Microbial Electrochemical Sensor and Its Preparation Method and Application

Abstract

This application belongs to the technical field of biosensors, and specifically discloses a microbial electrochemical sensor, its preparation method and application. The preparation method includes: inoculating Acinetobacter baylyi strain BD4 into a culture medium for activation culture to obtain a bacterial suspension; performing low-temperature pretreatment on the bacterial suspension; constructing an electrochemical system and connecting a potentiostat. The electrochemical system includes a microbial three-electrode cell, and the microbial three-electrode cell includes a working electrode, and the working electrode includes a double-layer structure formed by carbon cloth and carbon felt; connecting the bacterial suspension to the electrochemical system, filtering the bacterial suspension through the carbon cloth first and then through the carbon felt to form a carbon felt-carbon cloth double-layer material biofilm, and obtaining a microbial electrochemical sensor. This application significantly improves the sensitivity of the sensor, enabling the sensor to not only have a short start-up time and a toxicity detection response, but also reduce the lower limit of toxicity detection of the sensor.

Description

Technical Field

[0001] This application belongs to the technical field of biosensors, and particularly relates to a microbial electrochemical sensor, a preparation method thereof, and an application thereof. Background Art

[0002] The rapid and accurate detection of toxic substances in water pollution has become particularly important. However, traditional biosensors (such as fish, daphnia magna, luminescent bacteria, etc.) are often limited in the detection of trace toxic substances, mainly manifested as problems such as low sensitivity and poor signal stability. In recent years, microbial electrochemical sensors (MES) have been widely used in the fields of environmental monitoring, food safety, medical diagnosis, etc. due to their advantages such as high sensitivity, rapid response, and low cost.

[0003] In MES, electrochemically active bacteria (EAB) determine the function and application of the system and are the core elements of MES. EAB is a type of microorganism with the ability of extracellular electron transfer, which can oxidize substrates through its own respiratory metabolism, extract electrons from environmental organic matter, and convert chemical energy into electrical energy. Compared with traditional biosensors, biosensors based on EAB have more advantages in terms of stability, simplicity, adaptability, etc. The principle of MES toxicity detection is that when toxic substances are present, toxicological effects will occur, inhibiting the metabolic activity and extracellular electron transfer process of EAB on the biofilm as the bioanode, resulting in changes in the output electrical signal, and the information of the substance to be detected can be directly transduced into an electrical signal without an additional signal transduction process.

[0004] Traditional MES is basically based on a microbial fuel cell (MFC). However, such sensors need to be acclimated with sewage or EAB for a long time before toxicity testing to form a mature biofilm as a sensing element, which will lead to a long sensor startup process. This biofilm belongs to a natural mixed bacterial biofilm, which has the advantages of high power generation, stable performance, and being not easily interfered by the detection process; its disadvantages are a long startup process and strong resistance to toxic substances (i.e., low sensitivity to toxic substances).

[0005] Therefore, a large number of studies have turned to the method of instantaneously and rapidly preparing pure-bacteria EAB biofilms to shorten the sensor startup time and detection time. Compared with natural mixed-bacteria biofilms, pure-bacteria EAB biofilms are formed by rapid filtration, and the startup process is shortened significantly. However, pure-bacteria EAB biofilms have the problem of poor sensitivity. The poor sensitivity is reflected in that for the same toxic substance at the same concentration, within the same detection time, its inhibition rate is smaller; if the same inhibition rate is to be achieved, the detection time needs to be extended to increase its inhibition rate, and even extending the detection time may not be able to increase its inhibition rate. Therefore, it limits the accuracy and reliability in the practical application of some water bodies. Currently, there is no technology to solve the above problems. Summary of the Invention

[0006] This application aims to solve at least one of the technical problems in the related art to some extent. For this purpose, the object of this application is to provide a microbial electrochemical sensor, a preparation method thereof, and an application. By performing low-temperature pretreatment on Acinetobacter baylyi strain BD4, and at the same time passing the bacterial suspension through a double-layer structure formed by carbon cloth and carbon felt in sequence to form a biofilm, a sensor is prepared, which significantly improves the sensitivity of the sensor. As a result, the sensor not only has a short startup time and a toxic detection response, can instantaneously detect the water body toxicity pollution situation, but also can lower the toxic detection lower limit of the sensor, making the toxic detection standard approach or reach the water quality standard reported by the World Health Organization, and has good application prospects.

