Multilayer electrochemical active biological membrane, preparation method and microbial electrochemical sensor

Abstract

The invention belongs to the technical field of biosensors, and particularly discloses a multi-layer electrochemical active biological membrane, a preparation method and a microbial electrochemical sensor, the multi-layer electrochemical active biological membrane comprises a plurality of sheet-shaped conductive materials, the plurality of sheet-shaped conductive materials are stacked in sequence, the plurality of sheet-shaped conductive materials are fixed through metal wires, and the metal wires are arranged on the sheet-shaped conductive materials. Pure electrochemical active microorganisms are attached to the surfaces of the two sides of each sheet-shaped conductive material respectively. The multi-layer electrochemical active biological membrane comprises the multi-layer compact biological membrane formed from the inner layer to the outer layer, the amount of active EAB is effectively increased, the biological performance of the electrochemical active biological membrane is effectively more stable, the electricity generation performance is higher, and therefore the problems that a traditional pure bacterial biological membrane is thin, the amount of active EAB is low, and the electricity generation performance is not stable are solved. In addition, the multi-layer electrochemical active biological membrane is naturally formed by utilizing the automatic enrichment property of EAB, so that the problem that the biological membrane is instable after being passively formed under the promotion of external force is solved.

Description

Technical Field

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

[0002] Microbial electrochemical sensors (MES) with electrochemically active bacteria (EAB) as the core can directly transduce the substances to be tested in water into bioelectric signals. They have the advantages of rapid detection, high sensitivity, low detection cost, and strong anti-interference ability, and have good application prospects in the fields of biomedicine and environmental monitoring. EAB is widely present in nature. Most of the daily applications are based on the mixture of multiple strains of EAB and the formation of mature mixed biofilms after long-term laboratory domestication. Mature mixed biofilms are enriched with more than dozens of EAB species, with high electrical signal generation, easy detection, strong stress resistance, and rapid automatic repair after encountering toxic shocks. They have been widely used in water toxicity detection. However, the various EAB enriched in mixed biofilms are prone to dynamic changes due to differences in domestication environment and test environment (temperature, pH, oxygen, nutrient solution composition, and test water conditions), resulting in unstable electrical performance and significant changes in sensitivity to toxic substances. At the same time, mature mixed bacterial biofilms are unable to detect low concentrations of toxic substances due to their strong stress resistance. A single pure strain of EAB selected from mature mixed bacterial biofilms can solve this problem, but pure strains of EAB often have weak electricity production performance, the signal detection process is easily disturbed, and pure strains of EAB are very easy to die, further increasing the difficulty of detection.

[0003] In some existing technologies, the enriched suspended electroactive bacteria and an appropriate amount of adhesive solution are mixed evenly to prepare an electroactive bacteria composite preparation, and then the electroactive bacteria composite preparation is evenly coated on the collector by spin coating to quickly form it into an electrode biofilm. Its disadvantages are: 1) The use of adhesive chemical reagents may change the surface structure of microbial cells or even destroy the metabolic active sites inside the cells, affecting the subsequent electroactivity of the microorganisms. 2) The complexity of multiple steps. This method involves multiple steps such as preparing an electroactive bacteria composite preparation, spin coating, and pressing. Each step requires precise control, and there may be problems in the connection between the steps, which increases the risk of operational errors.

[0004] In other existing technologies, nanoscale magnetic particles are used as carriers of electrochemically active microorganisms, and the electrochemically active microorganism suspension attached to the surface of the magnetic particles is added to the electrochemical system, and a specific magnetic force is applied to drive the magnetic particles to deposit on the surface of the working electrode. Since the electrochemically active microorganisms are attached to the surface of the magnetic particles, the magnetic force also drives the electrochemically active microorganisms to move to the surface of the working electrode; the electrochemically active microorganisms on the surface of the working electrode can form a three-dimensional structure similar to a biofilm with the magnetic particles, in which the magnetic particles are both the framework of the three-dimensional structure and the fixed microorganisms; when the electrochemically active microorganisms respire extracellularly, the electrons are collected by the magnetic particles with good conductivity and transferred to the working electrode, and the electrochemical system generates current accordingly, which can be used as a microbial electrochemical sensor, avoiding the lengthy incubation process of the natural biofilm, that is, the biosensing element of the microbial electrochemical sensor is quickly constructed by magnetic force. Its disadvantages are: 1) Nanoscale magnetic particles need to be used as carriers of microorganisms, and the procurement cost of these magnetic particles may be high, especially for high-quality nanomagnetic particles with good biocompatibility and conductivity. 2) Specialized equipment is required to apply specific magnetic forces, which increases the complexity of the operation and equipment requirements. 3) Microorganisms attached to the surface of magnetic particles may produce stress responses to them, affecting their normal physiological functions and metabolic activities, resulting in a decrease in their electrical activity. Summary of the invention

[0005] The present application aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the purpose of the present application is to propose a multilayer electrochemically active biofilm, a preparation method and a microbial electrochemical sensor. The multilayer self-enriching electrochemically active biofilm of the present application includes a multilayer dense biofilm formed from the inner layer to the outer layer, which effectively increases the amount of active EAB, effectively makes the biological properties of the electrochemically active biofilm more stable and the electricity production performance higher, and effectively solves the problems of thin traditional pure bacteria biofilms, low active EAB content, and unstable electricity production performance. In addition, the multilayer self-enriching electrochemically active biofilm of the present application is naturally formed by utilizing the self-enrichment of EAB, retaining the original physiological metabolism and electrocatalytic activity of microorganisms to the greatest extent, and the active EAB biological community is evenly distributed on the conductive material and has a high adsorption strength, thereby solving the problem of instability of the passively formed biofilm under the influence of external forces.

