A rapid detection method of methanogens in water body

Abstract

The present application relates to a kind of methanogen rapid detection method in water body, belong to the field of microbiological detection technology.Based on the coenzyme of methanogen, different concentrations of methanogen standard sample are counted by blood cell plate to determine the methanogen concentration of each standard sample;Then the standard sample methanogen is heated at a certain temperature to separate and purify coenzyme, then high-speed centrifugation is removed to leave supernatant, and the relationship between coenzyme concentration and current response signal is determined by electrochemical detection to determine the current response signal of coenzyme.According to the coenzyme concentration and its current response signal and the methanogen concentration of plate counting, the corresponding relationship of methanogen concentration and current response signal and linear regression equation are determined, and finally the method for rapidly detecting bacterial concentration by coenzyme detection is realized.The present application can solve the related problems of low sensitivity and long cycle in the detection of methanogen concentration in water body.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganism detection, and more particularly relates to a rapid detection method of methanogens in water bodies, and especially relates to a detection method of high sensitivity and rapid detection of the number of methanogens in river sludge, industrial wastewater and submarine sediments. BACKGROUND

[0002] The number of microorganisms in water bodies such as river sludge, industrial wastewater and submarine sediments is an important biological indicator for evaluating the cleanliness of water bodies. At present, the counting method of microorganisms in water bodies is usually that an operator collects water samples, takes the water samples back to the laboratory, and obtains bacterial colonies that can be directly observed by separation, culture and purification of bacteria, and then obtains the total amount of bacteria by using the way of plate counting to realize counting. The operation is very cumbersome, the culture period is long, and the technical level of the experimental personnel is required to be high. So far, no more than 10% of the microorganisms in the external environment can be separated, cultured and purified. The components, chemical conditions and physical conditions of the culture medium for different microorganisms are different. However, the traditional bacterial count method needs the preparation of culture medium, the long period of plate culture and colony counting, and a large amount of work and manpower, which limits the application of microorganism detection. Therefore, the real-time monitoring of microorganisms in water environment urgently needs to establish a new type of simple, rapid and sensitive quantitative detection method.

[0003] The direct counting method of bacteria is limited and has poor sensitivity. The enzymes contained in the bacteria generally present a positive correlation with the number of bacteria, so the detection of the specific enzymes of bacteria can realize the detection of the number of bacteria. Methanogens are a kind of archaea that can convert inorganic or organic compounds into methane and carbon dioxide by anaerobic fermentation. They contain specific enzymes such as coenzyme F 420 , coenzyme M and methyl coenzyme M. The specific electrode material can produce an electric signal by redox reaction of these specific enzymes, which can realize the quantitative detection of the enzymes. The concentration of coenzyme is linearly related to the current response signal within a certain range. Therefore, after extracting the coenzyme of methanogens and determining the coenzyme content by electrochemical oxidation technology, the bacterial content can be calculated. The whole process is completed within 30 minutes. The detection of specific enzymes of this kind of bacteria does not have the problem of influence of other bacteria, and does not need the culture process, which is simple in operation, high in sensitivity and short in period. It has been rapidly developed in the quantitative detection of microorganisms, and many countries have used it as an effective means for environmental health field supervision and detection. However, it is still in the initial stage in China. SUMMARY

[0004] The present application aims to improve the sensitivity of methanogen detection, and provides a detection method for quantitatively detecting bacteria based on specific enzymes (coenzymes) of methanogens, which has specificity, avoids interference of other bacteria in counting, significantly improves the sensitivity of detection, and shortens the time required for detection, thereby solving the technical problems of poor sensitivity and long period of methanogen detection in water environment.

