Microbial electrochemical sensor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biosensors, and particularly discloses a microbial electrochemical sensor and a preparation method and application thereof.The preparation method comprises the steps that a culture medium is inoculated with an acinetobacter baini DB-4 strain for activation culture, and a bacterial suspension is obtained; performing low-temperature pretreatment on the bacterial suspension; an electrochemical system is constructed and connected with a constant potential rectifier, the electrochemical system comprises a microbial three-electrode pool, the microbial three-electrode pool comprises a working electrode, and the working electrode comprises a double-layer structure formed by carbon cloth and carbon felt; and connecting the bacterial suspension with an electrochemical system, filtering the bacterial suspension through carbon cloth and then filtering the bacterial suspension through carbon felt to form a carbon felt-carbon cloth double-layer material biological membrane, thereby obtaining the microbial electrochemical sensor. The sensitivity of the sensor is remarkably improved, so that the sensor not only has short-time starting and toxicity detection response, but also reduces the lower toxicity detection limit of the sensor.

Description

Technical Field

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

[0002] The rapid and accurate detection of toxic substances in water pollution has become particularly important. However, traditional biological sensors (such as fish, large fleas, luminous bacteria, etc.) are often limited in the detection of trace toxic substances, mainly manifested in low sensitivity and poor signal stability. In recent years, microbial electrochemical sensors (MES) have been widely used in environmental monitoring, food safety, medical diagnosis and other fields due to their 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 extracellular electron transfer ability. It can convert chemical energy into electrical energy by oxidizing substrates and extracting electrons from environmental organic matter through its own respiratory metabolism. Compared with traditional biosensors, EAB-based biosensors have advantages in stability, simplicity, and adaptability. 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 a biological anode, resulting in changes in the output electrical signal, and the information of the substance to be tested can be directly transduced into an electrical signal without the need for an additional signal transduction process.

[0004] Traditional MES is basically based on microbial fuel cells (MFCs), but such sensors need to be domesticated for a long time with sewage or EAB before toxicity testing to form a mature biofilm as a sensing element, which will lead to a lengthy sensor startup process. This biofilm is a natural mixed bacterial biofilm, which has the advantages of high power generation, stable performance, and is not easily disturbed 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 use of instant and rapid preparation of pure bacterial EAB biofilms to shorten the sensor startup time and detection time. Compared with natural mixed bacterial biofilms, pure bacterial EAB biofilms are formed by rapid filtration, and the startup process is much shorter. However, pure bacterial EAB biofilms have the problem of poor sensitivity. The poor sensitivity is reflected in the fact that for the same toxic substance at the same concentration, the inhibition rate is small within the same detection time; if the same inhibition rate is to be achieved, the detection time needs to be extended to increase the inhibition rate, and even extending the detection time may not increase the inhibition rate. Therefore, its accuracy and reliability in practical applications in some water bodies are limited. There is currently no technology that can solve the above problems. Summary of the invention

[0006] The present application aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the purpose of the present application is to propose a microbial electrochemical sensor and its preparation method and application. The present application prepares a sensor by pre-treating the DB-4 strain of Acinetobacter bayleyi at low temperature, and filtering the bacterial suspension through a double-layer structure formed by carbon cloth and carbon felt in sequence to form a biofilm, thereby significantly improving the sensitivity of the sensor. As a result, the sensor not only has a short start-up time and toxicity detection response, and can detect the toxicity pollution of water bodies in real time, but also can reduce the lower limit of the sensor's toxicity detection, so that the toxicity detection standard approaches or reaches the water quality standard reported by the World Health Organization, and has good application prospects.

[0007] 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, the method comprises: (1) inoculating the Acinetobacter bayenii DB-4 strain 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 it to a potentiostat, wherein the electrochemical system comprises a microbial three-electrode cell, wherein the microbial three-electrode cell comprises a working electrode, and wherein the working electrode comprises 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 double-layer material biofilm, thereby obtaining a microbial electrochemical sensor.

