Targeting microbial membranes with electrodes and their application in electrochemical detection of new pollutants

Abstract

The application relates to the technical field of electrochemical sensors and environmental monitoring, and discloses a targeted microbial membrane electrode and application of the microbial membrane electrode in electrochemical detection of new pollutants. The membrane electrode comprises a flexible substrate and a membrane layer on the surface of the flexible substrate, and the membrane layer is prepared by printing, curing and drying of a full-water-based bio-conductive ink. The ink comprises a buffer conductive base ink system and a bioactive recognition protection system. During preparation, the slow acid hydrolysis of gluconic acid-delta-lactone is used to stimulate the release of divalent calcium ions from calcium carbonate, so that sodium alginate, carbon nanotubes and an electronic mediator are crosslinked in coordination, the electronic mediator and microorganisms are fixed in the conductive network, and the loss of the mediator is limited. In combination with a step-by-step pulping, in-situ crosslinking and step vacuum dewatering process, and the physical protection of trehalose, the activity loss of the bacterial body is reduced. The application can be used for electrochemical detection of new pollutants in water bodies, quantitative analysis is carried out by extracting a characteristic peak current attenuation value, and the stability of detection and the storage period of the electrode are improved.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensors and environmental monitoring technology, specifically to targeted microbial membrane electrodes and their application in the electrochemical detection of new pollutants. Background Technology

[0002] With the development of modern industry and the chemical industry, the problem of emerging pollutants in the aquatic environment has received increasing attention. These emerging pollutants mainly include antibiotics (such as sulfonamides) and persistent organic pollutants (such as perfluorinated compounds), which, although present at low concentrations in water bodies, exhibit strong environmental persistence and potential biotoxicity. Traditional physicochemical analysis instruments typically require complex sample pretreatment processes and are costly. Whole-cell microbial electrochemical sensors, due to their ability to directly reflect the toxic and inhibitory effects of pollutants on organisms, are gradually becoming a research direction in the field of environmental monitoring of emerging pollutants. This technology mainly relies on electrons produced by specific microorganisms during metabolism and uses changes in electrical signals to invert the concentration of pollutants in the water.

[0003] However, existing microbial membrane electrodes still face certain technical limitations in practical preparation and application. In electrochemical systems, water-soluble electron mediators are typically doped into the electrode matrix to improve the electron transfer rate between microbial cells and inorganic electrodes. In the liquid environment of the water sample, these small-molecule electron mediators easily diffuse and leak from the fixed matrix into the surrounding water, causing a continuous drift in the electrode background signal and interfering with the stable quantitative detection of new pollutants. Simultaneously, existing membrane electrode slurry and molding processes struggle to balance material dispersibility and biocompatibility. Conventional physical stirring slurry processes generate significant mechanical shear forces, easily damaging the fragile cell walls of microorganisms. Furthermore, during the dehydration stage after electrode solidification, traditional thermal drying leads to rapid evaporation of water within the system, resulting in osmotic pressure fluctuations that disrupt cell membrane structures. This physical stress during processing not only reduces the initial survival rate of microorganisms but also causes the electrode's activity to rapidly decline during storage, limiting the product's shelf life and actual detection lifespan. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a targeted microbial membrane electrode and its application in the electrochemical detection of new pollutants. It solves the problems of rapid loss of microbial activity and easy leakage of electron mediators inside the electrode, which leads to unstable detection signals in existing whole-cell electrochemical sensors.

[0005] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a targeted microbial membrane electrode, which adopts the following technical solution: A targeted microbial membrane electrode includes a flexible substrate and a membrane layer attached to the surface of the flexible substrate. The membrane layer is made by printing, curing, and drying an all-water-based bioconductive ink comprising the following components in parts by weight: 53.6-74.3 parts deionized water, 0.3-0.8 parts tris(hydroxymethyl)aminomethane, and polysorbate-80. The composition includes: 0.1-0.3 parts sodium alginate, 1.5-3.0 parts sodium polyacrylate, 0.5-1.0 parts hydrophilic carbon black, 0.5-1.5 parts carboxylated multi-walled carbon nanotubes, 0.3-0.8 parts sodium anthraquinone-2,6-disulfonate as an electron mediator, and 1.0-2.0 parts calcium carbonate powder; the components of the bioactive recognition and protection system are: 15.0-25.0 parts concentrated mud of targeted functional strains, 2.0-4.0 parts trehalose, and 1.5-3.0 parts gluconate-δ-lactone as a slow-release acid activator; wherein, the tris(hydroxymethyl)aminomethane is used to absorb protons released in the later stage of the system to provide a pH operating window, and the calcium carbonate is used to release divalent calcium ions in situ to bridge and crosslink the sodium alginate, carboxylated multi-walled carbon nanotubes, and sodium anthraquinone-2,6-disulfonate.

[0006] By employing the above technical solution, an in-situ delayed cross-linking conductive gel network is constructed in an aqueous environment using a specific ratio of buffered conductive ink system and a bioactive recognition and protection system. The reaction mechanism is mainly as follows: gluconic acid-δ-lactone in the system undergoes ring-opening hydrolysis in the liquid phase, slowly releasing hydrogen ions. During the mixing and initial printing stages, tris(hydroxymethyl)aminomethane in the formulation preferentially binds these free hydrogen ions with its basic amino groups, playing a buffering role and maintaining the system pH at a near-neutral range, preventing irreversible damage to microbial cell membranes caused by localized over-acidity. As the reaction progresses, the buffer capacity gradually becomes saturated, and the environmental pH decreases accordingly. At this point, the weakly acidic conditions trigger the dissolution of calcium carbonate, releasing divalent calcium ions and generating carbon dioxide gas. During this process, the escape of microbubbles leaves a porous structure in the matrix; simultaneously, divalent calcium ions act as multidentate coordination centers, undergoing ion exchange and coordination with the carboxyl groups of sodium alginate molecular chains, the oxygen-containing functional groups on the surface of multi-walled carbon nanotubes, and the sulfonic acid groups of anthraquinone-2,6-disulfonate. This cross-linking reaction fixes the conductive filler, water-soluble electron mediator, and polymer backbone within the same network, thus limiting the free diffusion of the electron mediator into the outer aqueous phase to some extent. Therefore, this approach can obtain a relatively stable bioconductive composite film, mitigating signal drift caused by electron mediator loss and thereby improving the electrochemical stability of the electrode under testing conditions.

[0007] Preferably, the all-water-based bioconductive ink is made from the following components in parts by weight: 63.6 parts deionized water, 0.5 parts tris(hydroxymethyl)aminomethane, 0.2 parts polysorbate-80, 2.0 parts sodium alginate, 0.8 parts sodium polyacrylate, 4.0 parts hydrophilic carbon black, 1.0 part carboxylated multi-walled carbon nanotubes, 0.5 parts sodium anthraquinone-2,6-disulfonate, 1.5 parts calcium carbonate powder, 20.0 parts concentrated mud of targeted functional strains, 3.5 parts trehalose, and 2.4 parts glucono-δ-lactone.

[0008] By adopting the above technical solution, the chemical components with the above mass ratio basically match the stoichiometric balance of the crosslinking process. The concentration of free calcium ions can be well matched to the gelation requirements of sodium alginate, while also taking into account the net trapping effect on conductive materials, which helps to form a uniform solidified network and maintain appropriate electron transfer efficiency.

[0009] Preferably, the concentrated sludge of the targeted functional strain is prepared through the following steps: activated sludge is collected as an inoculum and placed in a bioreactor; a basic salt culture medium is used as a nutrient source, 1.0 mmol / L sodium acetate is added as a co-metabolic carbon source, and the target new pollutant is used as a screening pressure factor; during the acclimatization period, the target new pollutant is added in progressively increasing concentration gradients of 10 μg / L, 50 μg / L, 200 μg / L, and 500 μg / L, with each concentration gradient running for 7-9 days; the bacterial solution in the logarithmic growth phase is collected, centrifuged, washed, and the moisture content is adjusted to 75%-78% to obtain the final product; the target new pollutant is selected from sulfamethoxazole or perfluorooctanoic acid.

[0010] By employing the above technical solution, and through a combined acclimatization strategy that progressively increases the concentration of co-metabolite substrates and target pollutants, strains in the mixed microbial community capable of tolerating and responding to specific new pollutants are gradually enriched and become the dominant microbial community. Furthermore, by controlling the moisture content of the bacterial sludge within the range of 75%-78%, some free water is removed while the bound water of the microbial cells is retained, providing suitable solid content conditions for subsequent direct integration into the all-water-based ink system.

[0011] Preferably, the carboxylated multi-walled carbon nanotubes are prepared by the following steps: adding multi-walled carbon nanotubes to a mixed acid of concentrated sulfuric acid and concentrated nitric acid in a volume ratio of 3:1, and ultrasonically dispersing for 30-40 min to obtain a dispersion; refluxing and stirring the dispersion at 70-80℃ for 4-6 h; cooling after the reaction, washing and centrifuging until the pH of the supernatant reaches 6.5-7.0, and then vacuum drying the obtained precipitate at 60-70℃, followed by grinding and sieving for later use.

[0012] By employing the above technical solution, and through liquid-phase oxidation using a specific ratio of mixed acid and corresponding thermal reflux parameters, a large number of carboxyl functional groups are introduced into the defect sites on the surface of carbon nanotubes. This treatment not only improves the dispersibility of carbon nanotubes in water-based inks but also provides chemical bonding sites for subsequent coordination crosslinking with calcium ions.

[0013] Preferably, the flexible substrate is a polyethylene terephthalate film with conductive tracks pre-printed on its surface, and the conductive tracks are carbon conductive tracks or gold conductive tracks.