[0007] In one aspect of this application, a method for preparing a microbial electrochemical sensor is provided. According to the embodiments of this application, the method includes:

[0008] (1) Inoculating Acinetobacter baylyi strain BD4 into a culture medium for activation culture to obtain a bacterial suspension;

[0009] (2) Performing low-temperature pretreatment on the bacterial suspension;

[0010] (3) Constructing an electrochemical system and connecting a potentiostat. The electrochemical system includes a microbial three-electrode cell, and the microbial three-electrode cell includes a working electrode, and the working electrode includes a double-layer structure formed by carbon cloth and carbon felt;

[0011] (4) Connecting the bacterial suspension to the electrochemical system, passing the bacterial suspension through the carbon cloth first and then through the carbon felt to form a carbon felt-carbon cloth double-layer material biofilm, and obtaining a microbial electrochemical sensor.

[0012] The method for preparing a microbial electrochemical sensor according to the embodiments of the present application adopts the technology of precisely pre-treating the bacterial suspension at low temperature, significantly improving the sensitivity of the sensor. As a result, the time from the start of the sensor to toxicity detection can be within 35 minutes, with a short required time, meeting the requirements of rapid detection, and showing great potential in the development of portable microbial toxicity sensors. The principle of the above-mentioned effect of precisely pre-treating the bacterial suspension at low temperature is as follows: Treating the bacterial suspension in a low-temperature environment will slow down the metabolic activities of electrochemically active microorganisms, entering a relatively weak dormant state. In this state, the physiological state of bacteria is more stable, and the internal enzyme system and cell membrane structure can maintain better integrity; at the same time, the short-term cold treatment process acts as a natural screening process, and only those electrogenic bacteria with better adaptability to the low-temperature environment can survive. These surviving electrogenic bacteria have better stress resistance; moreover, it causes less damage to microbial cells. Therefore, the above-mentioned technology of precisely pre-treating the bacterial suspension at low temperature significantly improves the response of Acinetobacter baylyi BD4 cells to toxic substances, so that when the time from the start of the sensor to toxicity detection is within 35 minutes, the sensor still has high sensitivity to toxic substances, with a short required time, meeting the requirements of rapid detection.

[0013] In addition, the working electrode of the present application includes a double-layer structure formed by carbon cloth and carbon felt. The carbon cloth is on the inner side and the carbon felt is on the outer side. When the bacterial suspension passes through the double-layer material, microorganisms first come into contact with the inner carbon cloth. The dense structure of the carbon cloth can initially intercept some larger bacteria, playing a role of preliminary filtration and reducing the content of impurities and miscellaneous bacteria in the biofilm. When the bacterial suspension passes through the carbon cloth and enters the carbon felt layer, the fiber structures of the carbon felt are intertwined with each other, forming a large number of pores and surfaces. Its surface also contains functional groups such as hydroxyl groups and carboxyl groups. This unique pore structure, high specific surface area, and functional groups can adsorb and retain microorganisms again, providing good attachment sites for microorganisms, enabling microorganisms to firmly adhere to the carbon felt, promoting the formation of biofilms, and the carbon felt has good electrical conductivity, which can effectively conduct electrons, so that the electrons generated by microorganisms during metabolism and other processes can be quickly transferred to the external circuit through the carbon felt, thereby effectively improving the detection efficiency and sensitivity of the sensor.

[0014] In summary, the present application pre-treats the Acinetobacter baylyi BD4 strain at low temperature, and at the same time makes the bacterial suspension sequentially pass through the double-layer structure formed by carbon cloth and carbon felt to form a biofilm, and prepares a sensor, significantly improving the sensitivity of the sensor. As a result, the sensor not only has a short start-up time and toxicity detection response, can rapidly detect the water toxicity pollution situation, but also can lower the toxicity detection limit of the sensor, making the toxicity detection standard approach or reach the water quality standard reported by the World Health Organization, and has good application prospects.

