Working Electrode of CuS / Cu2S@PSS Self-Assembled Nanomaterial Bioelectrochemical Sensor: Preparation Method and Application

Abstract

This invention discloses a working electrode for a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor, its preparation method, and its application. The invention utilizes sulfur-containing bacterial broth generated by SRB metabolism as a sulfur source and reaction medium. Uniformly monodisperse CuS / Cu2S nanocomposite materials are in-situ self-assembled and deposited on the surface of a flexible carbon material modified with the conductive polymer poly(4-styrenesulfonic acid), constructing a microbial sensing material with CuS / Cu2S@PSS as the core material. Based on the highly selective and specific chemisorption of methanogenic enzymes by CuS / Cu2S and the excellent conductivity of PSS, non-destructive detection of the methanogenic enzyme F420 is achieved. The methanogenic concentration is calculated using a standard curve showing the relationship between the enzyme concentration and the electrochemical reduction current. The electrochemical sensor of this invention possesses the core characteristics of high specificity, fast response speed, and high detection accuracy.

Description

Technical Field

[0001] This invention relates to the field of microbial electrochemical sensor technology, and in particular to a CuS / Cu2S@PSS flexible electrochemical sensor constructed in situ based on sulfides generated by SRB metabolism, and its application in the quantitative detection of methanogenic bacteria, which are electroactive corrosive microorganisms in oil and gas fields. Background Technology

[0002] With the continuous expansion of oil and gas field development, corrosion problems of downhole tubing, surface pipelines, and equipment are becoming increasingly prominent. Corrosion-induced issues such as gas acidification, pipeline perforation, and natural gas leaks lead to decreased production and quality, resulting in severe economic losses, environmental pollution, and threats to production safety. Corrosion caused primarily by electroactive sulfate-reducing bacteria (SRB) and methanogenic bacteria is particularly severe. Compared to SRB, methanogenic bacteria are more corrosive to metals, and the coexistence of methanogenic bacteria and SRB results in a much higher corrosion rate for carbon steel than a single bacterium. Rapid and accurate detection of corrosive electroactive microorganisms provides crucial protection for the safe and stable operation of offshore oilfield pipelines and is a key pre-emptive measure to ensure oil and gas field production, environmental protection, and economic benefits.

[0003] Among existing microbial detection technologies, culture methods are accurate but time-consuming, microscopy is rapid but cannot distinguish between live and dead bacteria; immunoassays are rapid but have poor reproducibility, and fluorescence PCR is accurate and rapid but complex and costly. Electrochemical sensors are widely used in biochemical detection due to their advantages such as rapid response, ease of operation, low cost, and real-time detection. However, currently reported electrochemical sensors are mainly based on the redox electrochemical signals of catalytic target analytes. These sensors are susceptible to the accumulation of catalytic products on the sensor surface, have poor environmental adaptability, and lack selectivity. Summary of the Invention

[0004] To address the problems of time-consuming and inefficient traditional culture methods, and the high cost and inability to distinguish between living and dead cells in molecular biology methods, this invention provides a working electrode for a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor, along with its preparation method and applications. This invention utilizes metal nanomaterials obtained from microbial metabolites, which are then combined with conductive polymers to prepare a bioelectrochemical sensor. This sensor enables rapid, highly selective, and highly sensitive detection of the abundance of living methanogens, providing a new technical approach for the rapid detection of methanogens in environments such as oil and gas pipelines and produced water from oil and gas wells.

[0005] In a first aspect, the present invention provides a method for preparing a working electrode of a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor, which is achieved by the following technical solution.

[0006] A method for preparing the working electrode of a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor includes the following steps: S1. Culture sulfate-reducing bacteria to OD 600 =0.240 - 0.260, centrifuge to remove bacterial cells, and retain the culture supernatant rich in sulfide metabolites; S2. A poly(4-styrene sulfonic acid) aqueous solution is uniformly drop-coated onto the surface of the pretreated carbon paper electrode. After the solvent is evaporated, a thermosetting treatment is performed to obtain a PSS-modified flexible carbon paper material. S3. Immerse the flexible carbon paper material obtained in step S2 into the culture supernatant obtained in step S1, and add Cu dropwise. 2+ The solution was used to obtain CuS / Cu2S@PSS modified carbon paper material, which was then naturally dried to obtain the working electrode.

[0007] By adopting the above technical solution, flexible carbon paper is immersed in PSS solution, and a PSS film is formed on the surface of carbon paper by electrostatic adsorption using the negative charge characteristics of PSS, thus obtaining PSS modified carbon paper material.