[0008] According to the method for preparing a microbial electrochemical sensor in an embodiment of the present application, the technology of accurately pretreating the bacterial suspension at low temperature is used to significantly improve the sensitivity of the sensor, so that the sensor can be started from toxicity detection within 35 minutes, the time required is short, and instant detection is met, showing great potential in the development of portable microbial toxicity sensors. The principle of the above-mentioned effect of accurately pretreating the bacterial suspension at low temperature is that treating the bacterial suspension under a low temperature environment will slow down the metabolic activity of electrochemically active microorganisms and enter a relatively weak dormant state. In this state, the physiological state of the 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 plays a natural screening process, and only those electrogenic bacteria with good adaptability to low temperature environments can survive, and these surviving electrogenic bacteria have better stress resistance; and the damage to microbial cells is small; thus, the above-mentioned technology of accurately pretreating the bacterial suspension at low temperature significantly improves the response of the Acinetobacter bacillin DB-4 bacteria to toxic substances, so that the sensor still has a high sensitivity to toxic substances when the time from startup to toxicity detection is within 35 minutes, and the time required is short, which meets instant detection.

[0009] 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 inside and the carbon felt on the outside. When the bacterial suspension passes through the double-layer material, the microorganism first contacts the carbon cloth on the inside, and the dense structure of the carbon cloth can initially intercept some larger bacteria, play a role in preliminary filtration, and reduce 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 structure of the carbon felt is intertwined to form a large number of pores and surfaces, and its surface also contains functional groups such as hydroxyl and carboxyl. This unique pore structure and high specific surface area and functional groups can re-adsorb and intercept microorganisms, provide good attachment sites for microorganisms, enable microorganisms to firmly attach to the carbon felt, and promote the formation of biofilms, and the carbon felt has good conductivity and can effectively conduct electrons, so that the electrons generated by microorganisms in 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.

[0010] In summary, the present application prepares a sensor by performing low-temperature pretreatment on the DB-4 strain of Acinetobacter bayenii, and filtering the bacterial suspension in sequence through a double-layer structure formed by carbon cloth and carbon felt to form a biofilm, thereby significantly improving the sensitivity of the sensor. As a result, the sensor not only has a short startup time and toxicity detection response, and can instantly detect the toxicity pollution of water bodies, but also can reduce the lower limit of the sensor's toxicity detection, so that the toxicity detection standard approaches or reaches the water quality standard reported by the World Health Organization, and has good application prospects.

[0011] In addition, the method according to the above embodiment of the present application may also have the following additional technical features: In some embodiments of the present application, the activation culture temperature of the Acinetobacter baylyi DB-4 strain is set to T0, the temperature for low-temperature pretreatment of the bacterial suspension is set to T1, satisfying T1=T0-(10℃~25℃), and T1>0℃, and the time for low-temperature pretreatment of the bacterial suspension is 5h~48h.

[0012] In some embodiments of the present application, the activation culture temperature T0 of the Acinetobacter bayleyi DB-4 strain is 25°C to 35°C, and the activation culture time is 20h to 30h.

[0013] In some embodiments of the present application, the OD of the bacterial suspension is 600 It is 1.2~2.0.

[0014] In some embodiments of the present application, the culture medium comprises NaCl, tryptone and yeast extract.

[0015] In some embodiments of the present application, the concentration of NaCl in the culture medium is 5 g / L~15 g / L, the concentration of trypsin in the culture medium is 5 g / L~15 g / L, and the concentration of yeast extract in the culture medium is 2 g / L~10 g / L.

[0016] In some embodiments of the present application, the microbial three-electrode cell further includes a reference electrode and a counter electrode.

[0017] In some embodiments of the present application, the reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum wire electrode.

[0018] 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. As a result, 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 toxicity detection response, can instantly detect the toxicity pollution of water bodies, but also can reduce the lower limit of sensor toxicity detection, so that the toxicity detection standard is close to or reaches the water quality standard reported by the World Health Organization, and has good application prospects.

[0019] In the third aspect of the present application, the present application proposes the application of the method described in one or more embodiments or the microbial electrochemical sensor described in the above embodiments in the detection of heavy metal pollution in water bodies. As a result, in the process of heavy metal pollution detection 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 toxicity detection response, can instantly detect the toxicity pollution of water bodies, but also can reduce the lower limit of sensor toxicity detection, so that the toxicity detection standard is close to or reaches the water quality standard reported by the World Health Organization, and has good application prospects.

[0020] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 Schematic diagram of the process of preparing a microbial electrochemical sensor according to an embodiment of the present application.

[0022] Figure 2 This is a schematic diagram of the structure of the microbial electrochemical sensor according to an embodiment of the present application.

