A water quality toxicity detection method and device based on gradient pore biofilm electrode

Abstract

The application relates to a water quality toxicity detection method and device based on a gradient pore biofilm electrode, and the method comprises the following steps: preparing a gradient pore biofilm electrode, the gradient pore biofilm electrode comprises a bottom layer micropore area, a middle layer mesopore area and a surface layer macropore area; constructing a three-electrode detection cell based on the gradient pore biofilm electrode, a platinum pair electrode and a reference electrode, and injecting a water sample to be detected into the three-electrode detection cell; applying an alternating current disturbance voltage signal to the three-electrode detection cell, collecting an electrochemical impedance spectrum of the three-electrode detection cell; calculating a corresponding frequency band impedance change rate based on the electrochemical impedance spectrum; and calculating a toxic substance concentration in the water sample to be detected and a penetration depth of the toxic substance in the gradient pore biofilm electrode based on the impedance change rate. Through the gradient pore biofilm electrode, the toxic substance can penetrate from the surface to the inside in stages, the different depth pores and the microbial activity response matching can be realized, and the toxic substance concentration in the water sample to be detected and the penetration depth inside the electrode can be solved synchronously.

Description

Technical Field

[0001] This application relates to the field of water quality monitoring technology, specifically to a method and device for detecting water toxicity based on a gradient pore biofilm electrode. Background Technology

[0002] Water toxicity monitoring is of great significance for water resource protection and ecological security. Microbial electrochemical sensors using electroactive biofilms as sensing elements have become a research hotspot in water toxicity monitoring due to their advantages such as rapid response, ease of operation, and ability to perform online monitoring.

[0003] Traditional methods for detecting microbial toxicity primarily assess the concentration of toxic substances based on changes in a single electrochemical signal. This electrochemical signal can be current, voltage, or single-frequency impedance.

[0004] However, existing technologies have significant drawbacks: First, they cannot distinguish between the concentration of toxic substances and their depth of action within the biofilm. When toxic substances enter the water, they first contact the surface microorganisms of the biofilm and then gradually diffuse inward. A single electrochemical signal reflects the average response of the entire biofilm, making it difficult to differentiate between "low concentration, high permeability" and "high concentration, low permeability." For example, the traditional single-signal method may give the same response value when 0.5 mg / L of toxic substance completely permeates the biofilm and when 5.0 mg / L of toxic substance remains only on the surface, leading to misjudgment. Second, the sensor's lifetime warning capability is insufficient. When the concentration of toxic substances is high, surface microorganisms may be completely inactivated, but deeper microorganisms remain active. A single signal cannot determine the degree of biofilm damage and remaining lifespan, affecting the reliability of the sensor.

[0005] In recent years, electrochemical impedance spectroscopy (EIS) has been applied to the research of microbial sensors. According to AC impedance theory, AC signals of different frequencies have different characteristic diffusion lengths: high-frequency signals reflect the electrochemical processes on and near the electrode surface, while low-frequency signals reflect the diffusion and charge transfer characteristics in deeper regions. This physical principle provides a theoretical basis for stratified analysis. However, existing research mainly analyzes impedance spectra as a whole, failing to fully utilize the correlation between its multi-band information and biofilm structure. Furthermore, to effectively analyze the penetration depth of toxic substances, the biofilm electrode itself needs a structure conducive to stratified microbial colonization. Biofilm electrodes with gradient porosity structures allow microorganisms to colonize stratified along the thickness direction, providing an ideal structural basis for multi-band impedance analysis. Currently, there is no technology that combines gradient porosity structures with multi-band impedance spectroscopy analysis for water toxicity detection.

[0006] Therefore, this invention proposes a water toxicity detection method and device based on gradient pore biofilm electrodes. Summary of the Invention

[0007] To overcome the shortcomings of the existing technology, this application provides a water toxicity detection method and device based on a gradient pore biofilm electrode, which can be applied to drinking water source early warning, industrial wastewater discharge monitoring, and surface water ecotoxicity assessment. Its core component—the gradient pore biofilm electrode—can be mass-produced using a mild freeze-drying process, resulting in low cost. The detection device is small in size and low in power consumption, making it suitable for field and online monitoring. The specific technical solution is as follows.