[0015] In addition, the method according to the above embodiments of the present application may further have the following additional technical features:

[0016] In some embodiments of the present application, let the activation culture temperature of the Acinetobacter baylyi BD4 strain be T0, and let the temperature for low-temperature pretreatment of the bacterial suspension be T1, where T1 = T0 - (10°C to 25°C) and T1 > 0°C. The time for low-temperature pretreatment of the bacterial suspension is 5 h to 48 h.

[0017] In some embodiments of the present application, the activation culture temperature T0 of the Acinetobacter baylyi BD4 strain is 25°C to 35°C, and the activation culture time is 20 h to 30 h.

[0018] In some embodiments of the present application, the OD of the bacterial suspension 600 is 1.2 to 2.0.

[0019] In some embodiments of the present application, the culture medium includes NaCl, tryptone, and yeast extract.

[0020] In some embodiments of the present application, the concentration of NaCl in the culture medium is 5 g / L to 15 g / L, the concentration of tryptone in the culture medium is 5 g / L to 15 g / L, and the concentration of yeast extract in the culture medium is 2 g / L to 10 g / L.

[0021] In some embodiments of the present application, the microbial three-electrode cell further includes a reference electrode and a counter electrode.

[0022] In some embodiments of the present application, the reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum wire electrode.

[0023] In the second aspect of the present application, the present application provides a microbial electrochemical sensor. According to the embodiments of the present application, the microbial electrochemical sensor is prepared by the method described in the above embodiments. Thus, the present application significantly improves the sensitivity of the microbial electrochemical sensor, so that the microbial electrochemical sensor not only has a short start-up time and a toxicity detection response, can immediately detect the water toxicity pollution situation, but also can lower the toxicity detection lower limit of the sensor, making the toxicity detection standard approach or reach the water quality standard reported by the World Health Organization, and has good application prospects.

[0024] In the third aspect of the present application, the present application proposes an application of the method described in the above embodiments or the microbial electrochemical sensor described in the above embodiments in the detection of heavy metal pollution in water bodies. Thus, in the process of detecting heavy metal pollution in water bodies, the sensitivity of the microbial electrochemical sensor is significantly improved, so that the microbial electrochemical sensor not only has a short start-up time and a toxicity detection response, can instantly detect the toxicity pollution situation of water bodies, but also can lower the lower limit of toxicity detection of the sensor, making the toxicity detection standard approach or reach the water quality standard reported by the World Health Organization, and has good application prospects.

[0025] Additional aspects and advantages of the present application will be given in part in the following description, will become apparent in part from the following description, or will be understood through the practice of the present application. Brief Description of the Drawings

[0026] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0027] Figure 1 is a schematic flow chart of the method for preparing a microbial electrochemical sensor according to an embodiment of the present application.

[0028] Figure 2 is a schematic structural diagram of the microbial electrochemical sensor according to an embodiment of the present application.

[0029] Figure 3 is a schematic cross-sectional diagram of the microbial electrochemical sensor according to an embodiment of the present application.

[0030] Reference Signs:

[0031] 10 - reference electrode, 20 - water outlet, 30 - counter electrode, 40 - working electrode, 41 - carbon felt, 42 - carbon cloth, 50 - water inlet. Detailed Description of the Embodiments

[0032] The embodiments of the present application will be described in detail below. Examples of the embodiments are shown in the drawings, in which the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present application and should not be construed as limiting the present application.

[0033] In one aspect of the present application, the present application proposes a method for preparing a microbial electrochemical sensor. According to an embodiment of the present application, with reference to the attached Figure 1, The method includes: S100: Inoculate Acinetobacter baylyi strain BD4 into a culture medium for activation culture to obtain a bacterial suspension; S200: Perform low-temperature pretreatment on the bacterial suspension; S300: Construct an electrochemical system and connect a potentiostat. The electrochemical system includes a microbial three-electrode cell, and the microbial three-electrode cell includes a working electrode, and the working electrode includes a double-layer structure formed by carbon cloth and carbon felt; S400: Connect the bacterial suspension to the electrochemical system, filter the bacterial suspension through the carbon cloth first and then through the carbon felt to form a carbon felt-carbon cloth bilayer material biofilm, and obtain a microbial electrochemical sensor.

