the change in protein concentration in the mixed solution before and after deposition, the actual loading rate (enzyme loading rate) on the electrode was found to be 53%.

[0081] Performance testing

[0082] Detection Example 1

[0083] 400 μL of nitrite reductase solution with a protein concentration of 0.9 mg / mL was added to 10 mL of phosphate buffer solution and stirred at 4 °C until homogeneous. Then, 5 mL of reduced graphene oxide dispersion was added and stirred at room temperature for 30 minutes to obtain a mixed solution of nitrite reductase-reduced graphene oxide.

[0084] Using a mixed solution as the electrolyte, a 2.0*2.5cm... 2A cobalt phosphide-loaded carbon felt electrode was used as the working electrode, a platinum sheet as the counter electrode, and silver-silver chloride as the reference electrode. Deposition was carried out on an electrochemical workstation under an alternating electric field. Under positive current, the positive potential used was +0.7V with a single duration of 15s, and the negative potential used was -0.7V with a single duration of 10s. A total of 20 cycles of alternating positive and negative potentials were performed to obtain an electrode immobilized with nitrite reductase.

[0085] A nitrite reductase electrode was used as the cathode, and a platinum sheet electrode was used as the anode. 35 mL of nitrite with initial concentrations of 1, 2, and 3 mM was added to the electrolyte. The electrolyte was then subjected to an electrode at 1 mA cm⁻¹. -2 Enzyme electrocatalytic reduction experiments were conducted under low current density conditions, and the results showed... Figure 3 middle.

[0086] Depend on Figure 3 The results show that as the catalytic time increases from 15 minutes to 180 minutes, the treatment efficiency continuously improves, and the conversion rate of nitrate in the solution also increases accordingly, with the final nitrite conversion rates reaching 96.5%, 94.0%, and 84.6%, respectively. Based on the change in nitrite concentration, the reaction rate and efficiency at this point are mainly limited by the electron supply. It can be predicted that, with sufficient electrons provided by the external circuit, increasing the current density will further enhance the catalytic conversion effect of nitrite.

[0087] Detection Example 2

[0088] 400 μL of nitrite reductase solution with a protein concentration of 0.9 mg / mL was added to 10 mL of phosphate buffer solution and stirred at 4 °C until homogeneous. Then, 5 mL of reduced graphene oxide dispersion was added and stirred at room temperature for 30 minutes to obtain a mixed solution of nitrite reductase-reduced graphene oxide.

[0089] Using a mixed solution as the electrolyte, a 2.0*2.5cm... 2 A cobalt phosphide-loaded carbon felt electrode was used as the working electrode, a platinum sheet as the counter electrode, and silver-silver chloride as the reference electrode. Deposition was carried out on an electrochemical workstation under an alternating electric field. Under positive current, the positive potential used was +0.7V with a single duration of 15s, and the negative potential used was -0.7V with a single duration of 10s. A total of 20 cycles of alternating positive and negative potentials were performed to obtain an electrode immobilized with nitrite reductase.

[0090] A nitrite reductase electrode was used as the cathode, and a platinum sheet electrode was used as the anode. 35 mL of nitrite with an initial concentration of 1 mM was added to the electrolyte, and the electrolyte was subjected to an electrode at 1 mA cm⁻¹. -2 Enzyme electrocatalytic reduction experiments were conducted under low current density conditions. Figure 4The results show that as the catalytic time increases from 15 minutes to 180 minutes, the processing efficiency continuously improves, and the conversion rate of nitrite in the solution also increases accordingly. The final nitrite conversion rate reaches 96%, indicating that most of the nitrite substrate in the solution is efficiently converted by the nitrite reductase electrode.

[0091] Detection Example 3

[0092] Take 2.0*2.5cm 2 Commercially available polyacrylonitrile carbon felt is pretreated with 68% nitric acid to remove surface impurities, resulting in pretreated carbon felt.