[0014] By adopting the above technical solution, the polyethylene terephthalate film with printed conductive tracks is used to collect electrons generated by microbial metabolism and channel them into the external electrochemical test circuit, in addition to providing a physical support interface for the gel-state film layer.

[0015] Secondly, the present invention provides a method for preparing a targeted microbial membrane electrode, which adopts the following technical solution: A method for preparing a targeted microbial membrane electrode includes the following steps: S1. Add all components except for the concentrated mud of the targeted functional strain, trehalose and gluconate-δ-lactone to the reaction vessel and perform high shear dispersion at room temperature to obtain a homogeneous buffer conductive ink. S2. Cool the reaction vessel and slowly add the concentrated mud of the targeted functional strain, trehalose and gluconate-δ-lactone to the buffered conductive ink obtained in step S1. Mix under low temperature and vacuum conditions with low shear folding to obtain the all-water-based bioconductive ink. S3. Transfer the all-water-based bioconductive ink obtained in step S2 to a printing device and perform roll-to-roll screen printing on the flexible substrate to obtain a wet film electrode array. S4. Place the wet film electrode array obtained in step S3 in a constant temperature and humidity environment for delayed crosslinking and in-situ foaming and curing to obtain the cured electrode array. S5. Place the electrode array cured in step S4 in a vacuum drying oven and perform step-by-step low-temperature vacuum water-retaining drying to obtain the targeted microbial membrane electrode.

[0016] By employing the above technical solution, the two-step pulping process—combining high-shear non-biological component dispersion with low-temperature low-shear biological component compounding—reduces the physical damage to microbial cell walls caused by mechanical shear force. Based on the delayed crosslinking mechanism of the formulation design, the ink maintains its fluidity at room temperature to adapt to screen printing, and after printing, it solidifies by inducing internal chemical reactions through changes in ambient temperature and humidity. Combined with subsequent stepwise dehydration treatment, a relatively uniform thin-film electrode array can be obtained while preserving the biological activity of the strain, demonstrating potential for mass production.

[0017] Preferably, in step S1, the ambient temperature is 20-25℃, the high-shear dispersion speed is 1500-2000 rpm, and the stirring time is 45-60 min; in step S2, the temperature of the system after cooling is controlled to be 4-8℃, the low-shear folding mixing speed is 30-50 rpm, the vacuum condition is to maintain the system vacuum degree at -0.05MPa to -0.08MPa, and the mixing time is 15-20 min; in step S3, the wet film thickness of the all-water-based bioconductive ink after printing on the flexible substrate is controlled to be 50-100 μm.

[0018] By adopting the above technical solution, the high shear parameter set in step S1 disrupts the van der Waals forces between the nano-carbon materials, promoting the uniform dispersion of the conductive components. In step S2, the use of vacuum low shear combined with cooling treatment slows down the hydrolysis rate of gluconic acid-δ-lactone to maintain ink fluidity and helps to remove microbubbles trapped in the system. Controlling the wet film thickness within the range of 50-100 μm ensures that the final film layer maintains good mass transfer performance, which is beneficial for substrate diffusion in subsequent tests.

[0019] Preferably, step S4 is implemented as follows: the wet film electrode array is placed in a reaction chamber with an ambient temperature of 25-30℃ and a relative humidity of 85%-95% and left to stand continuously for 2.0-3.0h; through the above environmental control, gluconate-δ-lactone in the system is hydrolyzed, and sodium alginate, carboxylated multi-walled carbon nanotubes and anthraquinone-2,6-disulfonate in the system are cross-linked to form a gel network that fixes the targeted functional strain.

[0020] By employing the above technical solution, the rapid escape of water molecules within the membrane is suppressed due to the control of a specific temperature and high humidity environment, providing and maintaining relatively stable liquid phase conditions for the hydrolysis reaction of gluconate-δ-lactone. The static settling process allows the released calcium ions to gradually diffuse between polymer molecules and initiate cross-linking, while the accompanying carbon dioxide gas slowly expands within the membrane, contributing to the formation of microporous structures.

[0021] Preferably, step S5 is implemented as follows: the temperature of the first-stage drying control plate is 20-22℃, the vacuum degree is -0.05MPa to -0.06MPa, and it is maintained for 2-3 hours; the temperature of the second-stage drying control plate is reduced to 8-10℃, the vacuum degree is increased to -0.09MPa to -0.10MPa, and it is maintained for 3-5 hours; by controlling the above-mentioned step-like dehydration path, the trehalose is dehydrated to form an amorphous glassy matrix that encapsulates and fixes the targeted microorganisms.

[0022] By employing the above technical solution, the segmented vacuum dehydration method, which deepens the vacuum through cooling, reduces the mechanical stress damage to the cell membrane caused by the rapid escape of free water. During this process, as the water content of the system gradually decreases, trehalose molecules replace the escaped water molecules through their polyhydroxyl structures, forming alternative hydrogen bonds with phospholipid groups on the surface of the microbial cell membrane. This transforms the trehalose into a high-viscosity amorphous solid, protecting the cell matrix in a low-metabolic dormant state, thus helping to extend the shelf life of the electrode products.

[0023] Thirdly, the present invention provides an application of a targeted microbial membrane electrode in the electrochemical detection of novel pollutants, employing the following technical solution: An application of a targeted microbial membrane electrode in the electrochemical detection of new pollutants involves placing the targeted microbial membrane electrode as the working electrode in the water body to be tested, detecting the inhibitory effect of the target pollutant on the electron transfer process of microbial metabolism under the applied potential, and quantitatively analyzing the new pollutant in the water body by obtaining the oxidation peak current decay value of the differential pulse voltammetry characteristic.

[0024] By employing the above technical solution, and using the targeted microbial membrane electrode as the sensing element, during the testing process, trace amounts of new pollutants such as sulfamethoxazole or perfluorooctanoic acid in the water permeate into the microbial cell environment through the micropores within the electrode. This interferes with or inhibits enzymatic reactions in the respiratory chain of the strain, leading to hindered generation of reducing equivalent substances. This inhibitory effect further reduces the electron flux that shuttles to the immobilized electron mediator and ultimately reaches the surface of the current collector. Therefore, by extracting the attenuated macroscopic characteristic electrochemical signal parameters, a mapping relationship between the signal and concentration can be established, thereby enabling the calculation and analysis of the concentration of new pollutants in the water.

[0025] This invention provides a targeted microbial membrane electrode and its application in the electrochemical detection of novel pollutants. It offers the following advantages: 1. This invention utilizes an in-situ delayed crosslinking system composed of gluconate-δ-lactone, calcium carbonate, sodium alginate, and other components. The slow acid production from the hydrolysis of the lactone stimulates the release of calcium ions from the calcium carbonate, promoting coordination crosslinking between the polymer framework, carbon nanotubes, and electron mediators. This design immobilizes the water-soluble electron mediators and conductive fillers within the hydrogel network, restricting the free diffusion and loss of electron mediators to the external liquid phase, thereby improving the electrochemical signal stability of the membrane electrode during continuous testing.

[0026] 2. This invention combines high- and low-shear stepwise pulping with a stepped vacuum dehydration process, and introduces trehalose into the biological components. During the preparation stage, low-temperature, low-shear mixing conditions reduce physical damage to microbial cells caused by mechanical forces; during the drying stage, stepped dehydration promotes the formation of alternative hydrogen bonds between trehalose and the cell membrane surface, thereby transforming it into an amorphous matrix encapsulating the bacterial cells. Through the synergistic effect of process parameters and protective agents, microbial inactivation during processing and subsequent long-term storage is reduced, helping to extend the effective shelf life of the electrode products.

[0027] 3. The targeted functional strains of this invention are obtained through a combined domestication process using a progressively increasing concentration of co-metabolite substrates and target pollutants, resulting in the enrichment of a micro-ecological community within the membrane layer that specifically responds to novel pollutants. During detection applications, the target pollutant in the water, upon entering the cells, inhibits the respiratory chain metabolism of the bacterial community, leading to a corresponding decrease in the electron flux transferred to the peripheral circuit. Based on this correspondence between microscopic metabolic inhibition and macroscopic electrical signal attenuation, the electrode can reliably and quantitatively measure the concentration of novel pollutants in the aquatic environment. Detailed Implementation

[0028] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0029] Carbon black, CAS number 1333-86-4, is hydrophilic and has an average particle size of 20-50 nm.

[0030] Multi-walled carbon nanotubes, CAS number 308068-56-6, with an outer diameter of 10-20 nm and a length of 10-30 μm.

[0031] Sodium alginate, CAS number 9005-38-3, is a linear block copolymer with a weight-average molecular weight of 10-20 kDa, composed of β-D-mannuronic acid and α-L-guluronic acid linked by (1→4) glycosidic bonds.

[0032] Sodium polyacrylate, CAS number 9003-04-7, is a homopolymer with a weight-average molecular weight of 3-5 MDa.

[0033] Calcium carbonate powder, CAS number 471-34-1, with an average particle size of 1-5 μm.

[0034] Preparation Example 1: This preparation example provides a method for preparing carboxylated multi-walled carbon nanotubes, specifically including the following steps: (1) Acidification dispersion: Weigh 1g of multi-walled carbon nanotubes and add them to 45mL of mixed acid, which is a mixture of concentrated sulfuric acid and concentrated nitric acid in a volume ratio of 3:1. Then, ultrasonically disperse the mixture for 35min. (2) Reflux oxidation: The dispersion obtained in step (1) is placed in a heat-collecting magnetic stirring bath and refluxed and stirred at 75°C for 5 hours; (3) Washing and drying: After the reaction is completed, the suspension is cooled to room temperature and washed repeatedly by centrifugation with deionized water at a speed of 9000 rpm until the pH value of the supernatant reaches 6.8. Finally, the precipitate is placed in a vacuum drying oven at 65℃ and dried for 18 hours, then ground through a 200-mesh sieve for later use.