[0023] Figure 3 Schematic cross-sectional view of a microbial electrochemical sensor according to an embodiment of the present application.

[0024] Reference numerals: 10-reference electrode, 20-water outlet, 30-counter electrode, 40-working electrode, 41-carbon felt, 42-carbon cloth, 50-water inlet. DETAILED DESCRIPTION

[0025] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0026] In one aspect of the present application, the present application provides a method for preparing a microbial electrochemical sensor. Figure 1The method includes: S100: inoculating the DB-4 strain of Acinetobacter baylyi into a culture medium for activation culture to obtain a bacterial suspension; S200: performing low-temperature pretreatment on the bacterial suspension; S300: constructing an electrochemical system and connecting a constant potential instrument, the electrochemical system including a microbial three-electrode cell, the microbial three-electrode cell including a working electrode, and the working electrode including a double-layer structure formed by carbon cloth and carbon felt; S400: connecting the bacterial suspension to the electrochemical system, filtering the bacterial suspension first through the carbon cloth and then through the carbon felt to form a carbon felt-carbon cloth double-layer material biofilm to obtain a microbial electrochemical sensor.

[0027] According to the method for preparing a microbial electrochemical sensor in an embodiment of the present application, the technology of precise low-temperature pretreatment of bacterial suspension is adopted to significantly improve the response of Acinetobacter bayenii DB-4 bacteria to toxic substances, so that the sensor still has a high sensitivity to toxic substances when the time from startup to toxicity detection is within 35 minutes, which meets the requirements of instant detection and shows great potential in the development of portable microbial toxicity sensors. The principle of the above-mentioned effect of precise low-temperature pretreatment of bacterial suspension is: treating the bacterial suspension in a low-temperature environment will slow down the metabolic activity of electrochemically active microorganisms and enter a relatively weak dormant state. In this state, the physiological state of the 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 low-temperature environments can survive, and these surviving electrogenic bacteria have better stress resistance; and less damage to microbial cells; therefore, the above-mentioned technology of precise low-temperature pretreatment of bacterial suspension significantly improves the response of Acinetobacter bayenii DB-4 bacteria to toxic substances, so that the sensor still has a high sensitivity to toxic substances when the time from startup to toxicity detection is within 35 minutes, which meets the requirements of instant detection.

[0028] 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 inside and the carbon felt on the outside. When the bacterial suspension passes through the double-layer material, the microorganism first contacts the carbon cloth on the inside, and the dense structure of the carbon cloth can initially intercept some larger bacteria, play a role in preliminary filtration, and reduce 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 structure of the carbon felt is intertwined to form a large number of pores and surfaces, and its surface also contains functional groups such as hydroxyl and carboxyl. This unique pore structure and high specific surface area and functional groups can re-adsorb and intercept microorganisms, provide good attachment sites for microorganisms, enable microorganisms to firmly attach to the carbon felt, and promote the formation of biofilms, and the carbon felt has good conductivity and can effectively conduct electrons, so that the electrons generated by microorganisms in 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.

[0029] In summary, the present application prepares a sensor by performing low-temperature pretreatment on the DB-4 strain of Acinetobacter bayenii, and filtering the bacterial suspension in sequence through a double-layer structure formed by carbon cloth and carbon felt to form a biofilm, thereby significantly improving the sensitivity of the sensor. As a result, the sensor not only has a short startup time and toxicity detection response, and can instantly detect the toxicity pollution of water bodies, but also can reduce the lower limit of the sensor's toxicity detection, so that the toxicity detection standard approaches or reaches the water quality standard reported by the World Health Organization, and has good application prospects.

[0030] The method for preparing the microbial electrochemical sensor proposed in this application is described in detail below: Specifically, refer to the attached Figure 1 The above method for preparing a microbial electrochemical sensor comprises the following steps: S100: inoculating the Acinetobacter bayleyi DB-4 strain into a culture medium for activation culture to obtain a bacterial suspension; In this step, the Acinetobacter bayleyi DB-4 strain is inoculated into a culture medium for activation culture at a certain temperature to obtain a bacterial suspension of Shewanella leukemia that is already in a logarithmic growth phase or an early stage of a stable phase.