[0093] Pretreated carbon felt was used as the cathode, and a platinum sheet electrode was used as the anode. 35 mL of nitrite with an initial concentration of 1 mM was added to the electrolyte, and the electrolyte was subjected to an electrode at 1 mA cm⁻¹. -2 Enzyme electrocatalytic reduction experiments were conducted under low current density conditions. Figure 4 The results show that as the catalytic time increases from 15 minutes to 180 minutes, the treatment efficiency continuously improves, and the conversion rate of nitrite in the solution also increases accordingly. The final nitrite conversion rate reaches 58.5%, indicating that only a portion of the nitrite in the solution is completely converted, the nitrite residue is large, and the catalytic effect is poor.

[0094] Detection Example 4

[0095] Take 2.0*2.5cm 2 Commercially available polyacrylonitrile carbon felt was pretreated with 68% nitric acid to remove surface impurities. The pretreated carbon felt was then placed in a solution of 100 mg / L cobalt nitrate and 50 mg / L urea and reacted hydrothermally at 110°C for 5 h to obtain carbon felt supported by cobalt hydroxide precursor. Subsequently, using sodium hypophosphite as the phosphorus source, it was phosphated at 300°C for 2 h to obtain carbon felt supported by cobalt phosphide.

[0096] A cobalt phosphide-supported carbon felt was used as the cathode, and a platinum sheet electrode was used as the anode. 35 mL of nitrite with an initial concentration of 1 mM was added to the electrolyte, and the electrolyte was subjected to an electrode at 1 mA cm⁻¹. -2 Enzyme electrocatalytic reduction experiments were conducted under low current density conditions. Figure 4 The results show that as the catalytic time increases from 15 minutes to 180 minutes, the treatment efficiency continuously improves, and the conversion rate of nitrite in the solution also increases accordingly. The final nitrite conversion rate reaches 80.9%, and its catalytic effect is improved compared with carbon felt. However, the catalytic rate slows down significantly in the later stage of the reaction, and there is still nitrite residue in the solution.

[0097] Case 5

[0098] Without adding nitrite reductase, 5 mL of reduced graphene oxide dispersion was added to 10 mL of phosphate buffer solution and stirred at room temperature for 30 minutes to obtain a reduced graphene oxide solution.

[0099] Take 2.0*2.5cm 2 Commercially available polyacrylonitrile carbon felt was pretreated with 68% nitric acid to remove surface impurities. The pretreated carbon felt was then placed in a solution of 100 mg / L cobalt nitrate and 50 mg / L urea and reacted hydrothermally at 110°C for 5 h to obtain carbon felt supported by cobalt hydroxide precursor. Subsequently, using sodium hypophosphite as the phosphorus source, it was phosphated at 300°C for 2 h to obtain carbon felt supported by cobalt phosphide.

[0100] Using the above-mentioned reduced graphene oxide mixed solution as the electrolyte, a carbon felt electrode loaded with cobalt phosphide as the working electrode, a platinum sheet as the counter electrode, and silver-silver chloride as the reference electrode, deposition was carried out on an electrochemical workstation under an alternating electric field. Under positive current, the positive potential used was +0.7V, with a single duration of 15s, and the negative potential used was -0.7V, with a single duration of 10s. A total of 20 cycles of alternating positive and negative potentials were performed to obtain the reduced graphene oxide electrode.

[0101] The deposited reduced graphene oxide-cobalt phosphide electrode was removed and immersed in pure water for 20 minutes to remove the electrode pores and residual phosphate buffer on the surface. The electrode was then freeze-dried and stored at 4°C.

[0102] A reduced graphene oxide-cobalt phosphide electrode was used as the cathode, and a platinum sheet electrode was used as the anode. 35 mL of 1 mM nitrite was added to the electrolyte, and the electrolyte was subjected to an electrode at 1 mA cm⁻¹. -2 Enzyme electrocatalytic reduction experiments were conducted under low current density conditions. Figure 4 The results show that as the catalytic time increases from 15 minutes to 180 minutes, the treatment efficiency continuously improves, and the conversion rate of nitrite in the solution also increases accordingly. The final nitrite conversion rate reaches 79.2%. In the later stage of the reaction, the catalytic rate slows down significantly, and there is still nitrite residue in the solution.