[0035] Preparation Example 2: This preparation example provides a method for preparing carboxylated multi-walled carbon nanotubes, specifically including the following steps: (1) Acidification dispersion: Weigh 1g of multi-walled carbon nanotubes and add them to 50mL of mixed acid, which is a mixture of concentrated sulfuric acid and concentrated nitric acid in a volume ratio of 3:1. Then, ultrasonically disperse the mixture for 40min. (2) Reflux oxidation: The dispersion obtained in step (1) is placed in a heat-collecting magnetic stirring bath and refluxed and stirred at 80°C for 6 hours; (3) Washing and drying: After the reaction is completed, the suspension is cooled to room temperature and washed repeatedly by centrifugation with deionized water at a speed of 10,000 rpm until the pH value of the supernatant reaches 7.0. Finally, the precipitate is placed in a vacuum drying oven at 70℃ and dried for 24 hours, then ground through a 200-mesh sieve for later use.

[0036] Preparation Example 3: This preparation example provides a method for preparing carboxylated multi-walled carbon nanotubes, specifically including the following steps: (1) Acidification dispersion: Weigh 1g of multi-walled carbon nanotubes and add them to 40mL of mixed acid, which is a mixture of concentrated sulfuric acid and concentrated nitric acid in a volume ratio of 3:1. Then, ultrasonically disperse the mixture for 30min. (2) Reflux oxidation: The dispersion obtained in step (1) is placed in a heat-collecting magnetic stirring bath and refluxed and stirred at 70°C for 4 hours; (3) Washing and drying: After the reaction is completed, the suspension is cooled to room temperature and washed repeatedly by centrifugation with deionized water at a speed of 8000 rpm until the pH value of the supernatant reaches 6.5. Finally, the precipitate is placed in a vacuum drying oven at 60℃ and dried for 12 hours, then ground through a 200-mesh sieve for later use.

[0037] Preparation Example 4: This preparation example provides a method for preparing concentrated sludge of targeted functional strains, specifically including the following steps: (1) Inoculation and acclimatization: Activated sludge from the biochemical section of a pharmaceutical wastewater treatment plant was collected as the inoculation source and placed in a sequencing batch reactor; artificially prepared basic salt culture medium was used as the nutrient source, and 1.0 mmol / L sodium acetate was added to the culture medium as the co-metabolite carbon source, and sulfamethoxazole was used as the screening pressure and inducing factor; during the acclimatization period, the concentration of sulfamethoxazole was gradually increased according to the concentration gradient of 10 μg / L, 50 μg / L, 200 μg / L and 500 μg / L, and each concentration gradient was run for 8 days; (2) Centrifugation and washing: Collect the bacterial culture in the logarithmic growth phase in the acclimatization system, centrifuge at 5000 rpm for 12 min at 4℃, discard the supernatant, wash twice with pre-cooled 0.05 mol / L phosphate buffer, and centrifuge again to collect the obtained bacterial sludge; (3) Moisture content adjustment and preservation: Adjust the moisture content of the bacterial mud obtained in step (2) to 78% to obtain concentrated mud of targeted functional strains, and store it at 4℃ for later use.

[0038] Preparation Example 5: This preparation example provides a method for preparing concentrated sludge of targeted functional strains, specifically including the following steps: (1) Inoculation and acclimatization: Activated sludge from the biochemical section of a pharmaceutical wastewater treatment plant was collected as the inoculation source and placed in a sequencing batch reactor; artificially prepared basic salt culture medium was used as the nutrient source, and 1.0 mmol / L sodium acetate was added to the culture medium as the co-metabolite carbon source, and sulfamethoxazole was used as the screening pressure and inducing factor; during the acclimatization period, the concentration of sulfamethoxazole was gradually increased according to the concentration gradient of 10 μg / L, 50 μg / L, 200 μg / L and 500 μg / L, and each concentration gradient was run for 7 days; (2) Centrifugation and washing: Collect the bacterial culture in the logarithmic growth phase in the acclimatization system, centrifuge at 6000 rpm for 15 min at 4℃, discard the supernatant, wash twice with pre-cooled 0.05 mol / L phosphate buffer, and centrifuge again to collect the obtained bacterial sludge; (3) Moisture content adjustment and preservation: Adjust the moisture content of the bacterial mud obtained in step (2) to 75% to obtain concentrated mud of targeted functional strains, and store it at 4℃ for later use.

[0039] Preparation Example 6: This preparation example provides a method for preparing concentrated sludge of targeted functional strains, specifically including the following steps: (1) Inoculation and acclimatization: Activated sludge from the biochemical section of a pharmaceutical wastewater treatment plant was collected as the inoculation source and placed in a sequencing batch reactor; artificially prepared basic salt culture medium was used as the nutrient source, and 1.0 mmol / L sodium acetate was added to the culture medium as the co-metabolite carbon source, and perfluorooctanoic acid was used as the screening pressure and inducing factor; during the acclimatization period, the concentration of perfluorooctanoic acid was gradually increased according to the concentration gradient of 10 μg / L, 50 μg / L, 200 μg / L and 500 μg / L, and each concentration gradient was run for 9 days; (2) Centrifugation and washing: Collect the bacterial culture in the logarithmic growth phase in the acclimatization system, centrifuge at 5000 rpm for 12 min at 4℃, discard the supernatant, wash twice with pre-cooled 0.05 mol / L phosphate buffer, and centrifuge again to collect the obtained bacterial sludge; (3) Moisture content adjustment and preservation: Adjust the moisture content of the bacterial mud obtained in step (2) to 78% to obtain concentrated mud of targeted functional strains, and store it at 4℃ for later use. Example

[0040] This embodiment provides a method for preparing a targeted microbial membrane electrode. This embodiment prepares 100g of bioconductive ink, specifically including the following steps: (1) High-shear construction of buffered conductive ink at room temperature: Under an ambient temperature of 22°C, 63.6g of deionized water, 0.5g of tris(hydroxymethyl)aminomethane, 0.2g of polysorbate-80, 2.0g of sodium alginate, 0.8g of sodium polyacrylate, 4.0g of hydrophilic carbon black, 1.0g of carboxylated multi-walled carbon nanotubes obtained in Preparation Example 1, 0.5g of sodium anthraquinone-2,6-disulfonate and 1.5g of calcium carbonate powder were added sequentially to the reactor of a double planetary mixer; the spindle was turned on for high-shear dispersion, the speed was set to 1800 rpm, and the mixture was stirred continuously for 50 min to obtain a homogeneous conductive hydrogel ink. (2) Low-temperature low-shear biocomposite: Turn on the cooling circulation liquid of the reactor jacket to quickly reduce the temperature of the base ink system obtained in step (1) to 6°C; then slowly add 20.0g of the concentrated mud of the targeted functional strain obtained in Preparation Example 4, 3.5g of trehalose and 2.4g of gluconate-δ-lactone powder into the reactor; turn off the high-shear mode, reduce the stirring speed to 40rpm for low-speed folding and mixing, and turn on the vacuum pump to maintain the vacuum degree in the reactor at -0.06MPa. Continue the operation for 18min to obtain the all-water-based bioconductive ink; (3) Flexible substrate roll-to-roll screen printing: In a printing workshop with an ambient temperature of 18°C, the water-based bioconductive ink obtained in step (2) is transferred to the material tank of the screen printing machine; using a flexible polyethylene terephthalate film pre-printed with carbon conductive tracks as the substrate, a 150-mesh screen is selected for automated printing, and the wet film thickness is controlled to be 80μm. (4) Constant temperature and humidity delayed crosslinking and in-situ foaming curing: The wet film electrode array printed in step (3) is sent into the constant temperature and humidity reaction chamber, the ambient temperature is controlled at 28℃, the relative humidity is controlled at 90%, and it is kept static for 2.5h. (5) Step-type low-temperature vacuum water-retaining drying: Transfer the electrode array after solidification in step (4) to a step-type vacuum drying oven, control the temperature of the first drying partition to 20°C and the vacuum degree to -0.05MPa, and maintain for 2h; control the temperature of the second drying partition to drop to 10°C and the vacuum degree to -0.09MPa, and maintain for 4h; after drying, a new pollutant-targeted microbial membrane electrode suitable for storage is obtained. Example