[0006] In one aspect of the present application, the present application proposes a multilayer electrochemically active biofilm. According to an embodiment of the present application, the multilayer electrochemically active biofilm comprises: A plurality of sheet-like conductive materials are stacked in sequence, fixed with metal wires, and both sides of each sheet-like conductive material are respectively attached with pure electrochemically active microorganisms.

[0007] According to the multilayer electrochemically active biofilm of the embodiment of the present application, including the multilayer dense biofilm formed from the inner layer to the outer layer, the amount of active EAB can be effectively increased, and the biological performance of the electrochemically active biofilm can be effectively made more stable and the electricity production performance higher, thereby reducing the interference of other factors in the detection process, effectively solving the problems of thin traditional pure bacteria biofilm, low amount of active EAB, unstable electricity production performance, etc., and improving the sensitivity of MES to toxic pollutants. In addition, the multilayer automatic enrichment electrochemically active biofilm of the present application is formed naturally by utilizing the automatic enrichment of EAB, and is formed by promoting the natural aggregation and secretion of extracellular polymers of microorganisms under a mild physiological environment, which retains the original physiological metabolism and electrocatalytic activity of microorganisms to the greatest extent. The active EAB biological community is evenly distributed on the conductive material and has a high adsorption strength, thereby solving the problem of instability of the passively formed biofilm under external force (for example, using adhesives or magnetic particles). At the same time, the multilayer automatic enrichment electrochemically active biofilm provided by the present application can effectively combine the two stages of EAB culture and later biofilm preparation into one, greatly simplifying the operation process and improving work efficiency.

[0008] In addition, the multilayer electrochemically active biofilm according to the above embodiment of the present application may also have the following additional technical features: In some embodiments of the present application, 3 to 7 of the sheet-like conductive materials are stacked in sequence.

[0009] In some embodiments of the present application, the pure strain electrochemically active microorganism includes one of Loyshewanella PV-4, Loyshewanella MR-1 and Acinetobacter baylyi DB-4; and / or the sheet-like conductive material includes at least one of carbon felt and carbon cloth; and / or the metal wire includes at least one of platinum wire, silver wire, gold wire and titanium wire.

[0010] In some embodiments of the present application, the metal wire is fixed at the center or both ends of the plurality of sheet-shaped conductive materials.

[0011] In some embodiments of the present application, the multilayer electrochemically active biofilm further includes: a working electrode lead, wherein the working electrode lead is connected to the metal wire.

[0012] In the second aspect of the present application, the present application proposes a microbial electrochemical sensor. According to an embodiment of the present application, the above-mentioned microbial electrochemical sensor includes: a reference electrode, a counter electrode and a working electrode, and the working electrode includes a multilayer electrochemically active biofilm of the above embodiment. Therefore, the microbial electrochemical sensor of the present application uses a multilayer self-enriching electrochemically active biofilm as a working electrode, which effectively increases the amount of active EAB on the working electrode, making the biological properties of the electrochemically active biofilm more stable and the electricity production performance higher, thereby reducing the interference of other factors in the detection process, effectively solving the problems of thin traditional pure bacterial biofilms, low active EAB amounts, unstable electricity production performance, etc., and improving the sensitivity of the microbial electrochemical sensor MES to toxic pollutants.

[0013] In the third aspect of the present application, the present application proposes a method for preparing the multilayer electrochemically active biofilm of the above embodiment. According to the embodiment of the present application, the method comprises: (1) providing a plurality of sheet-shaped conductive materials, and disposing a metal wire on at least one of the sheet-shaped conductive materials; (2) placing a sheet-shaped conductive material provided with metal wires, a sheet-shaped conductive material not provided with metal wires, and a culture medium in a culture container; (3) inoculating pure electrochemically active microorganisms into the culture container and culturing them so that the pure electrochemically active microorganisms adhere to both sides of each of the sheet-like conductive materials; (4) Using the metal wires on the sheet-like conductive material, the sheet-like conductive material without the metal wires is sequentially stacked and fixed onto the sheet-like conductive material with the metal wires to obtain a multilayer electrochemically active biofilm.

[0014] According to the method for preparing a multilayer electrochemically active biofilm according to the embodiment of the present application, the multilayer dense biofilm formed from the inner layer to the outer layer can effectively increase the amount of active EAB, and can effectively make the biological performance of the electrochemically active biofilm more stable and the electricity production performance higher, thereby reducing the interference of other factors in the detection process, effectively solving the problems of thin traditional pure bacteria biofilm, low amount of active EAB, unstable electricity production performance, etc., and improving the sensitivity of MES to toxic pollutants. In addition, the method utilizes the automatic enrichment of EAB to naturally form a multilayer automatic enrichment electrochemically active biofilm, and promotes the natural aggregation and secretion of extracellular polymers of microorganisms under a mild physiological environment to form a multilayer automatic enrichment electrochemically active biofilm, which retains the original physiological metabolism and electrocatalytic activity of microorganisms to the greatest extent, thereby solving the problem of instability of passively formed biofilms under external force (for example, using adhesives or magnetic particles). At the same time, the method of the present application can effectively combine the two stages of EAB culture and later biofilm preparation into one, greatly simplifying the operation process and improving work efficiency.