[0005] According to the first aspect of the present application, a rapid detection method of methanogens in water bodies is provided, comprising the following steps:

[0006] (1) Plate counting of different concentrations of methanogen standard samples through a blood cell plate to determine the methanogen concentration of each standard sample;

[0007] (2) Heating and cracking of the different concentrations of methanogen standard samples obtained in step (1), centrifugation to obtain supernatant to separate coenzymes, the supernatant of the different concentrations of methanogen standard samples has the same pH, the coenzyme concentration in the different concentrations of methanogen samples is measured by a photometer to obtain a linear relationship between the plate counting methanogen concentration and the coenzyme concentration;

[0008] (3) Testing of the current response signals of different concentrations of coenzymes in the supernatant obtained in step (2) through a three-electrode system, the working electrode of the three-electrode system is obtained by dropping copper-based nanomaterials on a conductive material, and the linear relationship between the coenzyme concentration and the current response signal is determined;

[0009] (4) Establishing the linear relationship between the plate counting methanogen concentration and the current response intensity according to the linear relationship between the plate counting methanogen concentration and the coenzyme concentration obtained in step (2) and the linear relationship between the coenzyme concentration and the current response intensity obtained in step (3);

[0010] (5) Heating and cracking of the sample to be tested, centrifugation to obtain supernatant to separate coenzymes, and adjusting the pH to be the same as that of the supernatant of the standard sample in step (2), and then testing the current response signal of the supernatant through the three-electrode system in step (3), and calculating the methanogen concentration of the sample to be tested according to the linear relationship between the plate counting methanogen concentration and the current response intensity obtained in step (4).

[0011] Preferably, the copper-based nanomaterial is CuS nanomaterial, CuS nanomaterial, metal organic framework material of copper or copper alloy.

[0012] Preferably, the morphology of the CuS nanomaterial is star-shaped or flaky.

[0013] Preferably, in step (2), the temperature of the heating and cracking is 60-100℃.

[0014] Preferably, in step (2), the coenzyme is at least one of coenzyme F 420 , coenzyme M and methyl coenzyme M.

[0015] Preferably, in step (3), the counter electrode of the three-electrode system is a platinum sheet, the reference electrode is Ag and AgCl; a phosphate buffer solution is used as the electrolyte, and the current response signals corresponding to different concentrations of coenzyme are measured at a preset potential.

[0016] Preferably, the preset potential is 0.1-0.6 V.

[0017] Preferably, the concentration of the phosphate buffer solution is 0.01-0.05 M.

[0018] Preferably, in step (1), the concentration of the methanogen bacteria counted by the plate count method is 20-400 CFU / mL.

[0019] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages:

[0020] (1) The test substance used in the present application is a specific enzyme of methanogen bacteria, and the use of this enzyme can avoid the interference of other bacteria. In addition, the use of a specific catalyst also improves the specificity of the detection.

[0021] (2) The present application uses an electrochemical oxidation method to detect the content of the enzyme, and the prepared electrochemical sensor has low cost and high sensitivity. The detection concentration of the methanogen bacteria concentration by this method can reach 20 CFU / mL.

[0022] (3) The present application uses an electrochemical method for detection, which has fast response speed, and the whole process can be completed within 30 min, and the detection cycle is short.

[0023] (4) The present application detects the special enzyme contained in the bacteria by an electrochemical method, which does not need to culture the bacteria additionally, and the detection method is simple and has low operation difficulty, which greatly improves the work efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the current response step curve of methanogen bacteria of different concentrations at a potential of 0.1 V (vs. Ag / AgCl).

[0025] Figure 2 is Figure 1 is the linear relationship curve between the methanogen bacteria and the current response signal corresponding to different bacterial concentrations in the step curve.

[0026] Figure 3 is the anti-interference curve of CuS on other substances in the system.

[0027] Figure 4 The long-term stability curve of CuS for coenzyme detection.

[0028] Figure 5 The scanning electron microscope image of star-shaped CuS.

[0029] Figure 6 The scanning electron microscope image of flaky CuS. DETAILED DESCRIPTION

[0030] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not used to limit the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0031] The water body microorganism rapid detection method provided by the present application includes two parts. One is through standard bacterial sample dilution, plate counting and enzyme separation and extraction. The other is electrochemical detection of the coenzyme extracted from the bacteria using a catalyst.

[0032] The sensitivity of the present application can reach 20 CFU / mL, and the whole process can be completed within 30 minutes.