[0008] In the normal metabolic activities of SRB, sulfate ions (SO4) 2- It is sequentially reduced to sulfides via the dissimilatory sulfate reduction metabolic pathway. The resulting sulfides undergo an ionization equilibrium reaction in aqueous solution, forming a unique sulfur-containing mixture (H₂S, HS₂). - S 2- When exogenous divalent copper ions (Cu) are introduced into the bacterial culture after SRB removal... 2+ When Cu 2+ The sulfides produced by metabolism undergo an in-situ precipitation reaction, initially forming thermodynamically stable copper sulfide (CuS) nanocrystals. Due to S... 2- Exhibiting strong reducing power, under strictly anaerobic conditions, some of the formed CuS nanoparticles undergo further reduction transformation to generate the low-valence cuprous sulfide (Cu₂S) phase. This dual-phase nanomaterial, through electrostatic interactions, van der Waals forces, and the mediation of biomolecules (such as extracellular polysaccharides and proteins), ultimately forms a CuS / Cu₂S NPs binary composite nanomaterial system with heterogeneous structure. The generated CuS / Cu₂S NPs self-assemble and monodisperse on the surface of the PSS film, significantly increasing the density of electrochemically active sites.

[0009] Furthermore, in step S1, the sulfate-reducing bacteria culture medium contains 1 g / L yeast extract, 1 g / L ammonium chloride, 10 mL / L sodium lactate, 0.5 g / L potassium dihydrogen phosphate, 0.1 g / L calcium chloride dihydrate, and 0.05 mol / L sodium sulfate, with a pH of 7.0 ± 0.1.

[0010] Furthermore, in step S2, the concentration of the poly(4-styrenesulfonic acid) aqueous solution is 0.5-2 mg / mL, preferably 1 mg / mL.

[0011] Furthermore, in step S2, the solvent is slowly evaporated at 60-65°C; and a thermosetting treatment is performed under vacuum conditions at 80-85°C.

[0012] Furthermore, in step S3, Cu 2+ The solution may be selected from one or more of CuCl2, Cu(NO3)2, or CuSO4; Cu 2+ The concentration of the solution is 0.001-0.002 mol / L.

[0013] Furthermore, the loading of CuS / Cu2S NPs in the working electrode was controlled at 1.0~1.5 mg / cm³. 2 .

[0014] Secondly, the present invention provides a working electrode for a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor, which is achieved by the following technical solution.

[0015] A working electrode for a bioelectrochemical sensor made of CuS / Cu2S@PSS self-assembled nanomaterials prepared by the above method.

[0016] Thirdly, the present invention provides a microbial electrochemical sensor, which is achieved by the following technical solution.

[0017] A microbial electrochemical sensor is disclosed, wherein the working electrode of the electrochemical sensor is selected from the above-mentioned CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor, the counter electrode is a platinum mesh electrode, the reference electrode is an Ag / AgCl electrode, and the electrochemical performance is tested by differential pulse voltammetry (DPV).

[0018] Fourthly, the present invention provides an application of a microbial electrochemical sensor, which is achieved by the following technical solution.

[0019] Application of the above-mentioned microbial electrochemical sensor in the quantitative detection of methanogens.

[0020] Fifthly, the present invention provides a method for quantitative detection of methanogens, which is achieved by the following technical solution.

[0021] A method for quantitative detection of methanogens includes the following steps: S1. Water sample collection and pretreatment: Collect water samples to be tested, enrich microbial cells in the water samples by centrifugation or membrane filtration, and resuspend the collected bacterial precipitate or filter membrane residue with PBS buffer at pH 7.0. S2. Methanogen cell lysis and coenzyme F420 extraction: The bacterial resuspension obtained in step S1 was subjected to high-temperature cooking under strict anaerobic or inert gas protection to lyse the methanogen cells and release intracellular coenzyme F420; the lysate was centrifuged and the supernatant was collected to obtain the coenzyme F420 extract to be tested; S3. Electrochemical detection: Using the microbial electrochemical sensor described in claim 8, with PBS buffer solution at pH 7.0 as the electrolyte, differential pulse voltammetry is used to detect the coenzyme F420 extract obtained in step S2, and the corresponding current response value is obtained. S4. Calculation of methanogen concentration: Substitute the current response value obtained in step S3 into the correspondence between the current response value and the methanogen cell concentration to calculate the methanogen cell concentration in the water body to be tested, thereby realizing the quantitative detection of methanogens in the water body.