[0041] This embodiment provides a method for preparing a targeted microbial membrane electrode. This embodiment prepares 100g of bioconductive ink, specifically including the following steps: (1) High-shear construction of buffered conductive ink at room temperature: Under an ambient temperature of 20°C, 74.3g of deionized water, 0.3g of tris(hydroxymethyl)aminomethane, 0.1g of polysorbate-80, 1.5g of sodium alginate, 0.5g of sodium polyacrylate, 3.0g of hydrophilic carbon black, 0.5g of carboxylated multi-walled carbon nanotubes obtained in Preparation Example 2, 0.3g of sodium anthraquinone-2,6-disulfonate and 1.0g of calcium carbonate powder were added sequentially to the reactor of a double planetary mixer; the spindle was turned on for high-shear dispersion, the speed was set to 1500 rpm, and the mixture was stirred continuously for 45 min to obtain a homogeneous conductive hydrogel ink. (2) Low-temperature low-shear biocomposite: Turn on the cooling circulation liquid of the reaction vessel jacket to quickly reduce the temperature of the base ink system obtained in step (1) to 4°C; then slowly add 15.0g of the concentrated mud of the targeted functional strain obtained in Preparation Example 5, 2.0g of trehalose and 1.5g of gluconate-δ-lactone powder into the vessel; turn off the high-shear mode, reduce the stirring speed to 30rpm for low-speed folding and mixing, and turn on the vacuum pump to maintain the vacuum degree in the vessel at -0.05MPa. Continue the operation for 15min to obtain the all-water-based bioconductive ink; (3) Flexible substrate roll-to-roll screen printing: In a printing workshop with an ambient temperature of 15°C, the water-based bioconductive ink obtained in step (2) is transferred to the material tank of the screen printing machine; using a flexible polyethylene terephthalate film pre-printed with gold conductive tracks as the substrate, a 100-mesh screen is selected for automated printing, and the wet film thickness is controlled to be 50μm. (4) Constant temperature and humidity delayed crosslinking and in-situ foaming curing: The wet film electrode array printed in step (3) is sent into the constant temperature and humidity reaction chamber, the ambient temperature is controlled at 25℃, the relative humidity is controlled at 85%, and it is kept static for 2.0h. (5) Step-type low-temperature vacuum water-retaining drying: Transfer the electrode array after solidification in step (4) to a step-type vacuum drying oven, control the temperature of the first drying partition to 20°C and the vacuum degree to -0.05MPa, and keep it for 2 hours; control the temperature of the second drying partition to drop to 8°C and the vacuum degree to -0.09MPa, and keep it for 3 hours; after drying, a new pollutant-targeted microbial membrane electrode suitable for storage is obtained. Example

[0042] This embodiment provides a method for preparing a targeted microbial membrane electrode. This embodiment prepares 100g of bioconductive ink, specifically including the following steps: (1) High-shear construction of buffered conductive ink at room temperature: Under an ambient temperature of 25°C, 53.6g of deionized water, 0.8g of tris(hydroxymethyl)aminomethane, 0.3g of polysorbate-80, 3.0g of sodium alginate, 1.0g of sodium polyacrylate, 5.0g of hydrophilic carbon black, 1.5g of carboxylated multi-walled carbon nanotubes obtained in Preparation Example 3, 0.8g of sodium anthraquinone-2,6-disulfonate and 2.0g of calcium carbonate powder were added sequentially to the reactor of a double planetary mixer; the spindle was turned on for high-shear dispersion, the speed was set to 2000 rpm, and the mixture was stirred continuously for 60 min to obtain a homogeneous conductive hydrogel ink. (2) Low-temperature low-shear biocomposite: Turn on the cooling circulation liquid of the reactor jacket to quickly reduce the temperature of the base ink system obtained in step (1) to 8°C; then slowly add 25.0g of the concentrated mud of the targeted functional strain obtained in Preparation Example 4, 4.0g of trehalose and 3.0g of gluconate-δ-lactone powder into the reactor; turn off the high-shear mode, reduce the stirring speed to 50rpm for low-speed folding and mixing, and turn on the vacuum pump to maintain the vacuum degree in the reactor at -0.08MPa. Continue the operation for 20min to obtain the all-water-based bioconductive ink; (3) Flexible substrate roll-to-roll screen printing: In a printing workshop with an ambient temperature of 19°C, the water-based bioconductive ink obtained in step (2) is transferred to the material tank of the screen printing machine; using a flexible polyethylene terephthalate film pre-printed with carbon conductive tracks as the substrate, a 200-mesh screen is selected for automated printing, and the wet film thickness is controlled to be 100μm. (4) Constant temperature and humidity delayed crosslinking and in-situ foaming curing: The wet film electrode array printed in step (3) is sent into the constant temperature and humidity reaction chamber, the ambient temperature is controlled at 30℃, the relative humidity is controlled at 95%, and it is kept static for 3.0h. (5) Step-type low-temperature vacuum water-retaining drying: Transfer the electrode array after solidification in step (4) to a step-type vacuum drying oven, control the temperature of the first drying partition to 22°C and the vacuum degree to -0.06MPa, and maintain for 3h; control the temperature of the second drying partition to drop to 10°C and the vacuum degree to -0.10MPa, and maintain for 5h; after drying, a new pollutant-targeted microbial membrane electrode suitable for storage is obtained. Example

[0043] This embodiment provides a method for preparing a targeted microbial membrane electrode. This embodiment prepares 100g of bioconductive ink, specifically including the following steps: (1) High-shear construction of buffered conductive ink at room temperature: Under an ambient temperature of 22°C, 63.6g of deionized water, 0.5g of tris(hydroxymethyl)aminomethane, 0.2g of polysorbate-80, 2.0g of sodium alginate, 0.8g of sodium polyacrylate, 4.0g of hydrophilic carbon black, 1.0g of carboxylated multi-walled carbon nanotubes obtained in Preparation Example 1, 0.5g of sodium anthraquinone-2,6-disulfonate and 1.5g of calcium carbonate powder were added sequentially to the reactor of a double planetary mixer; the spindle was turned on for high-shear dispersion, the speed was set to 1800 rpm, and the mixture was stirred continuously for 50 min to obtain a homogeneous conductive hydrogel ink. (2) Low-temperature low-shear biocomposite: Turn on the cooling circulation liquid of the reaction vessel jacket to quickly reduce the temperature of the base ink system obtained in step (1) to 6°C; then slowly add 20.0g of the concentrated mud of the targeted functional strain obtained in Preparation Example 6, 3.5g of trehalose and 2.4g of gluconate-δ-lactone powder into the vessel; turn off the high-shear mode, reduce the stirring speed to 40rpm for low-speed folding and mixing, and turn on the vacuum pump to maintain the vacuum degree in the vessel at -0.06MPa. Continue the operation for 18min to obtain the all-water-based bioconductive ink; (3) Flexible substrate roll-to-roll screen printing: In a printing workshop with an ambient temperature of 18°C, the water-based bioconductive ink obtained in step (2) is transferred to the material tank of the screen printing machine; using a flexible polyethylene terephthalate film pre-printed with carbon conductive tracks as the substrate, a 150-mesh screen is selected for automated printing, and the wet film thickness is controlled to be 80μm. (4) Constant temperature and humidity delayed crosslinking and in-situ foaming curing: The wet film electrode array printed in step (3) is sent into the constant temperature and humidity reaction chamber, the ambient temperature is controlled at 28℃, the relative humidity is controlled at 90%, and it is kept static for 2.5h. (5) Step-type low-temperature vacuum water-retaining drying: Transfer the electrode array after solidification in step (4) to a step-type vacuum drying oven, control the temperature of the first drying partition to 20°C and the vacuum degree to -0.05MPa, and maintain for 2h; control the temperature of the second drying partition to drop to 10°C and the vacuum degree to -0.09MPa, and maintain for 4h; after drying, a new pollutant-targeted microbial membrane electrode suitable for storage is obtained.

[0044] Comparative Example 1: Compared with Example 1, the difference is that: calcium carbonate powder was not added in step (1), gluconate-δ-lactone powder was not added in step (2), and deionized water was added to make up to a total mass of 100g of bioconductive ink, and step (4) was changed to "standing treatment in a constant temperature and humidity room at 28°C and 90% relative humidity for 2.5h", the rest are the same, and in step (5), the electrode array after standing treatment is transferred to a stepped vacuum drying oven for drying.

[0045] Comparative Example 2: Compared with Example 1, the difference is that tris(hydroxymethyl)aminomethane was not added in step (1), and deionized water was added to make up to a total mass of 100g of bioconductive ink, while the rest were the same.

[0046] Comparative Example 3: Compared with Example 1, the difference is that polysorbate-80 was not added in step (1), and deionized water was added to make up to a total mass of 100g of bioconductive ink, while the rest were the same.

[0047] Comparative Example 4: Compared with Example 1, the difference is that in step (1), the "carboxylated multi-walled carbon nanotubes obtained in Example 1" are replaced with an equal mass of "commercially available ordinary multi-walled carbon nanotubes without any treatment", and the rest are the same.

[0048] Comparative Example 5: Compared with Example 1, the difference is that the “step-type low temperature deep vacuum water-retaining drying” in step (5) is replaced with “the cured electrode array is placed in a 60°C ordinary blower drying oven and dried for 4 hours”, and the rest are the same.

[0049] Test Example 1: This test case is used to examine the changes in pH and apparent viscosity of the ink system under varying temperature conditions, and to evaluate the effect of buffer components on the delayed crosslinking process.

[0050] The experimental subjects were the wet, water-based bioconductive inks prepared after step (2) of Example 1 and Comparative Example 2.

[0051] The ink samples to be tested were placed into the test plate of a rotational rheometer equipped with a temperature control module, and a miniature planar pH composite electrode was set at the edge of the test area of ​​the plate to synchronously collect the pH changes of the ink system.

[0052] The rheometer was set to constant shear rate rotation mode with a shear rate of 10 s. -1 The test temperature procedure is as follows: From 0 min to 60 min, the test system temperature is maintained at 15℃ to simulate the state of ink during low-temperature storage and printing; at 60 min, the test system is heated to 28℃ at a heating rate of 2℃ / min, and then maintained at a constant temperature of 28℃ during the subsequent test to simulate the constant temperature and humidity delayed crosslinking and in-situ foaming curing stages.

[0053] After starting the test, the apparent viscosity data output by the rheometer and the pH value output by the micro pH composite electrode were continuously recorded at a sampling interval of 1 minute. Data from key time points were selected and organized. The results are shown in Table 1.