[0033] The present application relates to a water body microorganism rapid detection method. Methanogen standard samples are diluted into five standard solutions with different concentrations (such as 0.9wt% sodium chloride solution) in sodium chloride solution step by step, and the bacterial concentration is determined by plate counting. Further, the diluted bacteria solution with different concentrations is heated and lysed, then high-speed centrifugation is performed to remove impurities, and the coenzyme (such as coenzyme M) in the bacteria is extracted. Then, the catalyst (such as copper sulfide) is used to perform electrochemical titration on the extracted solution with different concentrations, and the change of the current response signal is observed. The linear relationship between different coenzyme concentrations and current signals is measured, and the linear relationship between the bacterial concentration and the current response signal can be determined through the relationship between the bacterial concentration obtained by plate counting method and the coenzyme extract concentration. Specifically, the following steps are included:

[0034] (1) Take 0.1-1mL of methanogen standard sample, and dilute it into multiple different concentrations (such as 1:10, 1:100, 1:1000, etc.) with 0.6-1wt% sodium chloride solution step by step, and store it in a 0-4℃ refrigerator for standby; the total amount of bacteria in the standard diluted bacteria solution with different concentrations is counted under a microscope by plate counting method, and the bacterial concentration is 20-400 CFU / mL;

[0035] (2) The diluted bacteria solution of different concentrations in step (1) is heated to crack (60-100℃) for a period of time (5-10 min) to extract coenzyme specific to methanogens (such as coenzyme F 420 , coenzyme M or methyl coenzyme M), and further remove other impurities by high-speed centrifugation at a speed of 8000-12000 r / min to separate and purify the coenzyme. The supernatant containing different concentrations of coenzyme is placed in a refrigerator at 0-4℃ for cold storage for later use.

[0036] (3) The coenzyme supernatant in step (2) is tested for current response signal under constant potential (such as 0.1V) by using a three-electrode system, in which a carbon-based material with drop-coated catalyst is used as the working electrode, a platinum sheet electrode is used as the counter electrode, an Ag / AgCl electrode is used as the reference electrode, and a PBS of a certain concentration is used as the electrolyte solution, to obtain the correlation between enzyme concentration and bacterial concentration.

[0037] (4) The linear relationship between the determined coenzyme concentration and the current response signal is obtained, where I e is the current value, and C e is the coenzyme concentration; and the bacterial concentration obtained by plate counting method is used to finally obtain the correlation between bacterial concentration and current response signal.

[0038] (5) The actual river bottom sludge is treated in the same way as step (2) to extract coenzyme, and the current response signal is obtained by testing through a three-electrode system. The correlation between bacterial concentration and current response signal is finally obtained according to the bacterial concentration obtained by plate counting method in step (4), and the concentration of methanogens in the test solution is calculated.

[0039] In some embodiments, the methanogens are Methanosarcina barkeri.

[0040] In some embodiments, the low-temperature storage in steps (1), (2), and (5) is storage in a refrigerator at 0-4℃.

[0041] In some embodiments, the heating cracking time in step (3) is 30-50 min.

[0042] In some embodiments, the heating cracking temperature in step (3) is 60-100℃.

[0043] In some embodiments, the catalyst in step (4) is a copper-based nanocatalyst.

[0044] In some embodiments, the PBS concentration in step (4) is 0.01-0.1M.

[0045] The application investigates the influence of the acid and alkali resistance of CuS nanoparticles for detecting coenzyme M, investigates the sensitivity change of the detection of the coenzyme under different pH conditions, and further investigates the influence of CuS with different morphologies on the sensitivity of the detection of coenzyme M.