[0015] 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, in step (2), the culture medium includes at least one of LB culture medium, beef extract peptone culture medium, tryptic soy culture medium and tryptic yeast culture medium.

[0016] In some embodiments of the present application, step (3) includes: (3-1) inoculating pure electrochemically active microorganisms into the culture container and culturing on a shaking platform for 8 h to 15 h; (3-2) pouring out 50% to 80% of the culture medium in the culture container, adding an equal amount of fresh culture medium, and culturing on a shaking platform for 8 h to 15 h.

[0017] In a fourth aspect of the present application, the present application proposes another method for preparing the multilayer electrochemically active biofilm of the above embodiment. According to an embodiment of the present application, the method comprises: (a) providing a plurality of sheet-shaped conductive materials, and disposing a metal line on at least one of the sheet-shaped conductive materials; (b) placing a sheet-shaped conductive material provided with metal wires and a culture medium in a culture container; (c) inoculating pure strains of electrochemically active microorganisms into the culture container, culturing, and using the metal wires on the sheet-like conductive material to laminate and fix a sheet-like conductive material without a metal wire on both sides of the sheet-like conductive material with the metal wire, and continuing the culturing until all the sheet-like conductive materials without a metal wire are laminated and fixed on both sides of the sheet-like conductive material with the metal wire, thereby obtaining a multilayer electrochemically active biofilm.

[0018] According to the method for preparing a multilayer electrochemically active biofilm according to the embodiment of the present application, the multilayer dense biofilm formed from the inner layer to the outer layer can effectively increase the amount of active EAB, and can effectively make the biological performance of the electrochemically active biofilm more stable and the electricity production performance higher, thereby reducing the interference of other factors in the detection process, effectively solving the problems of thin traditional pure bacteria biofilm, low amount of active EAB, unstable electricity production performance, etc., and improving the sensitivity of MES to toxic pollutants. In addition, the method utilizes the automatic enrichment of EAB to naturally form a multilayer automatic enrichment electrochemically active biofilm, and promotes the natural aggregation and secretion of extracellular polymers of microorganisms under a mild physiological environment to form a multilayer automatic enrichment electrochemically active biofilm, which retains the original physiological metabolism and electrocatalytic activity of microorganisms to the greatest extent. The active EAB biological community is evenly distributed on the conductive material and has a high adsorption strength, thereby solving the problem of instability of the passively formed biofilm under external force (for example, using adhesives or magnetic particles). At the same time, the method of the present application can effectively combine the two stages of EAB culture and later biofilm preparation into one, greatly simplifying the operation process and improving work efficiency.

[0019] 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, in step (b), the culture medium includes at least one of LB medium, beef extract peptone medium, tryptic soytone medium and tryptic yeast medium.

[0020] In some embodiments of the present application, step (c) includes: (c-1) inoculating the pure electrochemically active microorganism into the culture container and culturing on a shaking table for 8 hours to 15 hours; (c-2) using the metal wire on the sheet conductive material to stack and fix a sheet conductive material without a metal wire on both sides of the sheet conductive material with a metal wire, and at the same time pouring out 50% to 80% of the culture medium in the culture container, adding an equal amount of fresh culture medium, and culturing on a shaking table for 8 hours to 15 hours; (c-3) repeating step (c-2) until all the sheet conductive materials without a metal wire are stacked and fixed on both sides of the sheet conductive material with a metal wire to obtain a multilayer electrochemically active biofilm.

[0021] 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

[0022] 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 structure of the multilayer electrochemically active biofilm according to an embodiment of the present application.

[0023] Figure 2 Schematic diagram of a process for preparing a multilayer electrochemically active biofilm according to some embodiments of the present application.

[0024] Figure 3 Schematic diagram of the process of preparing a multilayer electrochemically active biofilm according to some other embodiments of the present application.

[0025] 10-sheet conductive material, 20-metal wire, 30-pure electrochemically active microorganisms, 40-working electrode lead. DETAILED DESCRIPTION

[0026] 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.

[0027] In one aspect of the present application, the present application provides a multilayer electrochemically active biofilm. Figure 1 The multilayer electrochemically active biofilm comprises: a plurality of sheet-like conductive materials 10, the plurality of sheet-like conductive materials 10 are stacked in sequence, the plurality of sheet-like conductive materials 10 are fixed by metal wires 20, and pure electrochemically active microorganisms 30 are attached to the surfaces of both sides of each sheet-like conductive material 10, that is, the sheet-like conductive materials 10 serve as carriers of pure electrochemically active microorganisms 30, and pure electrochemically active microorganisms 30 are attached between adjacent sheet-like conductive materials 10 and to the surface of the outermost layer of the multilayer self-enriching electrochemically active biofilm.