[0031] According to some specific embodiments of the present application, the activation culture temperature T0 of the above-mentioned Acinetobacter bayleyi DB-4 strain is 25°C~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~30h (for example, it can be 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, etc.).

[0032] According to some further specific embodiments of the present application, the OD of the bacterial suspension is 600 The ratio of the carbonyl group to the carbonyl group is 1.2-2.0 (for example, 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 in the logarithmic growth phase or the early stage of the stable phase.

[0033] According to some further specific embodiments of the present application, the culture medium comprises NaCl, trypsin and yeast extract. Specifically, the concentration of NaCl in the culture medium is 5 g / L-15 g / L (for example, 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-15 g / L (for example, 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-10 g / L (for example, 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.).

[0034] S200: low temperature pretreatment of bacterial suspension; In this step, by performing low-temperature pretreatment of the above-mentioned bacterial suspension with precise temperature and time control, the response of Acinetobacter DB-4 bacteria to toxic substances is significantly improved, so that the sensor still has a high sensitivity to toxic substances when the time from startup to toxicity detection is within 35 minutes, and the time required is short, which meets the requirements of instant detection.

[0035] According to some specific embodiments of the present application, the activation culture temperature of the Acinetobacter bayleyi DB-4 strain is T0, the temperature of the bacterial suspension for low-temperature pretreatment is T1, T1=T0-(10°C~25°C), and T1>0°C, the time for low-temperature pretreatment of the bacterial suspension is 5h~48h (for example, it can be 5h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h, 48h, etc.), by activating the Acinetobacter bayleyi DB-4 bacterial suspension at a temperature lower than the culture temperature Treatment at 10℃-25℃ and above 0℃ for 5h-48h will slow down the metabolic activity of electrochemically active microorganisms and enter a relatively weak dormant state. In this state, the physiological state of the 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 plays a natural screening process, and only those electrogenic bacteria with good adaptability to low-temperature environments can survive, and these surviving electrogenic bacteria may have better stress resistance. Controlling T1 above 0℃ is to ensure that the bacterial solution does not freeze. If the bacterial solution freezes, even tiny ice crystals may cause internal damage to active bacteria and reduce EAB activity. If the temperature of low-temperature pretreatment is too low, it may cause internal damage to active bacteria and reduce EAB activity. If the temperature of low-temperature pretreatment is too high, the sensitivity of the sensor cannot be effectively improved; if the time of 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 low-temperature pretreatment within the above range can the technical effect of this application be achieved.

[0036] S300: construct an electrochemical system and connect a constant potential instrument, wherein the electrochemical system includes a microbial three-electrode cell, 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; In this step, an electrochemical system is constructed and connected to a constant potential instrument. 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. When the bacterial suspension passes through the double-layer material, the microorganism first contacts the carbon cloth on the inside. The dense structure of the carbon cloth can initially intercept some larger bacteria, play a role in preliminary filtration, and reduce the content of impurities and miscellaneous bacteria in the biofilm; when the bacterial suspension passes through the carbon cloth, it enters the carbon felt layer, and the fiber structure of the carbon felt is intertwined to form a large number of pores and surfaces. Its surface also contains functional groups such as hydroxyl and carboxyl. This unique pore structure and high specific surface area and functional groups can re-adsorb and intercept microorganisms, provide good attachment sites for microorganisms, enable microorganisms to firmly attach to the carbon felt, and promote the formation of biofilms. The carbon felt has good electrical conductivity and can effectively conduct electrons, so that the electrons generated by microorganisms in the process of metabolism can 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 that the bacterial suspension contacts first is the inner side, and the side that contacts later is the outer side, that is, the carbon cloth is on the inner side, and the carbon felt is on the outer side.

[0037] According to some other 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.

[0038] S400: Connecting the bacterial suspension to the electrochemical system, filtering the bacterial suspension through the carbon cloth first and then through the carbon felt, forming a carbon felt-carbon cloth double-layer material biofilm, and obtaining a microbial electrochemical sensor.

[0039] In this step, the bacterial suspension is connected to the electrochemical system, and the circular carbon cloth and the circular carbon felt are stacked to form a double-layer structure and fixed in the microbial three-electrode cell. After the battery is installed, a peristaltic pump can be used to filter the bacterial suspension through the carbon cloth and then through the carbon felt to form a carbon felt-carbon cloth double-layer material biofilm to obtain a microbial electrochemical sensor.