[0022] Furthermore, the method for obtaining the correspondence between the current response value and the concentration of methanogenic bacteria cells is as follows: S1. Culture of standard strains of methanogens: Methanogens were cultured in pure medium using Hungate medium under strict anaerobic conditions at a temperature of 35±2 ℃ for 20-40 days until the cell concentration reached a stable phase. S2. Quantitative microscopic counting of methanogenic bacteria cell density: The cells of cultured pure methanogenic bacteria were counted under a microscope using the hemocytometer method, and serial dilutions were performed. S3. Extraction and purification of coenzyme F420: The cell walls of methanogenic bacteria were destroyed by high-temperature cooking, and coenzyme F420 was extracted from the cells. After centrifugation, a coenzyme F420 solution was obtained. S4. Establishment of standard curve: Using the above-mentioned microbial electrochemical sensor, the electrolyte was PBS buffer solution with pH 7.0. The electrochemical response of different concentrations of coenzyme F420 was determined by DPV method, and the linear relationship between current density and coenzyme F420 concentration was established. S5. Establishment of a standard curve for quantitative detection of methanogens: The relationship between the concentration of cultured pure methanogens and the concentration of coenzyme was obtained by plate counting method, and the linear relationship between current density and coenzyme F420 concentration was established by DPV test. Finally, the quantitative relationship between methanogen concentration and current density was determined.

[0023] By adopting the above technical solution, this invention detects the changes in electrochemical signals generated by the specific chemical adsorption of the characteristic coenzyme F420 of methanogens by CuS / Cu2S NPs. By monitoring the change in reduction peak current before and after F420 adsorption by the sensor using differential pulse voltammetry (DPV), the relationship between coenzyme F420 concentration and current density is obtained. Combined with the relationship between F420 concentration and the number of active methanogens, a quantitative relationship between current density and methanogen concentration can be obtained.

[0024] This application has the following beneficial effects: This invention utilizes the reducing sulfide medium generated by the growth and metabolism of SRB (Syntrophic Biota synergisticis) as both a sulfur source and reaction medium. CuS / Cu2SNPs heterojunction materials are synthesized in situ via self-assembly on the surface of a flexible carbon material modified with conductive polymer PSS, overcoming the limitations of complex processes in traditional chemical synthesis. Modification with conductive polymer PSS prevents nanoparticle aggregation, increases electrochemical active sites, and significantly improves sensor sensitivity. Based on the unique chemisorption relationship between CuS / Cu2S and the characteristic coenzyme F420 of methanogens, specific recognition and activation of F420 are achieved. The rapid quantitative detection of methanogens overcomes the limitations of traditional culture methods, such as long culture time (20-40 days), inability of microscopy to distinguish between live and dead bacteria, and high cost of molecular biology methods. This sensor reduces the detection time to the hour level and has the advantages of strong specificity, short response time, simple operation, and low cost. Furthermore, the natural symbiotic relationship between SRB and methanogens gives the system good biocompatibility. This provides a new technical approach for the rapid detection and monitoring of methanogens, corrosive microorganisms in oil and gas field produced water and oil pipelines, and has important application value for establishing a microbial corrosion early warning and response system. Attached Figure Description

[0025] Figure 1 These are characterization images of CuS / Cu2S @PSS of the present invention (where A is a SEM image and B is an XRD pattern). Figure 2 The figures show the DPV curves (A) of CuS / Cu2S @PSS under different F420 concentrations and the linear relationship between current density and F420 concentration (B) of this invention. Figure 3 This is a taxonomic classification and genus attribution diagram of methanogens in water samples from a typical oil and gas field pipeline corrosion area according to the present invention. Figure 4 The present invention provides the DPV curves (A) of CuS / Cu2S@PSS under different concentrations of methanogens and the linear relationship between current density and methanogen count (B). Figure 5 This is a diagram showing the results of a spike recovery experiment on an actual water sample according to the present invention. Detailed Implementation

[0026] This invention utilizes a sulfur-containing bacterial solution produced by the metabolism of SRB (similar to methanogens) as a sulfur source and reaction medium. A uniformly monodisperse CuS / Cu2S NPs composite material is synthesized in situ on the surface of a flexible carbon material modified with conductive polymer poly(4-styrene sulfonic acid) (PSS). Leveraging the specific chemisorption between CuS / Cu2S and the characteristic enzyme F420 of methanogens, as well as the excellent conductivity of PSS, a CuS / Cu2S@PSS microbial sensing material is constructed for the rapid detection of methanogens. This sensor features a simple fabrication process, mild reaction conditions, avoids complex chemical modifications, and allows for large-scale production. The constructed electrochemical sensor exhibits high specificity, fast response (within 30 minutes), and high detection accuracy, making it suitable for rapid methanogenesis detection in environments such as oil and gas pipelines and produced water from oil and gas wells.