[0028] The multilayer electrochemically active biofilm proposed in this application is described in detail below: The multilayered self-enriching electrochemically active biofilm provided by the present application includes a plurality of sheet-like conductive materials stacked in sequence, and pure electrochemically active microorganisms are attached to both sides of each sheet-like conductive material. That is to say, the multilayered self-enriching electrochemically active biofilm of the present application includes a multilayered dense biofilm formed from the inner layer to the outer layer, which can effectively increase the amount of active EAB, and can effectively make the biological performance of the electrochemically active biofilm more stable and the electricity production performance higher, thereby reducing the interference of other factors in the detection process, effectively solving the problems of thin traditional pure bacteria biofilm, low amount of active EAB, unstable electricity production performance, etc., and improving the sensitivity of MES to toxic pollutants. In addition, the multilayered self-enriching electrochemically active biofilm of the present application is naturally formed by utilizing the self-enrichment of EAB, and is formed by promoting the natural aggregation and secretion of extracellular polymers of microorganisms under a mild physiological environment, retaining the original physiological metabolism and electrocatalytic activity of microorganisms to the greatest extent, and the active EAB biological community is evenly distributed on the conductive material and has a high adsorption strength, thereby solving the problem of instability of passively formed biofilms under external force (for example, using adhesives or magnetic particles). At the same time, the multilayered self-enriching electrochemically active biofilm provided in the present application can effectively combine the two-stage operations of EAB culture and subsequent biofilm preparation into one, greatly simplifying the operation process and improving work efficiency.

[0029] According to some specific embodiments of the present application, the multilayer self-enriching electrochemically active biofilm is stacked in sequence by 3 to 7 sheets of conductive materials, and pure electrochemically active microorganisms are attached between adjacent sheets of conductive materials and on the surface of the outermost sheet of conductive materials. In other words, the multilayer self-enriching electrochemically active biofilm of the present application includes 3 to 7 layers of dense biofilm formed from the inner layer to the outer layer, which can further effectively increase the amount of active EAB, and can further effectively make the biological performance of the electrochemically active biofilm more stable and the electricity production performance higher, and further effectively solve the problems of thin traditional pure bacteria biofilm, low amount of active EAB, and unstable electricity production performance.

[0030] According to some further specific embodiments of the present application, the above-mentioned pure electrochemically active microorganisms include one of Leujewanella PV-4, Leujewanella MR-1 and Acinetobacter bayenii DB-4. As mentioned above, pure electrochemically active microorganisms often have weak electricity production performance, the signal detection process is easily disturbed, and pure EAB is very easy to die, further increasing the difficulty of detection. The present application provides a multi-layer self-enriching electrochemically active biofilm, which effectively increases the amount of active EAB, effectively makes the biological performance of the electrochemically active biofilm more stable and the electricity production performance higher, thereby effectively solving the problems of thin traditional pure bacterial biofilms, low active EAB content, and unstable electricity production performance.

[0031] According to some further specific embodiments of the present application, a loose and porous sheet-like conductive material is selected as the carrier, and the sheet-like conductive material includes at least one of carbon felt and carbon cloth.

[0032] In the embodiments of the present application, the specific type of the above-mentioned metal wire is not particularly limited. As some preferred embodiments, the above-mentioned metal wire includes at least one of platinum wire, silver wire, gold wire and titanium wire.

[0033] According to some other specific embodiments of the present application, refer to the attached Figure 1 The metal wire 20 is fixed at the center or both ends of multiple sheet-like conductive materials 10. Specifically, the metal wire can be set at the center or both ends of one of the sheet-like conductive materials, and then the metal wire is used to fix other sheet-like conductive materials without metal wires, so as to form a multi-layer self-enriching electrochemically active biofilm.

[0034] According to some other specific embodiments of the present application, refer to the attached Figure 1 The multilayer self-enriching electrochemically active biofilm further includes: a working electrode lead 40, which is connected to the metal wire 20, and can be used as a connector for connecting other parts of the biosensor when the biosensor is subsequently manufactured. As a specific embodiment, the working electrode lead 40 and the metal wire 20 can be integrally formed, and the working electrode lead 40 can be formed by excess metal wire 20.

[0035] In the second aspect of the present application, the present application proposes a microbial electrochemical sensor. According to an embodiment of the present application, the above-mentioned microbial electrochemical sensor includes: a reference electrode, a counter electrode and a working electrode, and the working electrode includes a multilayer electrochemically active biofilm of the above embodiment. Therefore, the microbial electrochemical sensor of the present application uses a multilayer self-enriching electrochemically active biofilm as a working electrode, which effectively increases the amount of active EAB on the working electrode, making the biological properties of the electrochemically active biofilm more stable and the electricity production performance higher, thereby reducing the interference of other factors in the detection process, effectively solving the problems of thin traditional pure bacterial biofilms, low active EAB amounts, unstable electricity production performance, etc., and improving the sensitivity of the microbial electrochemical sensor MES to toxic pollutants.

[0036] In the third aspect of the present application, the present application proposes a method for preparing the multilayer electrochemically active biofilm of the above embodiment. Figure 2 The method includes: S100: providing a plurality of sheet-like conductive materials, and setting a metal wire on at least one sheet-like conductive material; S200: placing the sheet-like conductive material with the metal wire, the sheet-like conductive material without the metal wire, and a culture medium in a culture container; S300: inoculating pure electrochemically active microorganisms in the culture container and culturing them so that the pure electrochemically active microorganisms adhere to both sides of each sheet-like conductive material; S400: using the metal wire on the sheet-like conductive material to stack and fix the sheet-like conductive material without the metal wire on the sheet-like conductive material in sequence, so as to obtain a multi-layer self-enriching electrochemically active biofilm.