[0008] A water toxicity detection method based on gradient pore biofilm electrode includes the following steps.

[0009] A gradient pore biomembrane electrode is prepared, comprising a bottom microporous region, a middle mesoporous region, and a surface macroporous region; the middle mesoporous region is located between the bottom microporous region and the surface macroporous region.

[0010] A three-electrode detection cell is constructed based on the gradient pore biofilm electrode, platinum counter electrode, and reference electrode, and the water sample to be tested is injected into the three-electrode detection cell.

[0011] An AC perturbation voltage signal is applied to the three-electrode detection cell, and the electrochemical impedance spectroscopy of the three-electrode detection cell under the action of the water sample to be tested is collected.

[0012] The electrochemical impedance spectroscopy is divided into multiple frequency bands, and the impedance change rate of each frequency band is calculated. Based on the impedance change rate of each frequency band, the concentration of toxic substances in the water sample to be tested and the penetration depth of toxic substances in the gradient pore biofilm electrode are calculated.

[0013] The above technical solution involves preparing a gradient-pore biofilm electrode comprising a bottom microporous region, a middle mesoporous region, and a surface macroporous region. A three-electrode detection cell is constructed using this gradient-pore biofilm electrode, a platinum counter electrode, and a reference electrode, and the water sample to be tested is injected. An AC perturbation voltage signal is then applied to acquire an electrochemical impedance spectroscopy (EIS). The EIS is divided into multiple frequency bands, and the impedance change rate of each band is calculated. Finally, based on the impedance change rate of each frequency band, the concentration of toxic substances and the penetration depth of toxic substances in the water sample are calculated. This avoids interference from full-band signal coupling and allows for the acquisition of both toxic substance concentration and penetration depth, improving the comprehensiveness and accuracy of water toxicity detection.

[0014] As an optional embodiment of the present invention, the preparation of a gradient pore biomembrane electrode includes the following steps.

[0015] Prepare an electrodeposition solution containing electroactive microorganisms and film-forming materials.

[0016] An electrode with a hydrogel coating is formed by electrodeposition on the surface of a graphite rod using the electrodeposition solution.

[0017] The electrode with hydrogel coating was subjected to gradient temperature-controlled freeze-drying to obtain a freeze-dried electrode.

[0018] The freeze-dried electrode was placed in a sterile, pre-cooled calcium chloride solution for mild cross-linking enhancement treatment to obtain a cross-linked electrode.

[0019] The cross-linked electrode was placed in a sterile culture medium and cultured at a constant temperature to obtain the gradient pore biomembrane electrode.

[0020] The above technical solution stabilizes and forms an electrode substrate with a gradient pore structure, effectively retains the activity of electroactive microorganisms, ensures the electrochemical response performance of the gradient pore biofilm electrode, and provides a reliable electrode substrate for subsequent accurate detection of water toxicity.

[0021] As an optional embodiment of the present invention, the preparation of the electrodeposition solution containing electroactive microorganisms and film-forming materials includes the following steps.

[0022] Take 1.5%–2.5% sodium alginate as the main film-forming agent, add 2.5%–3.5% calcium disodium ethylenediaminetetraacetate, mix evenly to form the basic film-forming matrix.

[0023] Add 0.15% to 0.25% graphene solution to the basic film-forming matrix and stir until uniformly dispersed to obtain a reduced graphene composite dispersion.

[0024] According to the testing requirements, the corresponding electroactive microbial liquid is added to the reduced graphene composite dispersion to form a mixed system.

[0025] Add 0.05% to 0.1% cysteine ​​as a microbial protectant to the mixture, stir until homogeneous, and obtain the electrodeposition solution containing electroactive microorganisms and film-forming materials.