[0027] The present patent application will be further described below with reference to the accompanying drawings and embodiments.

[0028] The SRB bacteria used in the following embodiments of the present invention are derived from the strain Desulfovibrio (a type of Vibrio spp.) isolated and cultured from oilfield produced water. This strain is described in Living therapeutics of nonpathogenic bacteria as biosynthesis factory and active carriers for enhancing tumor-targeted therapy. Nature Communications (Nature Portfolio). Mengna Dong, Xinhui Yang, Wenqian Zhang, Yuzhi Qiu, Peng Song, Hongfang Liu, Yajiang Yang, Xiangliang Yang, Qin Wang. Vol.16 (1): 6532. DOI: 10.1038 / s41467-025-61675-4; The poly(4-styrenesulfonic acid) used in the following embodiments of the present invention has CAS number 28210-41-5, was purchased from Aladdin Reagent Company, catalog number: P169249-100g, purity: Mw~70000, 30wt.%inH2O.

[0029] 1. Bacterial culture Based on a total culture medium volume of 1 L, accurately weigh each component of the raw materials, including yeast extract (1 g / L), ammonium chloride (1 g / L), sodium lactate (10 mL / L), potassium dihydrogen phosphate (0.5 g / L), calcium chloride dihydrate (0.1 g / L), and sodium sulfate (0.05 mol / L). Dissolve the weighed raw materials in 1 L of ultrapure water and stir continuously on a magnetic stirrer until all components are completely dissolved to obtain a homogeneous culture medium solution. Transfer the culture medium to culture flasks and sterilize using a high-temperature autoclave (121 ℃, 20 min). After sterilization, cool the culture medium to room temperature, inoculate 1 mL of 3-day-old SRB bacterial suspension per 80 mL of culture medium, and incubate in a 37 ℃ anaerobic incubator for 72 h.

[0030] 2. Preparation of the working electrode: After culturing the bacteria for 72 h, the bacterial cells and supernatant were separated by centrifugation (6000 rpm, 8 min), and the supernatant rich in sulfides was collected. The carbon paper electrode was cut to a standard size of 1 cm × 1 cm and ultrasonically cleaned sequentially in acetone, anhydrous ethanol, and deionized water to remove surface oil and impurities. The cleaned electrode was dried in a vacuum oven at 60 ℃ to completely remove residual solvent. 150 μL of a 1 mg / mL PSS aqueous solution was uniformly drop-coated onto the surface of the pretreated carbon paper electrode (ensuring the droplet completely covers the effective working area). The electrode was then placed on a 60 ℃ heating platform to slowly evaporate the solvent. After forming a uniform polymer film, it was transferred to an 80 ℃ vacuum oven for thermosetting to completely remove residual solvent and enhance film adhesion, resulting in a PSS-modified flexible carbon paper material. This carbon paper material was then immersed in the bacterial solution, and 1 mL of 0.002 mol / L Cu was quantitatively added to the resulting supernatant. 2+ Solution, through Cu 2+ A chemical reaction with sulfides induces the in-situ deposition and self-assembly of CuS / Cu2S NPs on the surface of PSS-modified carbon paper. The CuS / Cu2S@PSS-modified carbon paper is then removed, air-dried, and used as the working electrode. The final loading of CuS / Cu2S NPs in the composite electrode is controlled at 1.0 mg / cm³. 2 .

[0031] 3. Characterization of CuS / Cu2S@PSS The microstructure and crystal structure of the CuS / Cu2S@PSS composite material were characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD). Figure 1As shown, the SEM images indicate that CuS / Cu2S NPs are uniformly monodisperse on the PSS substrate surface. The characteristic diffraction peaks in the XRD diffraction pattern are consistent with the standard cards for CuS and Cu2S, confirming the synthesis of the CuS / Cu2S NPs heterojunction material.

[0032] 4. Characterization of electrochemical behavior A microbial electrochemical sensor was constructed, wherein the working electrode was the CuS / Cu2S@PSS modified carbon paper material described above, and the counter electrode was 1 cm². 2 A platinum mesh electrode was used, with an Ag / AgCl electrode as the reference electrode. The electrochemical performance was tested using differential pulse voltammetry (DPV), with the following parameters: scan potential range of -1V to 0V, scan rate of 50 mV / s, and 1 scan.