[0037] Specifically, refer to the attached Figure 2 The method for preparing a multilayer electrochemically active biofilm comprises the following steps: S100: providing a plurality of sheet-shaped conductive materials, and disposing a metal wire on at least one sheet-shaped conductive material; In this step, a plurality of loose and porous sheet-like conductive materials are provided, a metal wire is arranged on at least one sheet-like conductive material (for example, a metal wire is fixed at both ends or at the center of the sheet-like conductive material), and no metal wire is arranged on the remaining sheet-like conductive materials.

[0038] S200: placing a sheet-shaped conductive material provided with metal wires, a sheet-shaped conductive material not provided with metal wires, and a culture medium in a culture container; In this step, the culture medium can be prepared first and placed in a culture container, and the sheet-shaped conductive material with fixed metal wires and the sheet-shaped conductive material without metal wires are placed therein, sterilized with high-temperature and high-pressure steam, and cooled for use.

[0039] In the embodiments of the present application, the specific type of the above-mentioned culture medium is not particularly limited. LB culture medium (i.e., Luria-Bertani culture medium) can be selected, or other conventional bacterial culture media can be selected, such as beef extract peptone culture medium, tryptic soy peptone culture medium, tryptic yeast culture medium, etc.

[0040] S300: inoculating pure electrochemically active microorganisms into a culture container and culturing them so that the pure electrochemically active microorganisms adhere to both sides of each sheet-like conductive material; In this step, the EAB strains frozen in a low-temperature (e.g. -80°C) refrigerator can be taken out first, thawed at room temperature, and then streaked on LB plate culture medium, and cultured at a constant temperature (e.g. 30°C) for activation. Select a single colony and inoculate it into LB liquid test tube culture medium, and culture it on a shaking table for 10h~12h. Take a small amount of test tube bacterial liquid and culture it in the culture container of step S200 to obtain a multi-layer self-enriching electrochemically active biofilm.

[0041] Specifically, step S300 includes: S310: inoculating the pure electrochemically active microorganisms into the culture container and culturing on a shaking table for 8h~15h (preferably 10h~12h), at which time the nutrients are almost exhausted; S320: Pour out 50% to 80% of the culture medium in the culture container, add an equal amount of fresh culture medium, and then culture on a shaking table for 8h to 15h (preferably 10h to 12h) so that both sides of each sheet-like conductive material are attached with pure electrochemically active microorganisms, that is, each sheet-like conductive material forms a layer of EAB biofilm.

[0042] The shaking culture can make the microorganisms constantly collide and contact with the biofilm during the culture process, which increases the chance of the microorganisms attaching to the biofilm, helps to form a more uniform and dense biofilm, and ensures the efficient performance of the biofilm in electrochemical sensing applications.

[0043] S400: using the metal wires on the sheet-like conductive material, the sheet-like conductive material without the metal wires is sequentially stacked and fixed onto the sheet-like conductive material with the metal wires, so as to obtain a multi-layer self-enriching electrochemically active biofilm.

[0044] In this step, after the cultivation in step S300 is completed, all the sheet-shaped conductive materials with active EAB enriched on the surface are taken out, and the sheet-shaped conductive materials without metal wires are stacked and fixed to the sheet-shaped conductive materials with metal wires in sequence using the metal wires on the sheet-shaped conductive materials to obtain a multi-layer self-enriching electrochemically active biofilm. Furthermore, the excess metal wires can form working electrode leads, which can be used as connectors for connecting other parts of the biosensor when the biosensor is subsequently manufactured.

[0045] According to the method for preparing a multilayer electrochemically active biofilm according to the embodiment of the present application, the multilayer dense biofilm formed from the inner layer to the outer layer can effectively increase the amount of active EAB, and can effectively make the biological performance of the electrochemically active biofilm more stable and the electricity production performance higher, thereby reducing the interference of other factors in the detection process, effectively solving the problems of thin traditional pure bacteria biofilm, low amount of active EAB, unstable electricity production performance, etc., and improving the sensitivity of MES to toxic pollutants. In addition, the method utilizes the automatic enrichment of EAB to naturally form a multilayer automatic enrichment electrochemically active biofilm, and promotes the natural aggregation and secretion of extracellular polymers of microorganisms under a mild physiological environment to form a multilayer automatic enrichment electrochemically active biofilm, which retains the original physiological metabolism and electrocatalytic activity of microorganisms to the greatest extent, thereby solving the problem of instability of passively formed biofilms under external force (for example, using adhesives or magnetic particles). At the same time, the method of the present application can effectively combine the two stages of EAB culture and later biofilm preparation into one, greatly simplifying the operation process and improving work efficiency.