[0040] 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. As a result, 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 toxicity detection response, can instantly detect the toxicity pollution of water bodies, but also can reduce the lower limit of sensor toxicity detection, so that the toxicity detection standard is close to or reaches the water quality standard reported by the World Health Organization, and has good application prospects.

[0041] Specifically, refer to the attached Figure 2 and 3The above-mentioned microbial electrochemical sensor includes a reference electrode 10, a counter electrode 30 and a working electrode 40, wherein the working electrode 40 includes a carbon felt 41 and a carbon cloth 42, 20 represents a water outlet, and 50 represents a water inlet, thereby allowing the bacterial suspension to be first filtered through the carbon cloth and then filtered through the carbon felt to form a carbon felt-carbon cloth double-layer material biofilm to obtain a microbial electrochemical sensor.

[0042] In the third aspect of the present application, the present application proposes the application of the method described in one or more embodiments or the microbial electrochemical sensor described in the above embodiments in the detection of heavy metal pollution in water bodies. As a result, in the process of heavy metal pollution detection 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 toxicity detection response, can instantly detect the toxicity pollution of water bodies, but also can reduce the lower limit of sensor toxicity detection, so that the toxicity detection standard is close to or reaches the water quality standard reported by the World Health Organization, and has good application prospects.

[0043] The embodiments of the present application are 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 should not be construed as limiting the present application. In addition, unless otherwise explicitly stated, all reagents used in the following embodiments are commercially available or can be synthesized according to this article or known methods, and the reaction conditions not listed are also easily available to those skilled in the art.

[0044] The following raw materials were used in the following examples and comparative examples: Luria-Bertani medium (LB medium), the composition of which is 10g NaCl, 10g tryptone, and 5g yeast extract per liter of medium.

[0045] Example 1 This embodiment provides a method for preparing a microbial electrochemical sensor, comprising the following steps: S1: Acinetobacter baylyi DB-4 was inoculated into LB medium and cultured for 24 hours at 30°C to obtain a bacterial suspension of Shewanella leukemia that was in the logarithmic growth phase or the early stage of the stationary phase (OD600 was 1.5).

[0046] S2: Place the above bacterial suspension in a 5°C environment for low temperature cold treatment for 20 hours.

[0047] S3: Construct an electrochemical system, including a microbial three-electrode cell (M3C). The M3C has a single-chamber structure and a working volume of 4 mL, including 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 constant potential instrument.

[0048] S4: Start the sensor and use a peristaltic pump to pass the inner 0.36mm×20mm circular carbon cloth and the outer 2mm×20mm circular carbon felt stacked on the M3C battery through the filtered bacterial suspension in sequence to form a double-layer structure biofilm (that is, the bacterial suspension is first filtered through the inner circular carbon cloth and then filtered through the outer circular carbon felt), to obtain a working electrode with a carbon felt-carbon cloth double-layer material biofilm attached with Acinetobacter baylyi DB-4, and to obtain a microbial electrochemical sensor.

[0049] Use electrolyte (not containing 0.2mg / L Hg 2+ ) to clean the pipeline, and then suck the blank solution (each liter of blank solution contains 2.5g NaHCO3, 1.0g NH4Cl, 0.08g CaCl2·H2O, 0.2g MgCl2·6H2O, 10g NaCl, 7.2g HEPES, 0.15g yeast extract and 2mM sodium lactate) into M3C through a peristaltic pump, and record its electrical signal with a constant potential instrument. After the signal stabilizes, the blank solution is emptied, and then the blank solution containing 0.2mg / L Hg 2+ The blank solution was sucked into M3C through a peristaltic pump, and its electrical signal was recorded using a constant potential instrument for heavy metal detection.

[0050] Example 2 The preparation method of this embodiment is basically the same as that of embodiment 1, except that the low temperature cold treatment in step S2 of embodiment 1 is replaced with 10°C.

[0051] Example 3 The preparation method of this embodiment is basically the same as that of embodiment 1, except that the low temperature cold treatment in step S2 of embodiment 1 is replaced with 15°C.

[0052] Example 4 The preparation method of this embodiment is basically the same as that of embodiment 1, except that the low-temperature cold treatment time in step S2 of embodiment 1 is changed to 10 hours.