[0046] The following is a specific embodiment

[0047] Example 1

[0048] Take 0.1 mL of standard sample of methanogen, dilute it into five different concentrations of methanogen standard solution using sodium chloride solution (0.9 wt%), and store it in a refrigerator at 0-4℃. Take 1 mL of the diluted sample solution after sufficient dilution, and spread it on a plate. Under a microscope, count the number of bacteria (20-400), and calculate the amount of methanogen in the five different concentrations of bacterial standard sample. Further, heat the five different concentrations of methanogen at 90℃ for 20 min by heating and cracking, to extract coenzyme (coenzyme M) in the bacteria, and then separate and purify the coenzyme M by using a high-speed centrifuge at a speed of 9500 r / min. Leave the supernatant containing different concentrations of coenzyme, and store it in a refrigerator at 0-4℃ for later use. Detect the coenzyme concentration by using a photometer, and obtain the relationship between the coenzyme concentration and the bacterial concentration as follows: C m = 3.724C e + 5.817, where C m is the bacterial concentration, the unit is CFU / mL, and C e is the coenzyme concentration, the unit is U / mL. Take 0.1 g of star-shaped CuSNPs as shown in Figure 5 , ultrasonically disperse it in 100 μL of 0.1% nafion aqueous solution, drop it on a glassy carbon electrode with a diameter of 3 mm, and test the current response signal intensity of different concentrations of coenzyme solution by using a three-electrode system with 0.01 M PBS (pH 7.4) as an electrolyte at a potential of 0.1 V (vs. Ag / AgCl). The relationship between the coenzyme concentration and the current response signal is as follows: I e = 4.211C e - 1.263, where I e is the current value, the unit is mA, and C e is the coenzyme concentration, the unit is U / mL. According to the relationship between the bacterial concentration and the enzyme concentration, the step curve of the bacterial concentration and the current value is obtained as shown in Figure 1 . The fitting calculation obtains the correlation between the bacterial concentration and the current signal as shown in Figure 2 , and the linear relationship between the bacteria and the current value is as follows: I e = 6.551C m - 0.148, where I e is the current value, the unit is mA, and Cm is the bacteria concentration, unit is CFU / mL.

[0049] Example 2

[0050] Take 0.1 mL of standard sample of methanogens, dilute it into five different concentrations of methanogen standard solution using sodium chloride solution (0.9 wt%), and store it in a refrigerator at 0-4°C for later use. Take 1 mL of the lowest concentration of the diluted sample and spread it on a blood cell plate. Under a microscope, observe and count the number of bacteria (20-400). Through the sample volume and dilution factor, the amount of methanogens in the five different concentrations of bacteria standard sample is calculated. Further, heat the five different concentrations of methanogens at 90°C for 20 min by heating lysis, and then separate and purify the coenzyme M (CoM) through a high-speed centrifuge at a speed of 9500 r / min. The supernatant containing different concentrations of coenzyme is stored in a refrigerator at 0-4°C for later use. The relationship between the coenzyme concentration and the bacteria concentration is obtained by detecting the coenzyme concentration through a photometer: C m = 2.115C e + 3.305, where C m is the bacteria concentration, unit is CFU / mL, and C e is the coenzyme concentration, unit is U / mL. Weigh 0.1 g of star-shaped CuS NPs and ultrasonically disperse it in 100 μL of 0.1% nafion aqueous solution, and drop it on a glassy carbon electrode with a diameter of 3 mm. Through a three-electrode system, use 0.1 M KOH (pH 13) as the electrolyte, and test the current response signal intensity of different concentrations of coenzyme solution at a potential of 0.1 V (vs. Ag / AgCl). The relationship between the coenzyme concentration and the current response signal is obtained: I e = 2.393C e - 0.717, where I e is the current value, unit is mA, and C e is the coenzyme concentration, unit is U / mL. The correlation between the bacteria concentration and the current signal is calculated: I e = 3.7C m - 0.084, where I e is the current value, unit is mA, and C m is the bacteria concentration, unit is CFU / mL.