[0026] The above technical solutions ensure good film formation of hydrogel and complete conductive pathway of electrode, while effectively inhibiting the damage of electric field to microorganisms during electrodeposition, stably preserving the metabolic activity of electroactive microorganisms, solving the problem of mutual constraints between film formation, pore formation and microbial survival, and ensuring that the prepared electrode has stable toxicity response capability.

[0027] As an optional embodiment of the present invention, the gradient temperature-controlled freeze-drying process for the electrode with hydrogel coating includes the following steps.

[0028] Pre-freezing stage: The electrode with hydrogel coating is cooled to below -50°C at a cooling rate of 0.5 to 2.0°C / min and kept at that temperature for 1.0 to 3.0 hours.

[0029] Ice crystal growth control stage: The electrode with hydrogel coating is heated to -20.0℃ to -10.0℃ at a heating rate of 0.4 to 0.6℃ / min and kept at that temperature for two to four hours.

[0030] First drying stage: the vacuum degree is lower than 10.0 Pa, the temperature of the electrode with hydrogel coating is controlled at -30.0℃ to -20.0℃, and the drying time is 10 to 15 hours.

[0031] Secondary drying stage: Heat to 20.0℃~30.0℃ and dry for five to ten hours.

[0032] By inducing the directional growth of ice crystals through temperature gradient, a pore gradient structure with a continuous and gradual change along the thickness direction is formed inside the electrode with hydrogel coating.

[0033] Through the above technical solution, a pore gradient structure that is continuously and gradually changes along the thickness direction is stably formed inside the electrode with hydrogel coating, forming a layered structure of bottom micropores, middle mesopores and surface macropores, ensuring that the electrode pore gradient is uniform and the structure is intact, providing a stable structural basis for microbial stratified colonization and multi-band impedance stratified response.

[0034] As an optional embodiment of the present invention, during the ice crystal growth control stage, under the condition of vacuum degree ≤10.0Pa, nitrogen gas is intermittently introduced at a flow rate of 50-80mL / min, with an interval of 30min / time, and each introduction lasting ten minutes, to suppress the oxidation of the surface of the electrode with hydrogel coating.

[0035] The above technical solutions effectively suppress the oxidation of the electrode surface with hydrogel coating, avoid pore collapse and structural damage during ice crystal growth, ensure the integrity and connectivity of the gradient pore structure, improve the consistency and repeatability of multiple batches of electrode preparation, and stabilize the electrode detection performance.

[0036] As an optional embodiment of the present invention, the pore size of the middle mesoporous region is smaller than that of the surface macroporous region; the pore size of the middle mesoporous region is larger than that of the bottom microporous region; the pore size of the surface macroporous region ranges from 20.1 to 50.0 μm, the pore size of the middle mesoporous region ranges from 5.0 to 20.0 μm, and the pore size of the bottom microporous region is less than 5.0 μm.

[0037] Through the above technical solution, by forming a gradient pore structure, it is possible to achieve stratified permeation of toxic substances in water samples and stratified colonization of microorganisms, so that different thickness regions of the electrode correspond to independent electrochemical response signals, providing structural support for multi-band impedance signal separation and stratified analysis of toxic permeation state.

[0038] As an optional embodiment of the present invention, in the step of obtaining the gradient pore biofilm electrode: two cross-linked electrodes are selected, one of which is placed in a Geobacterium culture medium containing 2.5g NaHCO3, 0.25g NH4Cl, 0.6g NaH2PO4·H2O, 0.1g KCl and 1.0g sodium acetate per liter, pH 6.8, and cultured under anaerobic conditions at 30.0℃ to cultivate anaerobic electroactive microorganisms adapted to the anodic gradient pore biofilm electrode; the other cross-linked electrode is placed in a lysine broth culture medium and cultured under aerobic conditions at 30℃ for twenty-four hours to cultivate aerobic electroactive microorganisms adapted to the gradient pore biofilm electrode as a cathode.

[0039] The above technical solutions can be used to specifically cultivate and adapt functional microbial communities to different pore levels, achieving stratified colonization of microbial communities and adaptation to the gradient pore structure of electrodes, thereby further improving the accuracy and stability of electrode stratified detection.