[0034] According to the method for preparing a microbial electrochemical sensor in the embodiments of the present application, by adopting the technique of precisely performing low-temperature pretreatment on the bacterial suspension, the response of Acinetobacter baylyi strain BD4 cells to toxic substances is significantly improved. When the time from the start of the sensor to toxicity detection is within 35 minutes, the sensor still has high sensitivity to toxic substances, meeting the requirements of rapid detection and showing great potential in the development of portable microbial toxicity sensors. The principle of the above-mentioned precisely performing low-temperature pretreatment on the bacterial suspension to produce the above effects is as follows: Treating the bacterial suspension in a low-temperature environment will slow down the metabolic activities of electrochemically active microorganisms, and they enter a relatively weak dormant state. In this state, the physiological state of bacteria is more stable, and its internal enzyme system and cell membrane structure can maintain good integrity; at the same time, the short-term cold treatment process acts as a natural screening process, and only those electrogenic bacteria with good adaptability to the low-temperature environment can survive. These surviving electrogenic bacteria have better stress resistance; and it causes less damage to microbial cells. Therefore, the above-mentioned technique of precisely performing low-temperature pretreatment on the bacterial suspension significantly improves the response of Acinetobacter baylyi strain BD4 cells to toxic substances. When the time from the start of the sensor to toxicity detection is within 35 minutes, the sensor still has high sensitivity to toxic substances, meeting the requirements of rapid detection.

[0035] In addition, the working electrode of the present application includes a double-layer structure formed by carbon cloth and carbon felt, with the carbon cloth on the inner side and the carbon felt on the outer side. When the bacterial suspension passes through the bilayer material, the microorganisms first come into contact with the inner carbon cloth. The dense structure of the carbon cloth can initially intercept some larger bacteria, playing a role of preliminary filtration and reducing the content of impurities and miscellaneous bacteria in the biofilm; when the bacterial suspension passes through the carbon cloth and enters the carbon felt layer, the fiber structures of the carbon felt are intertwined with each other, forming a large number of pores and surfaces, and its surface also contains functional groups such as hydroxyl groups and carboxyl groups. This unique pore structure, high specific surface area and functional groups can further adsorb and retain microorganisms, providing good attachment sites for microorganisms, enabling microorganisms to firmly adhere to the carbon felt, promoting the formation of the biofilm, and the carbon felt has good electrical conductivity and can effectively conduct electrons, enabling the electrons generated by microorganisms during metabolism and other processes to be quickly transferred to the external circuit through the carbon felt, thereby effectively improving the detection efficiency and sensitivity of the sensor.

[0036] In summary, in the present application, by performing low-temperature pretreatment on Acinetobacter baylyi strain BD4, and simultaneously passing the bacterial suspension through a double-layer structure formed by carbon cloth and carbon felt in sequence to form a biofilm, a sensor is prepared, significantly improving the sensitivity of the sensor. As a result, the sensor not only has a short startup time and a toxicity detection response, can immediately detect the toxicity pollution situation of water bodies, but also can lower the toxicity detection lower limit of the sensor, making the toxicity detection standard approach or reach the water quality standard reported by the World Health Organization, and has good application prospects.

[0037] The method for preparing a microbial electrochemical sensor proposed in the present application will be described in detail below:

[0038] Specifically, referring to the attached Figure 1 , the method for preparing a microbial electrochemical sensor includes the following steps:

[0039] S100: Inoculate Acinetobacter baylyi strain BD4 into a culture medium for activation culture to obtain a bacterial suspension;

[0040] In this step, at a certain temperature, inoculate Acinetobacter baylyi strain BD4 into a culture medium for activation culture to obtain a bacterial suspension of Leucobacter luti that is already in the logarithmic growth phase or the initial stage of the stationary phase.