[0054] Table 1. Changes in pH value and apparent viscosity during the temperature variation test in Example 1 and Comparative Example 2:

[0055] According to the data in Table 1, during the 15℃ test period from 0 min to 60 min, the pH value of the system in Example 1 remained between 7.45 and 7.62, and the apparent viscosity remained between 45.3 Pa·s and 48.6 Pa·s, with relatively small overall fluctuations, indicating that the system maintained a good flow state during this period. After heating to 28℃, the pH value of the system in Example 1 gradually decreased, and the apparent viscosity increased accordingly; at 90 min, the pH value of the system in Example 1 was 6.21, and the apparent viscosity was 2341.6 Pa·s, while at 120 min, the apparent viscosity was 3418.2 Pa·s, indicating that the system gradually reached a higher viscosity state after heating.

[0056] Compared to Example 1, Comparative Example 2 did not contain tris(hydroxymethyl)aminomethane. As shown in Table 1, Comparative Example 2 exhibited a decrease in pH and an increase in apparent viscosity during the 15°C testing phase. At 30 min, the pH of the Comparative Example 2 system was 6.32, and the apparent viscosity was 342.8 Pa·s. At 60 min, the pH decreased to 5.92, and the apparent viscosity increased to 1523.7 Pa·s. These results indicate that, without the addition of a buffer component, a significant viscosity-increasing process may occur in the system at low temperatures.

[0057] The test results from Example 1 and Comparative Example 2 show that the addition of tris(hydroxymethyl)aminomethane helps maintain the pH stability of the system at low temperatures and, to some extent, delays the effect of acid production from gluconate-δ-lactone hydrolysis on the system viscosity. Example 1 maintains a low viscosity at low temperatures, which is beneficial for meeting the flowability requirements of the printing process. After heating to 28°C, the pH value of the system decreases, accompanied by an increase in apparent viscosity, indicating that this ink system exhibits a tendency to delay thickening and film-forming curing under temperature variations.

[0058] The above results indicate that, through the combination of buffer components, gluconate-δ-lactone and calcium carbonate powder, the ink system can maintain a relatively stable flow state during the low-temperature processing stage and gradually form a high-viscosity network structure during the subsequent heating stage, thus taking into account both the applicability of printing and processing and the structural stability after film formation.

[0059] Test Example 2: This test example is used to examine the changes in the infrared characteristic absorption peaks of the ink system before and after curing and drying, and to evaluate the interaction changes of components such as sodium alginate, sodium anthraquinone-2,6-disulfonate, and carboxylated multi-walled carbon nanotubes during the curing process.

[0060] The experimental subjects included the wet all-water-based bioconductive ink sample obtained in step (2) of Example 1 and the electrode film sample obtained after completing step (5) of Example 1; at the same time, pure sodium alginate powder, pure sodium anthraquinone-2,6-disulfonate powder and carboxylated multi-walled carbon nanotube powder obtained in Preparation Example 1 were selected as reference samples.

[0061] For the powder reference sample and the cured and dried electrode film sample, appropriate amounts of the sample were mixed with dried spectrally pure potassium bromide powder at a mass ratio of approximately 1:100. After thorough grinding and homogenization, the mixture was placed in a tablet mold and pressed into a test sheet under a pressure of 20 MPa. For the wet, water-based bioconductive ink sample, an attenuating total reflection attachment was used to uniformly coat an appropriate amount of the sample onto the surface of zinc sulfide crystals before testing.

[0062] The above samples were sequentially tested using a Fourier transform infrared spectrometer. The wavenumber range was 4000 cm⁻¹. -1 Up to 400cm -1 The resolution is 4cm. -1 Each sample was scanned 32 times. After the test, the acquired infrared spectral data were baseline corrected and smoothed, and the wavenumbers of the characteristic absorption peaks corresponding to the relevant oxygen-containing functional groups in each sample were extracted. The results are shown in Table 2.

[0063] Table 2. Wavenumber distribution of characteristic absorption peaks before and after crosslinking of pure raw materials and Example 1:

[0064] According to the data in Table 2, the asymmetric stretching vibration peak of the carboxyl group in pure sodium alginate powder is located at 1612.4 cm⁻¹. -1 The peak of the carboxyl group's symmetric stretching vibration is located at 1416.8 cm⁻¹. -1 In pure anthraquinone-2,6-disulfonic acid sodium powder, the characteristic absorption peak of the sulfonic acid group is located at 1195.2 cm⁻¹. -1 In the carboxylated multi-walled carbon nanotube powder obtained in Example 1, the characteristic peak of the C=O stretching vibration is located at 1718.5 cm⁻¹. -1 .

[0065] In the wet, all-water-based bioconductive ink sample obtained in step (2) of Example 1, the corresponding absorption peaks mentioned above appeared at 1610.7 cm⁻¹. -1 1415.2cm -1 1193.8cm -1 and 1715.3cm -1 Compared with the pure reference samples, the characteristic absorption peaks at this stage showed only slight shifts, indicating that before curing and drying, the components were mainly in a mixed and dispersed state. The changes in infrared absorption peaks may be related to the dispersion medium, weak interactions, or differences in the sample testing conditions.

[0066] In the electrode film sample obtained after completing step (5) in Example 1, the asymmetric stretching vibration peak of the sodium alginate carboxyl group shifted to 1594.3 cm⁻¹. -1 The peak of the carboxyl group's symmetric stretching vibration shifted to 1428.1 cm⁻¹. -1 The characteristic absorption peak of the anthraquinone-2,6-disulfonic acid sodium sulfonic acid group shifted to 1178.6 cm⁻¹. -1 The C=O stretching vibration peak on the surface of carboxylated multi-walled carbon nanotubes shifted to 1698.2 cm⁻¹. -1 Compared with the wet-state all-water-based bioconductive ink sample, the characteristic absorption peaks in the cured and dried electrode film sample all showed obvious regular shifts. Specifically, the asymmetric stretching vibration peak of sodium alginate carboxyl group, the characteristic absorption peak of anthraquinone-2,6-disulfonate sodium sulfonic acid group, and the C=O stretching vibration peak of carboxylated multi-walled carbon nanotubes shifted towards lower wavenumbers, while the symmetric stretching vibration peak of sodium alginate carboxyl group shifted towards higher wavenumbers.

[0067] The above results indicate that during the curing and drying process of Example 1, the acidic environment formed after the hydrolysis of gluconic acid-δ-lactone may promote the release of divalent calcium ions from calcium carbonate powder. The released divalent calcium ions can coordinate or crosslink with the carboxyl groups on the sodium alginate molecular chain, and may further generate certain ion bridging or coordination interactions with the sulfonic acid groups in sodium anthraquinone-2,6-disulfonate and the oxygen-containing functional groups on the surface of carboxylated multi-walled carbon nanotubes.

[0068] Therefore, it can be seen that in the electrode film layer after curing and drying in Example 1, a multi-point interaction structure with calcium ions as bridging centers may have formed between different components. This structure helps to reduce the migration tendency of water-soluble electron mediators in the aqueous environment and helps to improve the dispersion stability and interfacial bonding stability of carbon-based conductive fillers in water-absorbing environments.

[0069] Test Example 3: This test case is used to examine the electrochemical response of the electron mediator in the novel pollutant-targeting microbial membrane electrode obtained in Example 1, and to evaluate the connectivity of the electron transport channels within the membrane.

[0070] The experimental subject was the new pollutant-targeting microbial membrane electrode prepared in Example 1 and subjected to step-type low-temperature vacuum water-retaining drying.

[0071] A membrane electrode of a specified area was used as the working electrode, a saturated calomel electrode was selected as the reference electrode, and a platinum sheet electrode was used as the counter electrode to assemble a three-electrode electrochemical testing system.

[0072] A phosphate buffer solution with a concentration of 0.1 mol / L and a pH of 7.0 was prepared as the test substrate. No target new pollutant or other external carbon sources were added to the test substrate solution.

[0073] The three-electrode electrochemical test system was placed in the above-mentioned phosphate buffer solution, and high-purity nitrogen was continuously introduced into the solution for 20 minutes to remove oxygen, so as to reduce the influence of dissolved oxygen on the redox response. The nitrogen atmosphere was maintained during the test.

[0074] The electrochemical workstation was connected, and cyclic voltammetry was used for testing. The potential scan window was set from -0.8V to 0.0V, with all potentials relative to a saturated calomel electrode; the scan rates were set to 10mV / s, 30mV / s, 50mV / s, and 100mV / s, respectively.

[0075] After the cyclic voltammetry curves at each scan rate stabilized, the test curves were recorded, and the corresponding oxidation peak potential, reduction peak potential, oxidation peak current, and reduction peak current were extracted. The results are shown in Table 3.

[0076] Table 3 Cyclic voltammetric characteristics of the electrode in Example 1 at different scan rates:

[0077] According to the data in Table 3, when the electrode of Example 1 was tested in a phosphate buffer solution free of the target contaminant and an added carbon source, a set of redox response peaks were observed in the potential range of -0.32V to -0.48V. This potential range is close to the redox response range of sodium anthraquinone-2,6-disulfonate in the ink system, indicating that the anthraquinone electron mediators in the electrode film can still participate in the electrochemical response process after curing and drying.

[0078] As the scan rate increased from 10 mV / s to 100 mV / s, the oxidation peak current increased from 14.2 μA to 52.7 μA, and the absolute value of the reduction peak current increased from 13.8 μA to 51.4 μA. This increase in peak current with increasing scan rate indicates that charge transfer can occur between the redox components within the electrode film and the current collector. Simultaneously, the difference between the oxidation and reduction peak potentials widened with increasing scan rate, suggesting that the system exhibits quasi-reversible redox characteristics.

[0079] As can be seen from the results in Table 3, after the electrode obtained in Example 1 underwent step-type low-temperature vacuum water-retaining drying treatment, redox responses related to anthraquinone electron mediators could still be detected within the film layer. This response may be influenced by a combination of electron transport, ion migration, and local diffusion processes within the film layer.