[0051] Example 3

[0052] Take 0.1 mL of standard sample of methanogen, dilute it into five different concentrations of methanogen standard solution using sodium chloride solution (0.9 wt%), store it in a refrigerator at 0-4°C for standby. Take 1 mL of the lowest dilution of the bacterial solution and spread it on a blood cell plate, observe it under a microscope, count the number of bacteria (20-400), calculate the amount of methanogen in the five different concentrations of bacterial standard sample by taking the sample volume and dilution factor. Further, heat the five different concentrations of methanogen at 90°C for 20 min by heating lysis, extract the coenzyme (coenzyme M) in the bacteria, then separate and purify the coenzyme M by high-speed centrifuge at a speed of 9500 r / min, leave the supernatant containing different concentrations of coenzyme, store it in a refrigerator at 0-4°C for standby.C m = 7.198C e + 4.853, where C m is the bacterial concentration, unit is CFU / mL, C e is the coenzyme concentration, unit is U / mL. Weigh 0.1 g of star-shaped CuS NPs and ultrasonically disperse it in 100 μL of 0.1% nafion aqueous solution, drop it on a glassy carbon electrode with a diameter of 3 mm, use 0.001 M HCl (pH 3) as electrolyte in a three-electrode system, test the current response signal intensity of different concentrations of coenzyme solution at a potential of 0.1 V (vs. Ag / AgCl), and get the relationship between coenzyme concentration and current response signal: I e = 8.164C e - 3.152, where I e is the current value, unit is mA, C e is the coenzyme concentration, unit is U / mL. Calculate the correlation between bacterial concentration and current signal, I e = 1.759C m - 0.039, where I e is the current value, unit is mA, C m is the bacterial concentration, unit is CFU / mL.

[0053] Example 4

[0054] Take 0.1 mL of standard sample of methanogen, dilute it into five different concentrations of methanogen standard solution using sodium chloride solution (0.9 wt%), and store it in a refrigerator at 0-4°C. Take 1 mL of the diluted sample and spread it on a plate, and under a microscope, count the number of bacteria (20-400) to calculate the amount of methanogen in the five different concentrations of bacterial standard sample. Further, heat the five different concentrations of methanogen at 90°C for 20 min by heating lysis, extract coenzyme (coenzyme M) from the bacteria, and then separate and purify the coenzyme M by a high-speed centrifuge at a speed of 9500 r / min. The supernatant containing different concentrations of coenzyme is stored in a refrigerator at 0-4°C for later use. The relationship between the concentration of bacteria and the concentration of coenzyme is C m = 2.554C e + 3.984, where C m is the concentration of bacteria, with a unit of CFU / mL, and C e is the concentration of coenzyme, with a unit of U / mL. Take 0.1 g of star-shaped CuS NPs and ultrasonically disperse it in 100 μL of 0.1% nafion aqueous solution, and drop it on a glassy carbon electrode with a diameter of 3 mm. Through a three-electrode system, use 10 -5 M of HCl (pH 5) as an electrolyte, and test the current response signal intensity of different concentrations of coenzyme solution at a potential of 0.1 V (vs. Ag / AgCl). The relationship is I e = 2.845C e - 0.853, where I e is the current value, with a unit of mA, and C e is the concentration of coenzyme, with a unit of U / mL. The correlation between the concentration of bacteria and the current signal is calculated as I e = 4.426C m - 0.105, where I e is the current value, with a unit of mA, and C m is the concentration of bacteria, with a unit of CFU / mL.