[0040] As an optional embodiment of the present invention, in the step of dividing the electrochemical impedance spectrum into multiple frequency bands and calculating the impedance change rate of the multiple frequency bands.

[0041] The electrochemical impedance spectrum is divided into multiple characteristic frequency bands.

[0042] The high-frequency band is 10.0kHz to 100kHz, corresponding to 0 to 150μm surface penetration; the impedance change rate in the high-frequency band was calculated.

[0043] The mid-frequency band is 100Hz to 10.0kHz, corresponding to the 150 to 350μm mid-layer permeation; the impedance change rate in the mid-frequency band was calculated.

[0044] The low-frequency band is 0.1Hz to 100Hz, corresponding to a 350 to 500μm sublayer penetration; the impedance change rate in the low-frequency band was calculated.

[0045] The above technical solution enables the independent separation of toxicity response signals at different penetration depths, binds frequency band signals to the electrode's layered penetration area, solves the problem of full-band signal coupling interference, and can characterize the changes in microbial activity in the electrode's surface, middle, and bottom layers.

[0046] As an optional solution of the present invention, in the process of calculating the concentration of toxic substances in the water sample to be tested and the penetration depth of toxic substances in the gradient pore biofilm electrode based on the impedance change rate, a toxic substance concentration prediction model is constructed to obtain the concentration of toxic substances in the water sample to be tested, and a toxic substance penetration depth prediction model is constructed to obtain the penetration depth of toxic substances in the water sample to be tested in the gradient pore biofilm electrode.

[0047] The toxic substance concentration prediction model takes the high-frequency impedance change rate, mid-frequency impedance change rate and low-frequency impedance change rate as inputs, and outputs the toxic substance concentration in the water sample to be tested.

[0048] The toxic substance penetration depth prediction model takes high-frequency impedance change rate, mid-frequency impedance change rate and low-frequency impedance change rate as inputs, and outputs the penetration depth of toxic substances in the water sample to be tested within the gradient pore biofilm electrode.

[0049] The above technical solution effectively weakens the coupling interference between concentration and penetration depth in traditional detection, and simultaneously improves the detection accuracy of toxic substance concentration in water samples and the analytical accuracy of toxic penetration depth inside the electrode.

[0050] This invention provides a water toxicity detection device based on a gradient pore biofilm electrode for water toxicity detection, comprising a three-electrode detection cell and an electrochemical workstation.

[0051] The three-electrode detection cell includes a gradient pore biomembrane electrode, a platinum counter electrode, a reference electrode, and a housing.

[0052] The gradient pore biomembrane electrode comprises a bottom microporous region, a middle mesoporous region, and a surface macroporous region; the middle mesoporous region is located between the bottom microporous region and the surface macroporous region.

[0053] A current collector is provided at the upper end of the gradient pore biofilm electrode.

[0054] The gradient pore biomembrane electrode, platinum counter electrode, and reference electrode are respectively connected to the working electrode, counter electrode, and reference electrode interfaces of the electrochemical workstation.

[0055] The above technical solutions can be used to construct a stable electrochemical detection system, which can achieve toxicity stratification response based on the electrode gradient pore structure, ensuring the stability and accuracy of the detection signal, and realizing automated stratified detection of water sample toxicity.

[0056] The technical solution of this application has achieved the following beneficial effects.

[0057] 1. This invention employs a gradient pore biofilm electrode formed by sequentially arranging a bottom microporous region, a middle mesoporous region, and a surface macroporous region, creating a gradient pore size structure of surface macropores > middle mesopores > bottom micropores. Combined with gradient temperature-controlled freeze-drying treatment, the temperature gradient induces the directional growth of ice crystals, forming a pore gradient structure that gradually changes along the electrode thickness direction. This allows toxic substances to penetrate from the surface to the interior step by step, achieving precise matching between pore depths and microbial activity responses, and providing a stable structural basis for multi-band layered impedance response.