[0080] In Example 1, during the ink curing process, the calcium ion bridging structure may restrict the migration of sodium anthraquinone-2,6-disulfonate in the film layer through ion association or coordination interactions, while retaining the redox responsiveness of its quinone structure. The cyclic voltammetric responses shown in Table 3 further demonstrate that a certain charge transfer pathway can be formed between the carbon-based conductive filler and the electron mediator in the film layer, providing an electrochemical basis for the electrode's subsequent participation in the extracellular electron transfer process of microorganisms.

[0081] Test Example 4: This test case is used to examine the changes in the storage modulus and loss modulus of the ink system over time under normal temperature processing conditions, and to evaluate the rheological stability of the ink during the printing process.

[0082] The experimental subjects were the wet, water-based bioconductive inks obtained after step (2) of Example 1 and Comparative Example 2.

[0083] The ink sample to be tested was loaded into the parallel plate testing system of the rotational rheometer, the test gap was set to 1.0 mm, and silicone oil was used to liquid seal the edges of the test sample to reduce the impact of moisture evaporation on the test results during long-term testing.

[0084] The rheometer was set to a test temperature of 20°C to simulate the ambient temperature processing environment of a printing workshop. A small-amplitude oscillation time-scan mode was used for testing, with an oscillation frequency of 1 Hz and a strain amplitude of 1%, which was located within the linear viscoelastic region of the sample.

[0085] After the test was started, the data of the storage modulus G′ and loss modulus G″ of the sample were continuously recorded over time. The total test duration was 300 min. The results are shown in Table 4.

[0086] Table 4. Evolution of energy storage modulus and loss modulus of Example 1 and Comparative Example 2 at 20°C over time:

[0087] According to the data in Table 4, within the 300-minute test period, the loss modulus G″ of Example 1 was consistently higher than the storage modulus G′. Specifically, the storage modulus of Example 1 increased from 285.4 Pa to 382.1 Pa, and the loss modulus increased from 751.2 Pa to 827.5 Pa. The changes in these two values ​​were relatively small, indicating that the ink system of Example 1 could maintain a rheological state dominated by viscous response at 20°C.

[0088] Comparative Example 2 also exhibited a higher loss modulus G″ than storage modulus G′ in the initial stage of testing. As the testing time increased, the storage modulus of Comparative Example 2 increased significantly; between 30 and 45 minutes, the storage modulus increased from 458.2 Pa to 1420.7 Pa, exceeding the loss modulus during the same time period. By 60 minutes, the storage modulus of Comparative Example 2 reached 4352.1 Pa, indicating that this system showed enhanced elastic response relatively early at 20°C.

[0089] In oscillatory rheological tests, the crossover of storage modulus and loss modulus can often be used as one of the criteria for judging the transition of the system from a flowing state to a gel state. According to the data in Table 4, the modulus crossover point of Comparative Example 2 is between 30 min and 45 min, indicating that its processable time window is relatively short. Compared with Comparative Example 2, Example 1 did not show G′ exceeding G″ within the 300 min test range, indicating that it has a longer flow retention time under room temperature conditions.

[0090] The differences in formulation between Example 1 and Comparative Example 2 show that the addition of tris(hydroxymethyl)aminomethane helps buffer the effects of acid production from gluconate-δ-lactone hydrolysis on the pH and crosslinking process of the system, thereby delaying the increase in modulus caused by calcium ion release. These results indicate that the ink system of Example 1 can maintain a relatively stable rheological state during room temperature processing, which is beneficial for meeting the requirements of roll-to-roll screen printing processes regarding the ink's operating window.

[0091] Test Example 5: This test case is used to investigate the changes in electrochemical impedance of the electrode under long-term immersion conditions and to evaluate the stability of the conductive network in the electrode film under water-absorbing conditions.

[0092] The experimental subjects were the solid electrode arrays obtained after completing all preparation processes in Example 1, Comparative Example 1, and Comparative Example 4.

[0093] Each group of electrodes was cut to have an effective working area of ​​1.0 cm². 2The test unit is encapsulated with insulating resin for non-working areas and electrode edges.

[0094] A phosphate buffer solution containing 0.1 mol / L potassium chloride, with a pH of 7.0, was prepared as the immersion solution for the simulated environment. Each packaged electrode sample was immersed in a sealed container containing 200 mL of the simulated environment immersion solution and placed in a constant temperature incubator at 25°C for continuous immersion.

[0095] Electrochemical impedance spectroscopy (EIS) tests were performed on the corresponding electrode samples at the initial soaking time, after 7 days of soaking, and after 14 days of soaking. During the tests, the electrode under test was used as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum mesh as the counter electrode. A solution containing 5 mmol / L [Fe(CN)6] was used. 3- / 4- A 0.1 mol / L potassium chloride solution was used as the redox probe substrate.

[0096] Connect to an electrochemical workstation and apply an AC sinusoidal voltage disturbance with an amplitude of 5mV at the electrode open circuit potential. The test frequency range is from 100kHz to 0.01Hz.

[0097] Real and imaginary impedance data were obtained at different frequencies, and equivalent circuit fitting was performed using electrochemical analysis software to extract the solution resistance Rs and charge transfer resistance Rct. Before each impedance test, the appearance integrity of each electrode film was observed to confirm that its working area had not been completely detached; for samples with slight edge swelling, the effective working area retained after encapsulation was used as the impedance test area. The test results are shown in Table 5.

[0098] Table 5. Equivalent fitting parameters of electrochemical impedance for each electrode at different immersion times:

[0099] According to the data in Table 5, the Rct of Example 1 was 38.6 Ω at the initial soaking time, 42.3 Ω after 7 days of soaking, and 45.7 Ω after 14 days of soaking. The Rct only increased slightly with the extension of soaking time, indicating that the electrode of Example 1 can maintain a relatively low interfacial charge transfer resistance under the soaking conditions.

[0100] Comparative Example 1 had an Rct of 52.8 Ω at the initial immersion time, which increased to 384.5 Ω after 7 days of immersion and to 1142.1 Ω after 14 days of immersion, indicating that the interfacial charge transfer resistance of the electrode increased significantly under long-term immersion conditions. Comparative Example 4 had an Rct of 44.2 Ω at the initial immersion time, which increased to 156.4 Ω after 7 days of immersion and to 345.8 Ω after 14 days of immersion, with its Rct increase falling between that of Example 1 and Comparative Example 1.

[0101] In electrochemical impedance spectroscopy (EIS), the change in Rct is typically related to factors such as the contact state of the conductive filler in the membrane, the degree of water absorption and swelling of the membrane, the pore structure, and the accessibility of the redox probe at the electrode interface. Considering the differences in formulations across groups, it can be seen that Comparative Example 1, which did not introduce the in-situ crosslinking system formed by calcium carbonate and gluconate-δ-lactone, is more prone to water absorption and swelling or localized structural loosening in an aqueous environment, potentially increasing the spacing between the conductive filler and affecting the charge transfer process.

[0102] Comparative Example 4, while retaining the crosslinking system, used ordinary multi-walled carbon nanotubes that had not undergone carboxylation treatment. Compared to carboxylated multi-walled carbon nanotubes, the interfacial bonding between ordinary multi-walled carbon nanotubes and the polymer crosslinking network is relatively weak. Therefore, it may be more susceptible to local deformation of the film layer during immersion in water, as evidenced by the increase in Rct with increasing immersion time.

[0103] Example 1 employs carboxylated multi-walled carbon nanotubes, and improves the bonding stability between the conductive filler and the polymer cross-linked network through coordination or ion bridging between calcium ions and carboxyl groups. This structure helps reduce the relative displacement of the conductive filler and the degree of interfacial contact degradation during immersion, enabling the electrode to maintain a relatively low charge transfer resistance in a long-term aqueous environment.

[0104] Test Example 6: This test case is used to examine the redox response retention of the electrode under long-term continuous potential cyclic scanning conditions and to evaluate the retention state of electron mediators in the electrode film in an aqueous electrochemical testing environment.

[0105] The experimental subjects were the electrode samples obtained after completing the corresponding preparation and drying processes of Example 1 and Comparative Example 1.

[0106] Electrode samples with the same cross-sectional area were used as working electrodes, with a platinum sheet as the counter electrode and a saturated calomel electrode as the reference electrode to assemble a three-electrode electrochemical testing system.

[0107] Prepare a 0.1 mol / L phosphate buffer solution with a pH of 7.0 as the test substrate. Before the test, purge the test substrate solution with high-purity nitrogen for 20 min to reduce the influence of dissolved oxygen on the redox response, and maintain a nitrogen atmosphere during the test.

[0108] The cyclic voltammetry test module of the electrochemical workstation was used, with the scan potential range set to -0.8V to 0.0V and the voltage scan rate set to 50mV / s.

[0109] Each test electrode underwent 1000 consecutive cyclic voltammetric scans. During the test, the oxidation peak current was recorded at each cycle number, and the absolute values ​​of the oxidation peak current corresponding to the 1st, 100th, and every subsequent 100 cycles were extracted. Based on the oxidation peak current value of the 1st cycle, the oxidation peak current retention rate at different cycle numbers was calculated, and the results are shown in Table 6.