[0055] Example 5

[0056] Take 0.1 mL of standard sample of methanogen, dilute it into five different concentrations of methanogen standard solution using sodium chloride solution (0.9 wt%), and store it in a refrigerator at 0-4°C. Take 1 mL of the diluted sample and spread it on a plate, and under a microscope, count the number of bacteria (20-400) to calculate the amount of methanogen in the five different concentrations of bacterial standard sample. Further, heat the five different concentrations of methanogen at 90°C for 20 min by heating lysis, extract coenzyme (coenzyme M) in the bacteria, and then separate and purify the coenzyme M by a high-speed centrifuge at a speed of 9500 r / min. The supernatant containing different concentrations of coenzyme is stored in a refrigerator at 0-4°C for later use. The relationship between the coenzyme concentration and the bacterial concentration is: C m = 6.814C e + 7.223, where C m is the bacterial concentration, with a unit of CFU / mL, and C e is the coenzyme concentration, with a unit of U / mL. Take 0.1 g of star-shaped CuS NPs and ultrasonically disperse it in 100 μL of 0.1% nafion aqueous solution, drop it on a glassy carbon electrode with a diameter of 3 mm, and test the current response signal intensity of different concentrations of coenzyme solution by using a three-electrode system with 0.001 M KOH (pH 11) as an electrolyte at a potential of 0.1 V (vs. Ag / AgCl). The relationship between the coenzyme concentration and the current response signal is: I e = 3.651C e - 4.239, where I e is the current value, with a unit of mA, and C e is the coenzyme concentration, with a unit of U / mL. The correlation between the bacterial concentration and the current signal is calculated as: I e = 9.132C m - 1.658, where I e is the current value, with a unit of mA, and C m is the bacterial concentration, with a unit of CFU / mL.

[0057] Example 6

[0058] Take 0.1 mL of standard sample of methanogen, dilute it into five different concentrations of methanogen standard solution using sodium chloride solution (0.9 wt%), and store it in a refrigerator at 0-4°C. Take 1 mL of the diluted sample and spread it on a plate, and under a microscope, count the number of bacteria (20-400) to calculate the amount of methanogen in the five different concentrations of bacterial standard sample. Further, heat the five different concentrations of methanogen at 90°C for 20 min by heating lysis, extract coenzyme (coenzyme M) in the bacteria, and then separate and purify the coenzyme M by a high-speed centrifuge at a speed of 9500 r / min. The supernatant containing different concentrations of coenzyme is stored in a refrigerator at 0-4°C for use. The relationship between the coenzyme concentration and the bacterial concentration is C m = 2.617C e + 9.325, where C m is the bacterial concentration, the unit is CFU / mL, and C e is the coenzyme concentration, the unit is U / mL. Take 0.1 g of CuS NPs in the form of sheets as shown in Figure 6 , ultrasonically disperse them in 100 μL of 0.1% nafion aqueous solution, drop them on a glassy carbon electrode with a diameter of 3 mm, and test the current response signal intensity of different concentrations of coenzyme solution by using a three-electrode system with 0.01 M PBS (pH 7.4) as the electrolyte at a potential of 0.1 V (vs. Ag / AgCl). The relationship between the coenzyme concentration and the current value is I e = 1.665C e - 2.348, where I e is the current value, the unit is mA, and C e is the coenzyme concentration, the unit is U / mL. The correlation between the bacterial concentration and the current signal is calculated as I e = 3.029C m - 4.635, where I e is the current value, the unit is mA, and C m is the bacterial concentration, the unit is CFU / mL.

[0059] Example 7

[0060] Take 10 g of river bottom sludge, add 50 mL of 0.9 wt% sodium chloride solution, heat the river bottom sludge by heating cracking at 90°C for 20 min, for extracting coenzyme (coenzyme M) in bacteria. Then separate and purify the coenzyme M by high-speed centrifuge at a speed of 9500 r / min, leaving the supernatant containing different concentrations of coenzyme, stored in a refrigerator at 0-4°C for use. Weigh 0.1 g of star-shaped CuS NPs and ultrasonically disperse them in 100 μL of 0.1% nafion aqueous solution, drop them on a glassy carbon electrode with a diameter of 3 mm, and test the current response signal intensity of the coenzyme solution in the supernatant by using a three-electrode system with 0.01 M PBS (pH 7.4) as the electrolyte at a potential of 0.1 V (vs. Ag / AgCl). The formula I obtained at pH 7.4 is e = 6.551C m -0.148, and the current value is brought in to calculate the bacterial concentration C m .