[0058] 2. This invention constructs a toxic substance concentration prediction model and a toxic substance penetration depth prediction model. The toxic substance concentration prediction model and the toxic substance penetration depth prediction model have the same structure. Both use high-frequency impedance change rate, mid-frequency impedance change rate and low-frequency impedance change rate as inputs, and couple contact time, ambient temperature and water sample conductivity as auxiliary feature variables. By setting differentiated training sample labels, two sets of independent weight coefficients are obtained respectively, which can simultaneously calculate the concentration of toxic substances in the water sample to be tested and the penetration depth of toxic substances inside the electrode, so as to achieve accurate quantitative detection of concentration-depth dual parameters. Attached Figure Description

[0059] Figure 1 This is a schematic flowchart of the water toxicity detection method based on gradient pore biofilm electrode in the embodiments of this application.

[0060] Figure 2 The images shown are SEM cross-sectional views of the gradient porosity electrode in this embodiment. a) shows the pore diameter distribution in the surface macroporous region; b) shows the pore diameter distribution in the middle mesoporous region; c) shows the pore diameter distribution in the bottom microporous region.

[0061] Figure 3 This is a schematic diagram of a water toxicity detection device based on a gradient pore biofilm electrode.

[0062] Figure 4 This is a schematic diagram of the three-electrode detection cell.

[0063] Figure 5 This is a schematic diagram of the structure of a gradient pore biomembrane electrode.

[0064] Figure reference numerals: 1. Three-electrode detection cell; 2. Electrochemical workstation; 3. Data processing unit; 4. Model update unit; 5. Display unit; 11. Gradient pore biofilm electrode; 12. Platinum counter electrode; 13. Reference electrode; 14. Sealing cap; 15. Box body; 111. Electrode substrate; 112. Bottom layer microporous region; 113. Middle layer mesoporous region; 114. Surface layer macroporous region; 115. Electroactive microorganisms; 116. Current collector. Detailed Implementation

[0065] The present application will now be further described with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present application and should not be construed as limiting the scope of protection of the present application. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present application.

[0066] like Figure 1 As shown, this invention discloses a water toxicity detection method based on a gradient pore biofilm electrode, comprising the following steps.

[0067] Step S1: Prepare a biofilm electrode with gradient pores and retain microbial activity: Prepare an electrodeposition solution containing electroactive microorganisms and film-forming materials, and form a hydrogel coating by electrodeposition on the surface of a graphite rod. Then, form a gradient pore structure by gradient temperature-controlled freeze drying. After gentle cross-linking with calcium chloride, it is cultured in layers at a constant temperature to obtain a biofilm electrode with three-layer gradient pores, layered microbial colonization, and stable metabolic activity.

[0068] Step S2: Establish a quantitative relationship model for toxicity response: Prepare multi-gradient standard toxicity solutions, and use the prepared gradient pore biomembrane electrode to repeatedly measure multi-band impedance spectra. With the impedance change rate of each band as the core input, combined with environmental auxiliary characteristic variables, train and construct a hierarchical response coupling model. After cross-validation and outlier removal, establish a precise quantitative mapping relationship between impedance response and toxicity concentration and penetration depth.

[0069] Step S3: Acquire full-band electrochemical impedance spectroscopy. A three-electrode detection cell is constructed based on a gradient pore biofilm electrode. An AC perturbation voltage signal with an amplitude of 10mV (0.1Hz–100kHz) is applied to the three-electrode detection cell via an electrochemical workstation. The complete electrochemical impedance spectrum of the water sample under test is acquired under constant temperature conditions of 20–25℃ to avoid temperature interference with the detection results. A segmented acquisition mode is adopted during the acquisition process: the acquisition step size is 1.0kHz for the high-frequency band (10.0kHz–100kHz), 100Hz for the mid-frequency band (100Hz–10.0kHz), and 10.0Hz for the low-frequency band (0.1Hz–100Hz). This balances acquisition efficiency and data accuracy, and avoids the loss of frequency response information due to excessively large step sizes.