[0046] In the fourth aspect of the present application, the present application proposes another method for preparing the multilayer electrochemically active biofilm of the above embodiment. According to the embodiment of the present application, referring to the attached Figure 3 The method includes: S1000: providing a plurality of sheet-like conductive materials, and setting a metal wire on at least one sheet-like conductive material; S2000: placing the sheet-like conductive material set with the metal wire and a culture medium in a culture container; S3000: inoculating pure electrochemically active microorganisms in the culture container, culturing, and using the metal wire on the sheet-like conductive material to stack and fix a sheet-like conductive material without a metal wire on both sides of the sheet-like conductive material set with the metal wire, and continuing to cultivate until all the sheet-like conductive materials without a metal wire are stacked and fixed on both sides of the sheet-like conductive material set with the metal wire, so as to obtain a multi-layer self-enriching electrochemically active biofilm.

[0047] Specifically, refer to the attached Figure 3 The method for preparing a multilayer electrochemically active biofilm comprises the following steps: S1000: providing a plurality of sheet-shaped conductive materials, and disposing a metal wire on at least one sheet-shaped conductive material; In this step, a plurality of loose and porous sheet-like conductive materials are provided, a metal wire is arranged on at least one sheet-like conductive material (for example, a metal wire is fixed at both ends or at the center of the sheet-like conductive material), and no metal wire is arranged on the remaining sheet-like conductive materials.

[0048] S2000: placing a sheet-shaped conductive material provided with metal wires and a culture medium in a culture container; In this step, a culture medium may be prepared first and placed in a culture container, and a sheet-shaped conductive material with a metal wire fixed therein may be placed therein, sterilized with high temperature and high pressure steam, and cooled for later use.

[0049] In the embodiments of the present application, the specific type of the above-mentioned culture medium is not particularly limited. LB culture medium (i.e., Luria-Bertani culture medium) can be selected, or other conventional bacterial culture media can be selected, such as beef extract peptone culture medium, tryptic soy peptone culture medium, tryptic yeast culture medium, etc.

[0050] S3000: Inoculate pure electrochemically active microorganisms into a culture container, culture, use metal wires on the sheet conductive material to stack and fix a sheet conductive material without a metal wire on both sides of the sheet conductive material with the metal wire, and continue to culture until all the sheet conductive materials without the metal wire are stacked and fixed on both sides of the sheet conductive material with the metal wire, so as to obtain a multi-layer self-enriching electrochemically active biofilm.

[0051] In this step, the EAB strains frozen in a low-temperature (e.g. -80°C) refrigerator can be taken out first, thawed at room temperature, and then streaked on LB plate culture medium, and cultured at a constant temperature (e.g. 30°C) for activation. Select a single colony and inoculate it into LB liquid test tube culture medium, and culture it on a shaking table for 10h~12h. Take a small amount of test tube bacterial liquid and culture it in the culture container of step S2000 to obtain a multi-layer self-enriching electrochemically active biofilm.

[0052] Specifically, step S3000 includes: S3100: Inoculate pure electrochemically active microorganisms in a culture container and culture them on a shaker for 8h~15h (preferably 10h~12h). The pure electrochemically active microorganisms adhere to the surfaces of both sides of the sheet-like conductive material provided with metal wires, and the nutrients are almost exhausted at this time; S3200: using the metal wire on the sheet-like conductive material to laminate and fix a sheet-like conductive material without a metal wire on both sides of the sheet-like conductive material with the metal wire, pouring out 50% to 80% of the culture medium in the culture container, adding an equal amount of fresh culture medium, and culturing on a shaking table for 8h to 15h (preferably 10h to 12h), pure electrochemically active microorganisms adhere to the surfaces of both sides of the newly added sheet-like conductive material, at which time the nutrients are almost exhausted; S3300: Repeat step S3200 until all sheet-like conductive materials without metal wires are stacked and fixed on both sides of sheet-like conductive materials with metal wires, and both sides of all sheet-like conductive materials are attached with pure electrochemically active microorganisms to obtain a multilayer self-enriching electrochemically active biofilm.

[0053] The shaking culture can make the microorganisms constantly collide and contact with the biofilm during the culture process, which increases the chance of the microorganisms attaching to the biofilm, helps to form a more uniform and dense biofilm, and ensures the efficient performance of the biofilm in electrochemical sensing applications.

[0054] According to the method for preparing a multi-layer self-enriching electrochemically active biofilm according to the embodiment of the present application, the multi-layer dense biofilm formed from the inner layer to the outer layer can effectively increase the amount of active EAB, and can effectively make the biological performance of the electrochemically active biofilm more stable and the electricity production performance higher, thereby reducing the interference of other factors in the detection process, effectively solving the problems of thin traditional pure bacteria biofilm, low amount of active EAB, unstable electricity production performance, etc., and improving the sensitivity of MES to toxic pollutants. In addition, the method utilizes the self-enrichment of EAB to naturally form a multi-layer self-enriching electrochemically active biofilm, and promotes the natural aggregation and secretion of extracellular polymers of microorganisms under a mild physiological environment to form a multi-layer self-enriching electrochemically active biofilm, which retains the original physiological metabolism and electrocatalytic activity of microorganisms to the greatest extent, thereby solving the problem of instability of passively formed biofilms under external force (such as using adhesives or magnetic particles). At the same time, the method of the present application can effectively combine the two stages of EAB culture and late biofilm preparation into one, greatly simplifying the operation process and improving work efficiency.

[0055] 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.