[0103] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A nitrite reductase electrode, characterized in that, It includes a base layer and alternating layers of n electron transport layers and n enzyme activity layers located on the base layer, where n is in the range of 2 to 30, wherein the base layer includes a carbon-based electrode loaded with transition metal phosphides, the electron transport layer includes reduced graphene oxide, the enzyme activity layer includes nitrite reductase, and the transition metal phosphides are cobalt phosphide or iron phosphide. In this process, under an alternating electric field, reduced graphene oxide and nitrite reductase in a mixed solution are sequentially electrodeposited onto the carbon-based electrode to form the nitrite reductase electrode.

2. The nitrite reductase electrode according to claim 1, characterized in that, The nitrite reductase electrode comprises a base layer and alternating layers of n electron transport layers and n-1 enzyme activity layers located above the base layer.

3. The nitrite reductase electrode according to claim 1, characterized in that, The loading rate of nitrite reductase on the carbon-based electrode is 10%~70%.

4. The nitrite reductase electrode according to claim 1, characterized in that, The loading of transition metal phosphides on the carbon-based electrode is 0.2~1.5 mg / cm³. 2 .

5. The nitrite reductase electrode according to any one of claims 1-4, characterized in that, The nitrite reductase is selected from any one of copper nitrite reductase, cytochrome cd1 nitrite reductase, polyheme c nitrite reductase, and ferrooxidase-dependent nitrite reductase; the reduced graphene oxide has a zeta potential in the range of -2 to -10 mV.

6. The nitrite reductase electrode according to any one of claims 1-4, characterized in that, The nitrite reductase is a cytochrome c type nitrite reductase with a zeta potential in the range of 2 to 10 mV.

7. The nitrite reductase electrode according to any one of claims 1-4, characterized in that, The carbon-based electrode is selected from one of glassy carbon electrode, graphite electrode, carbon paper, carbon cloth, and carbon felt.

8. The nitrite reductase electrode according to claim 7, characterized in that, The carbon felt is selected from one of polyacrylonitrile-based carbon felt, viscose-based carbon felt, pitch-based carbon felt, and wood fiber-based carbon felt.

9. A method for preparing a nitrite reductase electrode according to any one of claims 1-8, characterized in that, The method involves applying an alternating electric field for 2 to 30 cycles to sequentially electrodeposit reduced graphene oxide and nitrite reductase in a mixed solution onto a carbon-based electrode loaded with transition metal phosphides, thereby forming the nitrite reductase electrode with the nitrite reductase immobilized.

10. The method according to claim 9, characterized in that, The mixed solution is formed by stirring 200-800 μL of nitrite reductase solution with 10 mL of phosphate buffer solution, and then adding 5 mL of reduced graphene oxide dispersion and stirring.

11. The method according to claim 10, characterized in that, The nitrite reductase solution is prepared by dissolving 0.1 to 1.0 mg / mL of nitrite reductase in a buffer solution with a pH range of 6.5 to 7.

12. The method according to claim 10, characterized in that, The reduced graphene oxide dispersion is obtained by mixing graphene oxide powder in 0.1~1.0 mg / mL ascorbic acid and ammonia water, and then hydrothermally treating it at 90~120℃ for 1~5 h to obtain a reduced graphene oxide dispersion with a concentration of 0.5~1.0 mg / mL. The pH of the reduced graphene oxide dispersion is adjusted to 3~9.

13. The method according to claim 10, characterized in that, The total volume of the mixed solution is 20-40 mL.

14. The method according to claim 9, characterized in that, Applying an alternating electric field involves using the mixed solution as the electrolyte, a carbon-based electrode loaded with transition metal phosphides as the working electrode, a platinum sheet as the counter electrode, and silver-silver chloride as the reference electrode. Positive and negative potentials are applied alternately for a total of 2 to 30 cycles to sequentially deposit reduced graphene oxide and nitrite reductase onto the working electrode to form an initial electrode. The initial electrode is then freeze-dried to obtain the nitrite reductase electrode. The conditions for the alternating electric field include a positive potential of +0.7 to +1.0 V and a single duration of 10 to 20 s; The negative potential is -0.5 to -0.8 V, and the duration of a single event is 10 to 15 seconds.

15. The method according to claim 14, characterized in that, The last applied alternating electric field was at a positive potential.

16. An electrochemical system for detecting nitrite content in water, characterized in that, The electrochemical system includes a nitrite reductase electrode as a cathode according to any one of claims 1-8.

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