[0041] According to some specific embodiments of the present application, the activation culture temperature T0 of the above-mentioned Acinetobacter baylyi strain BD4 is 25°C to 35°C (for example, it can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, etc.), and the activation culture time is 20h to 30h (for example, it can be 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, etc.).

[0042] According to still some specific embodiments of the present application, the OD 600 of the above-mentioned bacterial suspension is 1.2 to 2.0 (for example, it can be 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, etc.), thereby ensuring that the obtained bacterial suspension is already in the logarithmic growth phase or the initial stage of the stationary phase.

[0043] According to some further specific embodiments of the present application, the above-mentioned culture medium comprises NaCl, tryptone and yeast extract. Specifically, the concentration of the above-mentioned NaCl in the culture medium is 5 g / L to 15 g / L (for example, it can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, etc.), the concentration of tryptone in the culture medium is 5 g / L to 15 g / L (for example, it can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, etc.), and the concentration of yeast extract in the culture medium is 2 g / L to 10 g / L (for example, it can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, etc.).

[0044] S200: Perform low-temperature pretreatment on the bacterial suspension;

[0045] In this step, by performing low-temperature pretreatment with precise temperature control and precise time control on the above-mentioned bacterial suspension, the response of Acinetobacter baylyi BD4 cells to toxic substances is significantly improved, so that when the time from the start of the sensor to toxicity detection is within 35 minutes, the sensor still has high sensitivity to toxic substances, requires a short time, and meets the requirements of instant detection.

[0046] According to some further specific embodiments of the present application, the activation culture temperature of Acinetobacter baylyi BD4 strain is set as T0, and the temperature for low-temperature pretreatment of the bacterial suspension is set as T1, satisfying T1 = T0 - (10°C to 25°C), and T1 > 0°C. The time for low-temperature pretreatment of the bacterial suspension is 5h to 48h (for example, it can be 5h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h, 48h, etc.). By treating the Acinetobacter baylyi BD4 bacterial suspension in an environment that is 10°C - 25°C lower than the culture temperature and higher than 0°C for 5h - 48h, the metabolic activities of electrochemically active microorganisms will slow down and enter a relatively weak dormant state. In this state, the physiological state of the bacteria is more stable, and its internal enzyme system, cell membrane structure, etc. can maintain good integrity. At the same time, the short-term cold treatment process acts as a natural screening process, and only those electrogenic bacteria with better adaptability to low-temperature environments can survive. These surviving electrogenic bacteria may have better stress resistance. Controlling T1 above 0°C is to ensure that the bacterial solution does not freeze, because even a tiny ice crystal in the bacterial solution may cause internal damage to the active bacteria and lead to a decrease in EAB activity. If the temperature of the low-temperature pretreatment is too low, it may cause internal damage to the active bacteria and lead to a decrease in EAB activity. If the temperature of the low-temperature pretreatment is too high, the sensitivity of the sensor cannot be effectively improved. If the time of the low-temperature pretreatment is too short or too long, the sensitivity of the sensor cannot be effectively improved. It can be seen that only by limiting the temperature and time of the low-temperature pretreatment within the above ranges can the technical effects of the present application be achieved.

[0047] S300: Construct an electrochemical system and connect a potentiostat. The electrochemical system includes a microbial three-electrode cell, and the microbial three-electrode cell includes a working electrode. The working electrode includes a double-layer structure formed by carbon cloth and carbon felt.

[0048] In this step, an electrochemical system is constructed and a potentiostat is connected. The electrochemical system includes a microbial three-electrode cell, and the microbial three-electrode cell includes a working electrode. The working electrode includes a double-layer structure formed by carbon cloth and carbon felt. When the bacterial suspension passes through the double-layer material, the microorganisms first come into contact with the inner carbon cloth. The dense structure of the carbon cloth can initially intercept some larger bacteria, playing a role of preliminary filtration and reducing the content of impurities and miscellaneous bacteria in the biofilm. When the bacterial suspension passes through the carbon cloth and enters the carbon felt layer, the fiber structures of the carbon felt are intertwined with each other, forming a large number of pores and surfaces. Its surface also contains functional groups such as hydroxyl groups and carboxyl groups. This unique pore structure, high specific surface area, and functional groups can adsorb and retain microorganisms again, providing good attachment sites for the microorganisms, enabling the microorganisms to firmly attach to the carbon felt, promoting the formation of the biofilm. Moreover, the carbon felt has good electrical conductivity and can effectively conduct electrons, enabling the electrons generated by the microorganisms during metabolism and other processes to be quickly transferred to the external circuit through the carbon felt, thereby effectively improving the detection efficiency and sensitivity of the sensor. It should be noted that the side where the bacterial suspension first comes into contact is the inner side, and the side where it comes into contact later is the outer side, that is, the carbon cloth is on the inner side and the carbon felt is on the outer side.