[0070] Step S4: Frequency band division and calculation of impedance change rate of each frequency band: The collected full-band electrochemical impedance spectrum is divided into three independent characteristic frequency bands: high, medium and low, according to frequency and penetration depth. After blank baseline calibration and isothermal normalization correction, the impedance change rate of each frequency band is calculated to separate the independent response signals of the corresponding electrode surface, middle layer and bottom layer.

[0071] Step S5: Simultaneously calculate toxicity concentration and penetration depth: Substitute the impedance change rate of each frequency band and auxiliary variables such as contact time, temperature, and conductivity into the trained hierarchical response coupling model to weaken the coupling interference between concentration and penetration depth, and simultaneously calculate and output the concentration of toxic substances in the water sample to be tested and the penetration depth inside the electrode.

[0072] Step S6, Determination of Toxicity Penetration Status and Sensor Lifetime Warning: By comparing the relative magnitudes of impedance change rates in three frequency bands, the toxicity penetration status at the surface, middle, and deep layers is determined. The electrode working status is determined based on the graded warning thresholds. Combined with the electrode aging and decay law and lifetime prediction logic, the remaining lifespan of the electrode is assessed and replacement is predicted in advance.

[0073] This invention provides a water toxicity detection device based on a gradient pore biofilm electrode, such as... Figure 4 , Figure 4 and Figure 5 As shown, this device enables rapid, accurate, and stable detection of water toxicity, and is suitable for toxicity screening of various water samples, including surface water, industrial wastewater, and drinking water. It comprises a three-electrode detection cell 1, an electrochemical workstation 2, a data processing unit 3, a model updating unit 4, and a display unit 5. All components work together to complete the entire process from water sample detection to result output and model optimization. Stable connections between components are achieved via shielded wiring harnesses, ensuring interference-free signal transmission and preventing external electromagnetic interference from affecting detection accuracy.

[0074] The three-electrode detection cell 1 is connected to the signal input / output interface of the electrochemical workstation 2 via a shielded wiring harness. The three-electrode detection cell 1 includes a gradient pore biofilm electrode, a platinum counter electrode, and a reference electrode 13. The gradient pore biofilm electrode, platinum counter electrode, and reference electrode 13 of the three-electrode detection cell 1 are respectively connected to the working electrode, counter electrode, and reference electrode interfaces of the electrochemical workstation 2, for transmitting the electrochemical signals from the three-electrode detection cell 1 to the electrochemical workstation 2 in real time, and simultaneously receiving the AC perturbation voltage signal output by the electrochemical workstation 2. The electrochemical signals include raw impedance spectroscopy data.

[0075] Electrochemical workstation 2 is connected to the signal input terminal of data processing unit 3 via a shielded cable harness, transmitting the acquired raw impedance spectrum data to data processing unit 3 for subsequent signal analysis and toxicity analysis. The control signal output terminal of data processing unit 3 is connected to electrochemical workstation 2 via a cable harness, allowing it to send parameter adjustment commands to electrochemical workstation 2 according to analytical needs, such as the frequency range and amplitude of the AC disturbance voltage signal, achieving dynamic adaptation of detection parameters. The raw impedance spectrum data includes key parameters such as amplitude and phase.

[0076] Electrochemical workstation 2 is connected to display unit 5 via a bidirectional shielded cable harness, enabling bidirectional data transmission: on one hand, electrochemical workstation 2 transmits the acquired raw impedance data and real-time detection status to display unit 5 for operators to view in real time; on the other hand, display unit 5 can act as an operating terminal, receiving detection parameters input by the operator, including detection duration, temperature threshold, and stirring rate, and transmitting these parameter commands to electrochemical workstation 2 to complete the manual setting and adjustment of detection parameters. Real-time detection status includes detection progress and electrode connection status.