[0056] Comparative Example 1 Step 1: Preparation of culture medium, sterilization of materials, and inoculation and culture. Prepare Luria-Bertani culture medium (LB culture medium) in a conical flask. Each liter of LB culture medium contains 10g NaCl, 10g tryptone, and 5g yeast extract. Each bottle of 300mL LB culture medium is sterilized at 121℃ high temperature and high pressure steam for 25min, and cooled to room temperature for use. Take out the EAB strain (Shewanella loihica PV-4) frozen in a -80℃ refrigerator, thaw at room temperature, and inoculate the strain on the LB plate culture medium by streaking, and activate it by constant temperature (30℃). Select a single colony and inoculate it into LB liquid test tube culture medium, and shake it for 12h. Take 2mL of test tube bacterial liquid and culture it in a conical flask containing 300mL LB culture medium for 12h to obtain EAB active bacterial suspension.

[0057] Step 2: Prepare artificial EAB biofilm by negative pressure filtration. Spread loose porous conductive material carbon felt (thickness 2mm) on the bottom of the filtration cup, pour in the EAB active bacterial suspension in step 1, connect the filtration bottle and vacuum pump for filtration, the EAB in the EAB active bacterial suspension is trapped in the carbon felt, and the liquid is pumped into the filtration bottle. The filtration can be repeated several times to allow more EAB to adhere to the carbon felt; the last time, pour in 0.9% NaCl solution for filtration to wash the residual culture medium in the carbon felt.

[0058] Step 3: Test application: The test method is the same as that of Example 1.

[0059] Example 1 Step 1: Preparation of multilayer biofilm carrier material. Select a loose and porous sheet-like conductive material (here, carbon cloth with a thickness of about 1.5 mm) as the carrier and cut it into discs with a diameter of 2 cm. A platinum wire with a diameter of 0.5 mm is fixed at each end of some of the discs of carbon cloth. The platinum wire at one end is 1 cm long and is used to fix the multilayer carbon cloth, and the platinum wire at the other end is 2 cm long. In addition to fixing the carbon cloth, it also serves as an electrode lead to connect other parts of the biosensor when the biosensor is made later.

[0060] Step 2: Culture medium preparation and material sterilization. Prepare Luria-Bertani culture medium (LB culture medium) in a conical flask. Each liter of LB culture medium contains 10g NaCl, 10g tryptone, and 5g yeast extract. Place the disc of carbon cloth in it. Place 300mL LB culture medium, 2 pieces of carbon cloth with electrode leads, and 8 pieces of carbon cloth without platinum wire in each bottle. Sterilize at 121℃ high temperature and high pressure steam for 25min, and cool to room temperature for use.

[0061] Step 3: Inoculation and preparation of biofilm. Take out the EAB strain (Shewanellaloihica PV-4) frozen in a -80℃ refrigerator, thaw at room temperature, and then streak the strain on the LB plate medium, and activate it by constant temperature (30℃). Select a single colony and inoculate it into the LB liquid test tube culture medium, and culture it on a shaking table for 12 hours. Take 2mL of the test tube bacterial liquid and culture it in the LB conical flask in step 2. After culturing for 12 hours, pour out about 200mL of the original culture liquid, add 200mL of fresh LB culture liquid, and continue to culture for another 12 hours. After the culture is completed, take out the carbon cloth disc with active EAB enriched on the surface, and stack 4 pieces of carbon cloth without platinum wire on 1 piece of carbon cloth with electrode leads in turn. Fix the two ends with platinum wire to form a set of multilayer EAB biofilms. Here, two sets of five-layer EAB biofilms are made.

[0062] Step 4: Test application. A group of multilayer EAB biofilms can be assembled into a microbial electrochemical sensor MES. Here, two MESs are assembled, marked as MES1 and MES2 respectively. The microbial electrochemical sensors MES1 and MES2 are connected to the peristaltic pump and the electrochemical workstation respectively. First, non-toxic DM electrolyte is introduced into MES1 and MES2 at the same time, and the initial electrical signal I is recorded by the electrochemical workstation. 01 ,I 02 ; Then introduce DM electrolyte containing toxic pollutants, and use an electrochemical workstation to record the test electrical signal I 11 ,I 12 ; Calculate the inhibition rate of toxic substances R1 = (I 01 -I 11 )I 01 *100%, R2=(I 02 -I 12 )I 02 *100%.

[0063] Non-toxic DM electrolyte composition: each liter contains 2.5g NaHCO3, 1.0g NH4Cl, 0.08g CaCl2·H2O, 0.2g MgCl2·6H2O, 10g NaCl, 7.2g HEPES, 0.12g yeast extract and 2mM sodium acetate.

[0064] DM electrolyte composition containing toxic pollutants: high concentration of Pb is added to non-toxic DM electrolyte 2+ Prepare the standard stock solution to a final concentration of 0.2 mg / L.

[0065] Example 2 The preparation method of this embodiment is basically the same as that of embodiment 1, except that: The EAB strain used in step 3 was replaced with Shewanella loihica MR-1, and other operations remained unchanged.