[0053] Example 5 The preparation method of this embodiment is basically the same as that of embodiment 1, except that the low-temperature cold treatment time in step S2 of embodiment 1 is changed to 40 hours.

[0054] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that step S2 is not included (ie, the bacterial suspension is not subjected to low-temperature cold treatment), and the other operations are all the same as those in Example 1.

[0055] Comparative Example 2 The preparation method of Comparative Example 2 is basically the same as that of Example 1, except that the low-temperature cold treatment time in step S2 of Example 1 is changed to 4 hours.

[0056] Comparative Example 3 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 filtered through the circular carbon cloth. The rest of the preparation method and detection method are the same as Example 1.

[0057] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the 0.36mm×20mm circular carbon cloth on the inner side and the 2mm×20mm circular carbon felt on the outer side superimposed on the M3C battery in step S4 are modified to a 2mm×20mm circular carbon felt installed on the M3C battery (i.e., no circular carbon cloth is included), and the rest of the preparation method and detection method are the same as those in Example 1.

[0058] test The performance of the sensors prepared in Examples 1-5 and Comparative Examples 1-4 for detecting heavy metals was measured respectively.

[0059] Test method: 1) Detection duration: record the time from sensor startup to the end of toxicity detection; 2) Contain 0.2mg / L Hg 2+ The electrolyte was sucked into the M3C of the sensors prepared in Examples 1-5 and Comparative Examples 1-4 respectively by a peristaltic pump, and the current signals were recorded by a potentiostat to perform a sensitivity test.

[0060] The test results are shown in Table 1.

[0061] Table 1 Performance test results and analysis of sensors prepared in Examples 1-5 and Comparative Examples 1-4

[0062] It can be seen from Table 1 that compared with Comparative Examples 1-4, the sensitivity IR of Examples 1-5 is significantly improved. Among them, the low-temperature cold treatment time of Examples 1-3 is 20 hours, and the output current is lower than that of Comparative Example 1, but the sensitivity is 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 culture temperature, the more obvious the sensitivity improvement.

[0063] 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 after treatment for 10 hours and 20 hours are close; but after long-term treatment (40 hours), the sensitivity and output current are lower than those of the appropriate time, but the sensitivity is still higher than that of Comparative Example 1 which has not been treated at low temperature.

[0064] It can be seen from Table 1 that after the EAB in Comparative Example 1 that has not been subjected to low-temperature treatment is prepared into a sensor, its sensitivity is significantly lower than that of Example 1.

[0065] It can be seen from Table 1 that the sensitivity of the sensor prepared from the EAB in Comparative Example 2 with too short a low-temperature treatment time is significantly lower than that in Example 1.

[0066] It can be seen from Table 1 that the sensitivity of Comparative Example 3 is significantly reduced after the position of the double-layer structure is replaced.

[0067] It can be seen from Table 1 that comparative example 4 only adopts a single-layer structure, and its output current and sensitivity are reduced.

[0068] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0069] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for preparing a microbial electrochemical sensor, characterized in that: include: (1) inoculating the Acinetobacter bayenii DB-4 strain 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 it to a potentiostat, wherein the electrochemical system comprises a microbial three-electrode cell, wherein the microbial three-electrode cell comprises a working electrode, and wherein the working electrode comprises 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 double-layer material biofilm, thereby obtaining a microbial electrochemical sensor.

2. The method according to claim 1, characterized in that The activation culture temperature of the Acinetobacter bayleyi DB-4 strain is T0, and the temperature for low-temperature pretreatment of the bacterial suspension is T1, satisfying T1=T0-(10°C~25°C), and T1>0°C, and the time for low-temperature pretreatment of the bacterial suspension is 5h~48h.

3. The method according to claim 2, characterized in that The activation culture temperature T0 of the Acinetobacter bayleyii DB-4 strain is 25° C. to 35° C., and the activation culture time is 20 h to 30 h.

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

0.

5. The method according to any one of claims 1 to 3, characterized in that The culture medium included NaCl, tryptone and yeast extract.

6. The method according to claim 5, characterized in that 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.

7. The method according to any one of claims 1 to 3, characterized in that The microbial three-electrode cell also includes a reference electrode and a counter electrode.

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

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

10. Use of the method according to any one of claims 1 to 8 or the microbial electrochemical sensor according to claim 9 in the detection of heavy metal pollution in water bodies.

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