[0110] Table 6. Evolution data of oxidation peak current and retention rate in long-cycle cycling for Example 1 and Comparative Example 1:

[0111] According to the data in Table 6, under continuous electrochemical polarization operation, the redox response signals of the two electrode samples exhibited different maintenance states. After 1000 potential cycles, the characteristic oxidation peak current of Example 1 changed from an initial 37.4 μA to 32.5 μA, with a current retention rate maintained at 86.9%. The current decay curve was relatively flat throughout the cycle, exhibiting normal amplitude fluctuations consistent with diffusion control. The initial oxidation peak current of Comparative Example 1 was 38.6 μA, at the same level as Example 1, but its response current showed a non-linear decrease in the early stages with increasing cycle count. By the 300th cycle, the peak current of Comparative Example 1 had dropped to 16.3 μA (retention rate 42.2%), and after 1000 cycles, the peak current decreased to 4.8 μA, with a final retention rate of only 12.4%, reflecting a significant loss of the substances involved in the electrochemical reaction during the test.

[0112] This separation in the test results stems from the difference in the spatial fixation of components within the electrode. The presence of highly polar sulfonic acid groups in the sodium anthraquinone-2,6-disulfonate structure gives it high water solubility. In the system of Comparative Example 1, the lack of a cross-linking phase capable of in-situ dissolution results in sodium anthraquinone-2,6-disulfonate in a physically doped, free state in the aqueous environment. Based on the data in Table 6, after 1000 consecutive potential cycles, the oxidation peak current of the electrode in Example 1 changed from 37.4 μA in the first cycle to 32.5 μA in the 1000th cycle, corresponding to an oxidation peak current retention rate of 86.9%. Throughout the test, the oxidation peak current of the electrode in Example 1 remained at a high level, indicating that its electrochemical response exhibits good retention under long-cycle conditions.

[0113] The oxidation peak current of the electrode in Comparative Example 1 was 38.6 μA during the first cycle, which was similar to that of Example 1. With increasing cycle number, the oxidation peak current of Comparative Example 1 gradually decreased; at the 300th cycle, its oxidation peak current was 16.3 μA, with a retention rate of 42.2%; at the 1000th cycle, its oxidation peak current was 4.8 μA, with a retention rate of 12.4%. These results indicate that the redox response of the electrode in Comparative Example 1 decayed significantly during long-term potential cycling.

[0114] The differences in formulation between Example 1 and Comparative Example 1 show that Example 1 includes an in-situ crosslinking system formed by calcium carbonate powder and gluconate-δ-lactone, while Comparative Example 1 does not. Sodium anthraquinone-2,6-disulfonate has strong hydrophilicity; in the absence of an effective confined structure, it may migrate or be lost to some extent in an aqueous environment and under potential cycling, thus affecting the redox response retention of the electrode.

[0115] In Example 1, during the curing process, the divalent calcium ions released from the calcium carbonate powder can form a cross-linked structure with the carboxyl groups on the sodium alginate molecular chain. They may also interact with the sulfonic acid groups in sodium anthraquinone-2,6-disulfonate and the oxygen-containing functional groups on the surface of carboxylated multi-walled carbon nanotubes through ion bridging or coordination interactions. These network confinement and ion bridging effects help reduce the tendency of electron mediators to migrate into the external test solution, thereby enabling the electrode of Example 1 to maintain a relatively stable redox response during long-cycle potential scanning.

[0116] Therefore, it can be seen that the cross-linked network and carbon-based conductive framework in the electrode film obtained in Example 1 may play a certain confinement role on the electron mediator, which is beneficial to improving the signal retention capability of the electrode under aqueous electrochemical testing conditions.

[0117] Test Example 7: This test case is used to examine the changes in microbial activity-related indicators in the electrode film after different drying processes, and to evaluate the activity retention status of targeted biological elements within the electrode.

[0118] The experimental subjects included the electrode sample obtained by the stepped low-temperature vacuum water-retaining drying process in Example 1, the electrode sample obtained by drying in a 60℃ ordinary forced-air drying oven for 4 hours in Comparative Example 5, and the wet-state cured film sample of Example 1 after the constant temperature and humidity standing period in step (4) but before drying. The wet-state cured film sample served as the initial active reference sample before drying.

[0119] Using a sterile cutting tool, cut working areas of 2.0 cm² from each of the three samples mentioned above. 2 For each sample, six test slices were cut in parallel from the effective membrane layer area.

[0120] The cut membrane pieces were transferred to homogenization tubes containing 5.0 mL of sterile phosphate buffer (PBS) with a pH of 7.4. An appropriate amount of sterile glass beads were added to the homogenization tubes.

[0121] The homogenizing tube was placed in a low-temperature tissue homogenizer and homogenized at a frequency of 60 Hz for 3 minutes to break down the polymer network in the electrode membrane and release the bacterial cells embedded in the membrane into the buffer solution.

[0122] Add an equal volume of bacterial cell lysis buffer to the obtained suspension and sonicate for 10 min under ice bath conditions to lyse bacterial cells and release intracellular adenosine triphosphate.

[0123] The mixture was centrifuged at 10,000 r / min for 15 min at 4 °C. The supernatant was collected, and the relative luminescence intensity of the system was measured using a multifunctional microplate reader combined with an ATP bioluminescence detection kit. The ATP concentration per unit area of ​​each electrode film was calculated based on the pre-plotted ATP standard curve. The results are shown in Table 7.

[0124] Table 7. Intracellular ATP concentration distribution data of electrode membranes after different drying processes, in nmol / cm³. 2 .

[0125]

[0126] According to the data in Table 7, the ATP concentration at six parallel sampling points in the initial wet-cured film sample before drying was 74.28 nmol / cm³. 2 Up to 85.12 nmol / cm 2 The average value is 80.31 nmol / cm. 2 This value can be used as a reference for the activity level of biological components before drying.

[0127] Comparative Example 5, after being dried in a conventional blast furnace at 60℃, showed an ATP concentration of 3.84 nmol / cm³ at six parallel sampling points. 2 Up to 7.26 nmol / cm 2 The average value is 5.33 nmol / cm. 2 The ATP concentration decreased by approximately 93.4% compared to the initial wet-cured film sample. This result indicates that the detectable ATP level in the electrode film layer significantly decreased after high-temperature drying. In Example 1, after step-type low-temperature vacuum water-retaining drying, the ATP concentration at six parallel sampling points was 58.74 nmol / cm³. 2 Up to 68.17 nmol / cm 2 The average value is 63.18 nmol / cm. 2The retention rate was approximately 78.7% relative to the initial wet-cured membrane sample. Compared to Comparative Example 5, the ATP level detected in the dried membrane sample of Example 1 was higher, indicating that the low-temperature vacuum drying process helps to improve the retention of microbial activity-related indicators in the electrode membrane.

[0128] The differences in drying conditions between Example 1 and Comparative Example 5 show that higher-temperature forced-air drying may accelerate membrane moisture loss and adversely affect cell membrane structure, intracellular enzyme systems, or retention of intracellular contents, thereby reducing ATP detection values. The step-type low-temperature vacuum water-retaining drying process used in Example 1, which performs decompression dehydration at a lower temperature, helps reduce the impact of rapid high-temperature dehydration on microbial cells.

[0129] Furthermore, the ink system of Example 1 contains polyhydroxy components formed by the hydrolysis of trehalose and glucono-δ-lactone. These polyhydroxy components may form hydrogen bonds with polar groups on the cell membrane surface and form a protective matrix with a certain viscosity during the drying process, thereby helping to reduce the impact of dehydration treatment on cell activity-related indicators.

[0130] The above results indicate that the step-type low-temperature vacuum water-retaining drying process in Example 1 is beneficial to improving the retention of ATP levels in the electrode film after drying, providing a bioactive basis for the subsequent response of the electrode to pollutants after revival and activation.

[0131] Test Example 8: This test case is used to examine the electrochemical response of the electrode to a target new pollutant in water and to evaluate its applicability for the detection of target pollutant concentrations.

[0132] The experimental subjects were the targeted microbial membrane electrodes prepared in Examples 1 and 4 and subjected to resuscitation and activation treatment. Specifically, the electrode from Example 1 was used for electrochemical response testing of sulfamethoxazole, and the electrode from Example 4 was used for electrochemical response testing of perfluorooctanoic acid.

[0133] An electrode sample of a specified working area was taken as the working electrode, a saturated calomel electrode was used as the reference electrode, and a platinum sheet was used as the counter electrode to assemble a three-electrode electrochemical testing system.

[0134] Prepare a 0.1 mol / L phosphate buffer solution containing 5 mmol / L sodium acetate at pH 7.0 as a background solution to maintain the basic metabolism of microorganisms.

[0135] Sulfamethoxazole and perfluorooctanoic acid (PFOA) were selected as the new target pollutants for testing. A standard stock solution for the target pollutant was prepared using a mixture of methanol and pure water. This stock solution was then serially diluted and added to the background buffer to prepare a series of test solutions with pollutant concentrations of 0.5 μg / L, 1.0 μg / L, 2.5 μg / L, 5.0 μg / L, 10.0 μg / L, 25.0 μg / L, and 50.0 μg / L. The final methanol concentration in all test solutions and the solvent blank background buffer was kept consistent and did not exceed 0.1% to minimize the impact of solvent differences on the test results.

[0136] Before testing, each working electrode was placed in a blank background solution containing an equal amount of methanol but without the target contaminant, and reacted at a constant temperature for 30 minutes to obtain the basic electrochemical signal of the corresponding electrode.

[0137] Subsequently, the independently prepared and reactivated working electrodes from the same batch were placed in a series of test solutions of different concentrations. Each concentration was reacted at a constant temperature for 30 minutes to ensure sufficient contact between the microbial system in the electrode membrane and the test solution. Differential pulse voltammetry was then used for scanning. Using independently prepared working electrodes to test samples of different concentrations reduced the potential cumulative effects that might occur during the concentration increment testing process.