[0049] According to some further specific embodiments of the present application, the microbial three-electrode cell further includes a reference electrode and a counter electrode. Further, the reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum wire electrode.

[0050] S400: Connect the bacterial suspension to the electrochemical system, first filter the bacterial suspension through the carbon cloth and then through the carbon felt to form a biofilm of the carbon felt-carbon cloth double-layer material, and obtain a microbial electrochemical sensor.

[0051] In this step, connect the bacterial suspension to the electrochemical system, stack the circular carbon cloth and the circular carbon felt to form a double-layer structure and fixedly place it in the microbial three-electrode cell. After installing the battery, a peristaltic pump can be used to first filter the bacterial suspension through the carbon cloth and then through the carbon felt to form a biofilm of the carbon felt-carbon cloth double-layer material, and obtain a microbial electrochemical sensor.

[0052] In the second aspect of the present application, the present application proposes a microbial electrochemical sensor. According to the embodiments of the present application, the microbial electrochemical sensor is prepared by the method described in the above embodiments. Thus, the present application significantly improves the sensitivity of the microbial electrochemical sensor, so that the microbial electrochemical sensor not only has a short start-up time and a toxicity detection response, can instantaneously detect the water toxicity pollution situation, but also can reduce the toxicity detection lower limit of the sensor, making the toxicity detection standard approach or reach the water quality standard reported by the World Health Organization, and has good application prospects.

[0053] Specifically, refer to the appendix Figure 2 and 3, the above-mentioned microbial electrochemical sensor includes a reference electrode 10, a counter electrode 30 and a working electrode 40. The working electrode 40 includes a carbon felt 41 and a carbon cloth 42. 20 represents the water outlet, and 50 represents the water inlet. Thus, the bacterial suspension can be filtered through the carbon cloth first and then through the carbon felt to form a biofilm of a double-layer material of carbon felt-carbon cloth, and a microbial electrochemical sensor is obtained.

[0054] In the third aspect of the present application, the present application proposes an application of the method described in the above embodiments or the microbial electrochemical sensor described in the above embodiments in the detection of heavy metal pollution in water bodies. Thus, in the process of detecting heavy metal pollution in water bodies, the sensitivity of the microbial electrochemical sensor is significantly improved. As a result, the microbial electrochemical sensor not only has a short start-up time and a toxicity detection response, can immediately detect the toxicity pollution situation of water bodies, but also can lower the toxicity detection lower limit of the sensor, making the toxicity detection standard approach or reach the water quality standard reported by the World Health Organization, and has good application prospects.

[0055] The embodiments of the present application will be described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present application and cannot be understood as a limitation of the present application. In addition, if not specified clearly, all reagents used in the following embodiments are commercially available or can be synthesized according to the methods described in this article or known methods. For the reaction conditions not listed, they are also easily obtained by those skilled in the art.

[0056] The following raw materials are used in the following examples and comparative examples:

[0057] Luria-Bertani medium (LB medium), the components are 10 g of NaCl, 10 g of tryptone, and 5 g of yeast extract per liter of the medium.

[0058] Example 1

[0059] This example provides a method for preparing a microbial electrochemical sensor, including the following steps:

[0060] S1: At 30 °C, Acinetobacter baylyi BD4 is inoculated into the LB medium and cultured for 24 h to obtain a bacterial suspension of Leucobacter luti in the logarithmic growth phase or the initial stage of the stationary phase (OD600 is 1.5).