[0066] Example 3 The preparation method of this embodiment is basically the same as that of embodiment 1, except that: Step 3: Inoculation and preparation of biofilm. (1) The EAB strain type, plate activation, test tube culture and other operations are the same as in Example 1, except for the process of automatic enrichment to form EAB biofilm. (2) Remove the excess carbon cloth from the conical flask, leaving only 2 pieces of carbon cloth with platinum wire fixed in each flask, add 2mL of test tube bacterial solution and culture for 12h under the same conditions; (3) After 12h of culture, pour out about 200mL of the original culture solution, add 200mL of fresh LB culture solution, and fix another piece of carbon cloth on both sides of the original carbon cloth with platinum wire fixed, fix tightly, and continue to culture for 12h; after 12h of culture, repeat step (3) once to obtain two groups of five-layer EAB biofilms.

[0067] The test results of Comparative Example 1 and Examples 1 to 3 are shown in Table 1.

[0068] Table 1

[0069] As can be seen from Table 1, the electrical signals and toxicity inhibition rates of Examples 1 to 3 are significantly improved compared with Comparative Example 1. It can also be seen from Table 1 that, compared with Example 1, the electrical signal of Example 3 is more significantly improved, but the inhibition rate is slightly lower.

[0070] 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.

[0071] 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 multilayer electrochemically active biofilm, characterized in that: include: A plurality of sheet-like conductive materials are stacked in sequence, fixed with metal wires, and both sides of each sheet-like conductive material are respectively attached with pure electrochemically active microorganisms.

2. The multilayer electrochemically active biofilm according to claim 1, characterized in that 3 to 7 of the sheet-like conductive materials are stacked in sequence.

3. The multilayer electrochemically active biofilm according to claim 1, characterized in that: The pure electrochemically active microorganism includes one of Leucovorus PV-4, Leucovorus MR-1 and Acinetobacter bayleyi DB-4; And / or, the sheet-like conductive material includes at least one of carbon felt and carbon cloth; And / or, the metal wire includes at least one of platinum wire, silver wire, gold wire and titanium wire.

4. The multilayer electrochemically active biofilm according to claim 1, characterized in that The metal wire is fixed at the center or both ends of the plurality of sheet-shaped conductive materials.

5. The multilayer electrochemically active biofilm according to claim 1, characterized in that Also includes: A working electrode lead is connected to the metal wire.

6. A microbial electrochemical sensor, characterized in that: include: A reference electrode, a counter electrode, and a working electrode, wherein the working electrode comprises the multilayer electrochemically active biofilm according to any one of claims 1 to 5.

7. A method for preparing the multilayer electrochemically active biofilm according to any one of claims 1 to 5, characterized in that: include: (1) providing a plurality of sheet-shaped conductive materials, and disposing a metal wire on at least one of the sheet-shaped conductive materials; (2) placing a sheet-shaped conductive material provided with metal wires, a sheet-shaped conductive material not provided with metal wires, and a culture medium in a culture container; (3) inoculating pure electrochemically active microorganisms into the culture container and culturing them so that the pure electrochemically active microorganisms adhere to both sides of each of the sheet-like conductive materials; (4) Using the metal wires on the sheet-like conductive material, the sheet-like conductive material without the metal wires is sequentially stacked and fixed onto the sheet-like conductive material with the metal wires to obtain a multilayer electrochemically active biofilm.

8. The method according to claim 7, characterized in that In step (2), the culture medium includes at least one of LB culture medium, beef extract peptone culture medium, tryptic soy culture medium and tryptic yeast culture medium; And / or, step (3) includes: (3-1) inoculating the pure electrochemically active microorganisms into the culture container and culturing on a shaking table for 8 h to 15 h; (3-2) Pour out 50% to 80% of the culture medium in the culture container, add an equal amount of fresh culture medium, and culture on a shaking platform for 8 h to 15 h.

9. A method for preparing the multilayer electrochemically active biofilm according to any one of claims 1 to 5, characterized in that: include: (a) providing a plurality of sheet-shaped conductive materials, and disposing a metal line on at least one of the sheet-shaped conductive materials; (b) placing a sheet-shaped conductive material provided with metal wires and a culture medium in a culture container; (c) inoculating pure strains of electrochemically active microorganisms into the culture container, culturing, and using the metal wires on the sheet-like conductive material to laminate and fix a sheet-like conductive material without a metal wire on both sides of the sheet-like conductive material with the metal wire, and continuing the culturing until all the sheet-like conductive materials without a metal wire are laminated and fixed on both sides of the sheet-like conductive material with the metal wire, thereby obtaining a multilayer electrochemically active biofilm.

10. The method according to claim 9, characterized in that In step (b), the culture medium includes at least one of LB culture medium, beef extract peptone culture medium, tryptic soy culture medium and tryptic yeast culture medium; and / or, step (c) comprises: (c-1) inoculating the pure electrochemically active microorganism into the culture container and culturing on a shaking table for 8 h to 15 h; (c-2) using the metal wire on the sheet-like conductive material to laminate and fix a sheet-like conductive material without a metal wire on both sides of the sheet-like conductive material with the metal wire, and at the same time pouring out 50% to 80% of the culture medium in the culture container, adding an equal amount of fresh culture medium, and culturing on a shaking platform for 8 hours to 15 hours; (c-3) Repeat step (c-2) until all the sheet-like conductive materials without metal wires are stacked and fixed on both sides of the sheet-like conductive materials with metal wires to obtain a multilayer electrochemically active biofilm.

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