[0033] To establish the current density (mA / cm) 2 A linear standard curve was generated between the concentration of coenzyme F420 and the concentration of coenzyme F420 (nmol / L). The electrochemical response of the electrode to different concentrations of coenzyme F420 was determined using the DPV method. Figure 2 As shown, the sensor's detection limit is 0.01 nmol / L. The current density decreases with increasing F420 concentration, demonstrating that CuS / Cu2S exhibits specific chemisorption of F420. Linear fitting was performed on the relationship between F420 concentration and current density; when the F420 concentration is 0.01-400 nmol / L, The coefficient of determination of the equation is 0.992, which indicates that there is a good linear relationship between F420 concentration and current density. The sensor can be used to accurately detect F420 concentration.

[0034] 5. Cultivate pure cultures of methanogens Microorganisms were isolated and cultured from water samples in typical oil and gas field pipeline corrosion areas using anaerobic culture techniques. The cultures were kept at a constant temperature of 37 °C in a hungarian environment and passaged periodically until sufficient bacterial cell mass was obtained. 16S rRNA gene sequencing analysis was performed on the expanded cultured methanogens, and phylogenetic identification was performed by comparing with the NCBI database to determine the taxonomic position and species affiliation of the strains. The results are as follows: Figure 3 As shown, this bacterium is classified as Methanococcus maripaludis.

[0035] The bacterial cell concentration was determined using a cell counting chamber method. Cells were counted directly under a microscope using a cell counting chamber. Each sample was counted three times, and the average value was taken as the final result. The final result was 3.7 × 10⁻⁶. 9 CFU / mL. The bacteria were then serially diluted with sterile PBS buffer (pH 7.2) to a concentration of 10⁻⁶ CFU / mL. 810 7 10 6 10 5 10 4 10 3 CFU / mL was used to obtain six different concentration gradients of bacterial suspensions. 80 mL of bacterial suspension was taken from each concentration gradient, followed by extraction and purification of coenzyme F420.

[0036] 6. Extraction and purification of coenzyme F420 Accurately transfer 80 mL of pure methanogenic bacterial culture medium, dilute with 160 mL of 0.9% (w / v) sterile physiological saline, and collect cells by centrifugation at 6000 rpm for 15 min at room temperature. Discard the supernatant, resuspend the bacterial pellet in 40 mL of sterile physiological saline, and repeat the centrifugation process for a second washing to remove residual culture medium components. Discard the supernatant, resuspend the washed bacterial pellet in 30 mL of 0.9% (w / v) sterile physiological saline, transfer to a 95 °C constant temperature water bath, and perform thermal lysis for 30 min under continuous mechanical stirring to disrupt the cell wall and release intracellular coenzyme F420, while ensuring uniform heating of the sample. After lysis, allow the lysis buffer to cool naturally to 25 °C at room temperature, and accurately record the final volume. Add isopropanol at a volume ratio of 2:1 to the lysis buffer for extraction, and stir to mix thoroughly. The mixture was centrifuged at 10,000 rpm for 8 min to achieve phase separation. The upper layer, which was bright yellow isopropanol (containing F420), was collected, transferred to a brown sample bottle, and stored at 4 °C in the dark for later use.

[0037] 7. Study on the detection performance of methanogens Differential pulse voltammetry was used to measure the coenzyme F420 in methanogenic bacterial extracts at different concentrations using a CuS / Cu2S@PSS modified electrode. Based on the known current density of the coenzyme extracted from methanogenic bacterial concentrations, a method was established for determining the cell concentration (10T) of the coenzyme. 3 ~10 8 A linear standard curve between the CFU / mL concentration and the corresponding coenzyme peak current density. This standard curve can be used as a calibration basis for quantitative analysis of the abundance of methanogens in actual environmental water samples. Figure 4 As shown, the equation of the standard curve is (y is the current density, x is the logarithm of the methanogen count, CFU / mL), linear regression correlation coefficient R 2 = 0.994, indicating that there is a good linear correlation between current density and methanogen count within the measured concentration range. The above standard curve can be used to quantitatively determine the concentration of methanogens in environmental samples.