[0061] S2: The above bacterial suspension is placed in an environment at 5 °C for low-temperature cold treatment for 20 h.

[0062] S3: Construct an electrochemical system, including a microbial three-electrode cell (M3C). The M3C has a single-chamber structure with a working volume of 4 mL and includes a reference electrode (Ag / AgCl electrode), a counter electrode (platinum wire electrode), and a working electrode (carbon cloth + carbon felt electrode). Then connect the M3C to a peristaltic pump and a potentiostat.

[0063] S4: Start the sensor. Use the peristaltic pump to pass a 0.36 mm × 20 mm circular carbon cloth installed on the inner side and a 2 mm × 20 mm circular carbon felt installed on the outer side of the M3C battery successively through the filtered bacterial suspension to form a bilayer biofilm (i.e., the bacterial suspension first passes through the inner circular carbon cloth and then through the outer circular carbon felt), obtaining a working electrode with a carbon felt-carbon cloth bilayer material biofilm attached with Acinetobacter beijerinckii BD4, and obtaining a microbial electrochemical sensor.

[0064] Use the electrolyte (without 0.2 mg / L Hg 2+ ) to clean the pipeline, and then suck the blank solution (each liter of the blank solution contains 2.5 g of NaHCO3, 1.0 g of NH4Cl, 0.08 g of CaCl2·H2O, 0.2 g of MgCl2·6H2O, 10 g of NaCl, 7.2 g of HEPES, 0.15 g of yeast extract, and 2 mM of sodium lactate) into the M3C through the peristaltic pump, record its electrical signal with a potentiostat. After the signal is stable, drain the blank solution, and then suck the blank solution containing 0.2 mg / L Hg 2+ into the M3C through the peristaltic pump, record its electrical signal with a potentiostat, and perform heavy metal detection.

[0065] Example 2

[0066] The preparation method of this example is basically the same as that of Example 1, except that: the low-temperature cold treatment in step S2 of Example 1 is replaced with 10 °C.

[0067] Example 3

[0068] The preparation method of this example is basically the same as that of Example 1, except that: the low-temperature cold treatment in step S2 of Example 1 is replaced with 15 °C.

[0069] Example 4

[0070] The preparation method of this example is basically the same as that of Example 1, except that: the low-temperature cold treatment time in step S2 of Example 1 is replaced with 10 h.

[0071] Example 5

[0072] The preparation method of this example is basically the same as that of Example 1, except that: the low-temperature cold treatment time in step S2 of Example 1 is replaced with 40 h.

[0073] Comparative Example 1

[0074] The difference between Comparative Example 1 and Example 1 is that step S2 is not included (i.e., the bacterial suspension is not subjected to low-temperature cold treatment), and all other operations are the same as those in Example 1.

[0075] Comparative Example 2

[0076] The preparation method of Comparative Example 2 is basically the same as that of Example 1, and the difference is only that: the low-temperature cold treatment time in step S2 of Example 1 is changed to 4 h.

[0077] Comparative Example 3

[0078] The difference between Comparative Example 3 and Example 1 is that the positions of the circular carbon cloth and the circular carbon felt in step S4 are interchanged, that is, the bacterial suspension is first filtered through the circular carbon felt and then through the circular carbon cloth, and the remaining preparation method and detection method are the same as those in Example 1.

[0079] Comparative Example 4

[0080] The difference between Comparative Example 4 and Example 1 is that the 0.36 mm × 20 mm circular carbon cloth on the inner side and the 2 mm × 20 mm circular carbon felt on the outer side stacked and installed on the M3C battery in step S4 are modified to a 2 mm × 20 mm circular carbon felt installed on the M3C battery (i.e., the circular carbon cloth is not included), and the remaining preparation method and detection method are the same as those in Example 1.

[0081] Test

[0082] The sensors prepared in Examples 1-5 and Comparative Examples 1-4 were respectively tested for the performance of detecting heavy metals.