[0138] The differential pulse voltammetry test potential window was set to -0.6V to -0.1V, with a pulse amplitude of 50mV and a pulse width of 0.05s. Before testing, a recovery and activation electrode with a similar blank response current was selected for subsequent detection to reduce the influence of initial metabolic activity differences between different electrodes on the concentration response results. At least three independent parallel electrodes were used for each concentration. The data listed in Table 8 are the average values ​​of the parallel test results, and the relative standard deviation between parallel samples does not exceed 5%. The absolute values ​​of the characteristic oxidation peak current corresponding to each concentration condition were recorded, and the results are shown in Table 8.

[0139] Table 8. Average electrochemical response current data of electrodes in Examples 1 and 4 under different concentrations of new pollutants:

[0140] According to the data in Table 8, in a solvent blank background solution containing sodium acetate but free of the target contaminant, the average characteristic oxidation peak current of the electrodes in Examples 1 and 4 was 56.4 μA. This response can be used as the basic electrochemical signal under the corresponding test conditions.

[0141] When sulfamethoxazole was added to the system, the peak response current of the electrode in Example 1 decreased with increasing sulfamethoxazole concentration; within the range of 0.5 μg / L to 50.0 μg / L, its peak response current decreased from 48.7 μA to 15.8 μA. When perfluorooctanoic acid (PFOA) was added to the system, the peak response current of the electrode in Example 4 also decreased with increasing PFOA concentration; within the same concentration range, its peak response current decreased from 52.1 μA to 27.6 μA.

[0142] Regression analysis of data ranging from 0.5 μg / L to 50.0 μg / L revealed a linear negative correlation between the commonly used logarithmic value of the target pollutant concentration and the response peak current. This result indicates that, under the aforementioned test conditions, the electrode in Example 1 exhibits a concentration-dependent electrochemical response to sulfamethoxazole, and the electrode in Example 4 exhibits a concentration-dependent electrochemical response to perfluorooctanoic acid.

[0143] Based on the electrode composition and test results, it can be seen that the targeted acclimatized microbial membrane can serve as a response unit for the target pollutant. When the target pollutant enters the test system, it may affect the microbial basal metabolism, cell membrane function, or respiratory chain electron transport processes, thereby causing changes in the oxidation peak current collected by the electrode.

[0144] Furthermore, the carbon-based conductive filler and anthraquinone electron mediators in the electrode membrane are confined within the membrane through polymer networks, calcium ion bridging structures, and interfacial interactions, which helps reduce the impact of mediator loss and conductive network reconstruction on the detection signal. Therefore, the electrochemical response signal output by the electrode can be used to characterize the effect of changes in target pollutant concentration on the metabolic state of the microbial membrane.

[0145] The above results demonstrate that the targeted microbial membrane electrodes obtained in Examples 1 and 4 can convert changes in the concentration of the target new pollutant into a detectable electrochemical response signal under the test conditions, and can be used for the electrochemical detection and evaluation of the corresponding target pollutant.

[0146] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A targeted microbial membrane electrode, characterized in that, It includes a flexible substrate and a film layer attached to the surface of the flexible substrate, the film layer being made by printing, curing and drying an all-water-based bioconductive ink comprising the following components in parts by weight: The components of the buffered conductive ink system are: 53.6-74.3 parts deionized water, 0.3-0.8 parts tris(hydroxymethyl)aminomethane, 0.1-0.3 parts polysorbate-80, 1.5-3.0 parts sodium alginate, 0.5-1.0 parts sodium polyacrylate, 3.0-5.0 parts hydrophilic carbon black, 0.5-1.5 parts carboxylated multi-walled carbon nanotubes, 0.3-0.8 parts sodium anthraquinone-2,6-disulfonate as an electron mediator, and 1.0-2.0 parts calcium carbonate powder; wherein, the tris(hydroxymethyl)aminomethane is used to absorb protons released by the system in the later stage to provide a pH operating window, and the calcium carbonate is used to release divalent calcium ions in situ to bridge and crosslink the sodium alginate, carboxylated multi-walled carbon nanotubes and sodium anthraquinone-2,6-disulfonate; As components of the bioactive recognition and protection system: 15.0-25.0 parts of concentrated mud from targeted functional strains, 2.0-4.0 parts of trehalose, and 1.5-3.0 parts of gluconate-δ-lactone as a slow-release acid activator.

2. The targeted microbial membrane electrode according to claim 1, characterized in that, The all-water-based bioconductive ink is made from the following components in parts by weight: 63.6 parts deionized water, 0.5 parts tris(hydroxymethyl)aminomethane, 0.2 parts polysorbate-80, 2.0 parts sodium alginate, 0.8 parts sodium polyacrylate, 4.0 parts hydrophilic carbon black, 1.0 part carboxylated multi-walled carbon nanotubes, 0.5 parts sodium anthraquinone-2,6-disulfonate, 1.5 parts calcium carbonate powder, 20.0 parts concentrated mud containing targeted functional strains, 3.5 parts trehalose, and 2.4 parts glucono-δ-lactone.

3. The targeted microbial membrane electrode according to claim 1, characterized in that, The concentrated mud containing the targeted functional strains was prepared through the following steps: Activated sludge was collected as an inoculum and placed in a bioreactor. A basal salt culture medium was used as the nutrient source, with 1.0 mmol / L sodium acetate added as a co-metabolic carbon source. A new target pollutant was used as a screening stress factor. During the acclimation period, the new target pollutant was added in progressively increasing concentrations of 10 μg / L, 50 μg / L, 200 μg / L, and 500 μg / L, with each concentration gradient running for 7-9 days. The bacterial culture in the logarithmic growth phase was collected, centrifuged, washed, and its moisture content adjusted to 75%-78%. The new target pollutant was selected from sulfamethoxazole or perfluorooctanoic acid (PFOA).

4. The targeted microbial membrane electrode according to claim 1, characterized in that, The carboxylated multi-walled carbon nanotubes were prepared by the following steps: Multi-walled carbon nanotubes were added to a mixture of concentrated sulfuric acid and concentrated nitric acid in a volume ratio of 3:1 and ultrasonically dispersed for 30-40 min to obtain a dispersion. The dispersion was then refluxed and stirred at 70-80℃ for 4-6 h. After the reaction was completed, the mixture was cooled, washed, and centrifuged until the pH of the supernatant reached 6.5-7.

0. The resulting precipitate was vacuum dried at 60-70℃, then ground and sieved for later use.

5. The targeted microbial membrane electrode according to claim 1, characterized in that, The flexible substrate is a polyethylene terephthalate film with conductive tracks pre-printed on its surface, and the conductive tracks are carbon conductive tracks or gold conductive tracks.

6. The method for preparing the targeted microbial membrane electrode according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Add all components except for the concentrated mud of the targeted functional strain, trehalose and gluconate-δ-lactone to the reaction vessel and perform high shear dispersion at room temperature to obtain a homogeneous buffer conductive ink. S2. Cool the reaction vessel and slowly add the concentrated mud of the targeted functional strain, trehalose and gluconate-δ-lactone to the buffered conductive ink obtained in step S1. Mix under low temperature and vacuum conditions with low shear folding to obtain the all-water-based bioconductive ink. S3. Transfer the all-water-based bioconductive ink obtained in step S2 to a printing device and perform roll-to-roll screen printing on the flexible substrate to obtain a wet film electrode array. S4. Place the wet film electrode array obtained in step S3 in a constant temperature and humidity environment for delayed crosslinking and in-situ foaming and curing to obtain the cured electrode array. S5. Place the electrode array cured in step S4 in a vacuum drying oven and perform step-by-step low-temperature vacuum water-retaining drying to obtain the targeted microbial membrane electrode.

7. The method for preparing the targeted microbial membrane electrode according to claim 6, characterized in that, In step S1, the ambient temperature is 20-25℃, the high shear dispersion speed is 1500-2000 rpm, and the stirring time is 45-60 min; In step S2, the temperature of the system after cooling is controlled to be 4-8℃, the rotation speed of the low-shear folding mixing is 30-50 rpm, the vacuum condition is to maintain the system vacuum degree at -0.05MPa to -0.08MPa, and the mixing time is 15-20 min; In step S3, the thickness of the wet film after the all-water-based bioconductive ink is printed and formed on the flexible substrate is controlled to be 50-100μm.

8. The method for preparing the targeted microbial membrane electrode according to claim 6, characterized in that, The specific implementation method of step S4 is as follows: place the wet film electrode array in a reaction chamber with an ambient temperature of 25-30℃ and a relative humidity of 85%-95%, and let it stand continuously for 2.0-3.0h; Through the above environmental control, gluconate-δ-lactone in the system is hydrolyzed, and sodium alginate, carboxylated multi-walled carbon nanotubes and anthraquinone-2,6-disulfonate in the system are cross-linked to form a gel network that fixes the targeted functional strain.

9. The method for preparing the targeted microbial membrane electrode according to claim 1, characterized in that, The specific implementation method of step S5 is as follows: First stage of drying: Control the temperature of the partition at 20-22℃, the vacuum degree at -0.05MPa to -0.06MPa, and maintain for 2-3 hours; Second stage drying: Control the temperature of the partition to drop to 8-10℃, deepen the vacuum to -0.09MPa to -0.10MPa, and maintain for 3-5 hours; By controlling the aforementioned step-like dehydration path, the trehalose, after dehydration, forms an amorphous glassy matrix that encapsulates and fixes the target microorganisms.

10. The application of a targeted microbial membrane electrode according to any one of claims 1-5 in the electrochemical detection of novel pollutants, characterized in that, The targeted microbial membrane electrode is placed in the water body to be tested as the working electrode. The inhibitory effect of the target pollutant on the electron transfer process of microbial metabolism is detected under the applied potential. The new pollutants in the water body are quantitatively analyzed by the oxidation peak current decay value of the differential pulse voltammetry characteristic.

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