[0061] Example 8

[0062] Extract the coenzyme (coenzyme M) in the standard methanogen, then separate and purify the coenzyme M by high-speed centrifuge at a speed of 9500 r / min, leaving the supernatant containing different concentrations of coenzyme, stored in a refrigerator at 0-4°C for use. Weigh 0.1 g of star-shaped CuS NPs and ultrasonically disperse them in 100 μL of 0.1% nafion aqueous solution, drop them on a glassy carbon electrode with a diameter of 3 mm, and test the current response signal intensity of different substances by using a three-electrode system with 0.01 M PBS (pH 7.4) as the electrolyte at a potential of 0.1 V (vs. Ag / AgCl), first drop coenzyme M, then drop CH3COO - , CO3 2- , SO4 2- , SCN - , C2O4 2- , SO3 2- , and CH3CH2OH, and then drop coenzyme M again, and the current response curve is shown in Figure 3 .

[0063] Example 9

[0064] Coenzyme M in standard methanogen was extracted, and then separated and purified by high-speed centrifuge at a speed of 9500 r / min. The supernatant containing different concentrations of coenzyme M was stored in a refrigerator at 0-4°C for later use. 0.1 g of star-shaped CuS NPs was ultrasonically dispersed in 100 μL of 0.1% nafion aqueous solution, and then dropped and coated on a glassy carbon electrode with a diameter of 3 mm. The current response signal intensity of different days was tested at a potential of 0.1 V (vs. Ag / AgCl) by using a three-electrode system and 0.01 M PBS (pH 7.4) as an electrolyte. The current response signal was tested for thirty days to evaluate the stability of the experimental material. The stability curve is shown in Figure 4

[0065] Those skilled in the art will easily understand that the above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A method for rapid detection of methanogens in water, characterized in that: The following steps are involved: (1) performing plate counts on standard samples of methanogens of different concentrations using a hemocytometer to determine the methanogen concentration of each standard sample; (2) The methanogen standard samples of different concentrations obtained in step (1) are respectively subjected to heating and lysis, and the supernatants are obtained by centrifugation to separate the coenzyme, wherein the pH of the supernatants of the methanogen standard samples of different concentrations is the same, and the coenzyme concentrations in the methanogen samples of different concentrations are measured by photometry to obtain a linear relationship between the plate count methanogen concentration and the coenzyme concentration; the coenzyme is coenzyme F 420 , at least one of coenzyme M and methyl-coenzyme M; (3) testing the current response signals of the coenzyme at different concentrations in the supernatant obtained in step (2) using a three-electrode system, wherein the working electrode of the three-electrode system is obtained by drop-coating a copper-based nanomaterial on a conductive material, and determining the linear relationship between the coenzyme concentration and the current response intensity; the copper-based nanomaterial is a star-shaped or sheet-shaped CuS nanomaterial; (4) establishing a linear relationship between the plate count methanogen concentration and the current response intensity by combining the linear relationship between the plate count methanogen concentration and the coenzyme concentration obtained in step (2) and the linear relationship between the coenzyme concentration and the current response intensity obtained in step (3); (5) The sample to be tested is heated and lysed, and the supernatant is obtained by centrifugation to separate the coenzyme, and the pH is adjusted to be the same as the pH of the supernatant of the standard sample in step (2). Then, the current response signal of the supernatant is obtained by the three-electrode system test described in step (3). Based on the linear relationship between the plate count methanogen concentration and the current response intensity obtained in step (4), the methanogen concentration of the test solution is calculated.

2. The method for rapid detection of methanogens in water according to claim 1, wherein: In step (2), the temperature of the heating cracking is 60-100°C.

3. The method for rapid detection of methanogens in water according to claim 1, wherein: In step (3), the counter electrode of the three-electrode system is a platinum sheet, and the reference electrodes are Ag and AgCl; a phosphate buffer solution is used as the electrolyte, and the current response intensity corresponding to different concentrations of the coenzyme is measured at a preset potential.

4. The method for rapid detection of methanogens in water according to claim 3, wherein: The preset potential is 0.1-0.6V.

5. The method for rapid detection of methanogens in water according to claim 3, wherein: The concentration of the phosphate buffer solution is 0.01-0.05M.

6. The method for rapid detection of methanogens in water according to claim 1, wherein: In step (1), the concentration of methanogens counted on the plate is 20 to 400 CFU / mL.

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