[0083] Test method:

[0084] 1) Detection duration: Record the duration from the start of the sensor to the end of the toxicity detection;

[0085] 2) The electrolyte containing 0.2 mg / L Hg 2+ was respectively sucked into the M3C of the sensors prepared in Examples 1-5 and Comparative Examples 1-4 through a peristaltic pump, and a potentiostat was used to record its current signal for sensitivity testing.

[0086] The test results are shown in Table 1.

[0087] Table 1 Performance test results and analysis of the sensors prepared in Examples 1-5 and Comparative Examples 1-4

[0088]

[0089] As can be seen from Table 1, compared with Comparative Examples 1-4, the sensitivity IR of Examples 1-5 has been significantly improved. Among them, the low-temperature cold treatment time of Examples 1-3 is 20h, and the output current is lower than that of Comparative Example 1, but the sensitivity has been significantly improved; and it can be seen from Examples 1-3 that above 0°C, the greater the difference between the treatment temperature and the cultivation temperature, the more obvious the improvement in its sensitivity.

[0090] As can be seen from Table 1, the low-temperature cold treatment temperature of Example 1 and Examples 4-5 is 5°C, and the sensitivity and output current are close for 10h and 20h of treatment; but after long-term treatment (40h), its sensitivity and output current are lower than those at moderate time, but its sensitivity is still higher than that of Comparative Example 1 without low-temperature treatment.

[0091] As can be seen from Table 1, after the EAB without low-temperature treatment in Comparative Example 1 is prepared into a sensor, its sensitivity is significantly lower than that of Example 1.

[0092] As can be seen from Table 1, after the EAB with too short low-temperature treatment time in Comparative Example 2 is prepared into a sensor, its sensitivity is significantly lower than that of Example 1.

[0093] As can be seen from Table 1, after the double-layer structure position is exchanged in Comparative Example 3, its sensitivity is significantly reduced.

[0094] As can be seen from Table 1, when only a single-layer structure is used in Comparative Example 4, both its output current and sensitivity will decrease.

[0095] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without conflict, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0096] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present application.

Claims

1. A method for preparing a microbial electrochemical sensor, characterized in that, Comprising: (1) Inoculating Acinetobacter baylyi strain BD4 into a culture medium for activation culture to obtain a bacterial suspension; (2) Performing low-temperature pretreatment on the bacterial suspension; (3) Constructing an electrochemical system and connecting a potentiostat. The electrochemical system includes a microbial three-electrode cell, and the microbial three-electrode cell includes a working electrode, and the working electrode includes a double-layer structure formed by carbon cloth and carbon felt; (4) Connecting the bacterial suspension to the electrochemical system, filtering the bacterial suspension through the carbon cloth first and then through the carbon felt to form a carbon felt-carbon cloth bilayer material biofilm, and obtaining a microbial electrochemical sensor; Wherein, setting the activation culture temperature of the Acinetobacter baylyi strain BD4 as T0, and setting the low-temperature pretreatment temperature of the bacterial suspension as T1, satisfying T1 = T0 - (10°C to 25°C), and T1 > 0°C, and the time for performing low-temperature pretreatment on the bacterial suspension is 5h to 48h.

2. The method according to claim 1, wherein The activation culture temperature T0 of the Acinetobacter baylyi strain BD4 is 25°C to 35°C, and the activation culture time is 20h to 30h.

3. The method according to any one of claims 1 to 2, characterized in that The OD of the bacterial suspension 600 is 1.2 to 2.

0.

4. The method according to any one of claims 1 to 2, characterized in that, The culture medium includes NaCl, tryptone, and yeast extract.

5. The method according to claim 4, wherein The concentration of NaCl in the culture medium is 5g / L to 15g / L, the concentration of tryptone in the culture medium is 5g / L to 15g / L, and the concentration of yeast extract in the culture medium is 2g / L to 10g / L.

6. The method according to any one of claims 1 to 2, characterized in that The microbial three-electrode cell further includes a reference electrode and a counter electrode.

7. The method according to claim 6, characterized in that, The reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum wire electrode.

8. A microbial electrochemical sensor, characterized in that, Prepared by the method according to any one of claims 1 to 7.

9. Use of the microbial electrochemical sensor according to claim 8 in the detection of heavy metal pollution in water bodies.

Images