[0038] 8. Performance Study of Actual Samples To evaluate the applicability of the established method in actual sample analysis, a parallel control analysis was conducted on actual water samples (sampled from East Lake, Wuhan) using electrochemical detection and qPCR technology. Spike recovery experiment results ( Figure 5 Table 1 shows that in 10 3 Up to 10 8 Within the CFU / mL concentration range, the recovery rate of the electrochemical method was 95.12%–104.88%, with an average recovery rate of 99.51% and relative deviations of less than 5%, demonstrating good accuracy and precision. The electrochemical detection results showed a high degree of consistency with the qPCR measurements, further confirming the reliability of this method in practical applications, and indicating that the interference of complex water sample matrices on detection performance is negligible.

[0039] Table 1 The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing the working electrode of a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor, characterized in that: Includes the following steps: S1. Culture sulfate-reducing bacteria to OD 600 =0.240 - 0.260, centrifuge to remove bacterial cells, and retain the culture supernatant rich in sulfide metabolites; S2. A poly(4-styrene sulfonic acid) aqueous solution is uniformly drop-coated onto the surface of the pretreated carbon paper electrode. After evaporating the solvent, a thermosetting treatment is performed to obtain a PSS-modified flexible carbon paper material. S3. Immerse the flexible carbon paper material obtained in step S2 into the culture supernatant obtained in step S1, and add Cu dropwise. 2+ The solution was used to obtain CuS / Cu2S@PSS modified carbon paper material, which was then naturally dried to obtain the working electrode.

2. The method for preparing the working electrode of a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor according to claim 1, characterized in that: In step S1, the sulfate-reducing bacteria culture medium contains 1 g / L yeast extract, 1 g / L ammonium chloride, 10 mL / L sodium lactate, 0.5 g / L potassium dihydrogen phosphate, 0.1 g / L calcium chloride dihydrate, and 0.05 mol / L sodium sulfate, with a pH of 7.0 ± 0.

1.

3. The method for preparing the working electrode of a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor according to claim 1, characterized in that: In step S2, the concentration of the poly(4-styrenesulfonic acid) aqueous solution is 0.5-2 mg / mL.

4. The method for preparing the working electrode of a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor according to claim 1, characterized in that: In step S2, the solvent is slowly evaporated at 60-65℃; and thermosetting is performed under vacuum at 80-85℃.

5. The method for preparing the working electrode of a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor according to claim 1, characterized in that: In step S3, Cu 2+ The solution may be selected from one or more of CuCl2, Cu(NO3)2, or CuSO4; Cu 2+ The concentration of the solution is 0.001-0.002 mol / L.

6. The method for preparing the working electrode of a CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor according to claim 1, characterized in that: The loading of CuS / Cu2S NPs in the working electrode was controlled at 1.0~1.5 mg / cm³. 2 .

7. A working electrode for a bioelectrochemical sensor made of CuS / Cu2S@PSS self-assembled nanomaterials prepared by any of the preparation methods described in claims 1-6.

8. A microbial electrochemical sensor, characterized in that: The working electrode in the electrochemical sensor is the CuS / Cu2S@PSS self-assembled nanomaterial bioelectrochemical sensor working electrode described in claim 7, the counter electrode is a platinum mesh electrode, the reference electrode is an Ag / AgCl electrode, and the electrochemical performance is tested using differential pulse voltammetry.

9. The application of the microbial electrochemical sensor of claim 8 in the quantitative detection of methanogens.

10. A method for quantitative detection of methanogens, characterized in that: Includes the following steps: S1. Water sample collection and pretreatment: Collect water samples to be tested, enrich microbial cells in the water samples by centrifugation or membrane filtration, and resuspend the collected bacterial precipitate or filter membrane residue with PBS buffer at pH 7.

0. S2. Methanogen cell lysis and coenzyme F420 extraction: The bacterial resuspension obtained in step S1 was subjected to high-temperature cooking under strict anaerobic or inert gas protection to lyse the methanogen cells and release intracellular coenzyme F420; the lysate was centrifuged and the supernatant was collected to obtain the coenzyme F420 extract to be tested; S3. Electrochemical detection: Using the microbial electrochemical sensor described in claim 8, with PBS buffer solution at pH 7.0 as the electrolyte, differential pulse voltammetry is used to detect the coenzyme F420 extract obtained in step S2, and the corresponding current response value is obtained. S4. Calculation of methanogen concentration: Substitute the current response value obtained in step S3 into the correspondence between the current response value and the methanogen cell concentration to calculate the methanogen cell concentration in the water body to be tested, thereby realizing the quantitative detection of methanogens in the water body.

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