[0077] Both the data processing unit 3 and the display unit 5 are connected to the model update unit 4 via a bidirectional shielded cable harness, enabling bidirectional data transmission: the data processing unit 3 transmits error data generated during the detection process, deviation data of toxicity analysis results, and detection data of new water samples to the model update unit 4, providing data support for model adaptive correction; the model update unit 4 transmits the corrected optimized model parameters and model update status to the data processing unit 3, ensuring that the data processing unit 3 always uses the optimal model for toxicity analysis; at the same time, the model update unit 4 transmits the model update log and correction results to the display unit 5, allowing operators to view the model's operating status, and issuing early warning prompts through the display unit 5 if the model malfunctions.

[0078] Reference Figure 4 The three-electrode detection cell 1 includes a gradient pore biomembrane electrode 11, a platinum counter electrode 12, a reference electrode 13, and a housing 15. These components work together to form a complete detection reaction system.

[0079] The housing 15, as the main structure of the testing pool, is made of polytetrafluoroethylene in one piece. Its inner wall is smooth and treated with anti-adsorption to prevent impurities and toxic substances in the water sample from adsorbing onto the pool wall and affecting the test results. The side wall of the housing 15 is provided with an inlet and an outlet. The inlet is used to inject the water sample to be tested, and the outlet is used to discharge the water sample after the test is completed. Both the inlet and outlet are equipped with sealing valves to ensure the airtightness of the housing 15 when closed. A magnetic stir bar is provided at the bottom of the housing 15. The stirring vortex range is controlled in the bottom 1 / 3 area of ​​the housing, which, together with the built-in magnetic stirring device, achieves water sample stirring.

[0080] The built-in magnetic stirring device is a miniature embedded magnetic stirrer, integrated on the outer bottom of the housing 15, corresponding to the internal stir bar. It is driven by a DC brushless motor with a power of 5.0-10.0W and a speed adjustment range of 50-80rpm, with a default speed of 60rpm. This magnetic stirring device is electrically connected to the electrochemical workstation 2 and can receive speed adjustment commands from the workstation, enabling adaptive adjustment of the stirring rate. Its embedded installation design avoids contact with the water sample inside the housing, preventing contamination of the detection system and ensuring uniform stirring while preventing damage to the biofilm structure on the electrode surface during stirring. The housing 15 contains a detachable and fixed gradient pore biofilm electrode 11, a platinum counter electrode 12, and a reference electrode 13, all evenly distributed in a triangular pattern. The electrode tips are all immersed in the water sample, and the spacing between the electrodes is controlled at 2.0-3.0cm, ensuring smooth electrochemical signal transmission between the electrodes while avoiding mutual interference.

[0081] The sealing cap 14 is made of polytetrafluoroethylene material that matches the box body 15. It is detachably fixed inside the upper part of the box body 15. A fluororubber sealing ring is provided at the connection between the sealing cap 14 and the box body 15 to further improve the sealing performance and prevent water sample evaporation and outside air from entering the detection cell and affecting the stability of the detection system. The sealing cap 14 is provided with three electrode through holes for the gradient pore biofilm electrode 11, platinum counter electrode 12 and reference electrode 13 to pass through. A sealing gasket is provided between the through hole and the electrode to prevent water sample leakage from the through hole and to fix the electrode position to prevent the electrode from shaking during the detection process.

[0082] The platinum counter electrode 12 is made of high-purity platinum wire with a purity of ≥99.99%. The surface of the platinum counter electrode 12 is polished, which gives it good conductivity and chemical stability. During the detection process, it acts as a counter electrode, forming a current loop with the gradient pore biofilm electrode 11 to assist in the electrochemical reaction. It does not participate in the reaction itself and can be reused, thus reducing the detection cost.

[0083] The reference electrode 13 is an Ag / AgCl reference electrode based on a saturated KCl system. It exhibits stable potential and good reproducibility, with a standard potential of 0.197V at 25℃. During detection, it provides a stable reference potential for calibrating the electrode potential of the gradient pore biofilm electrode 11, ensuring the accuracy of electrochemical signal acquisition and avoiding detection errors caused by potential drift. A ceramic sand core is provided at the salt bridge end of the reference electrode 13 to prevent mixing of the reference solution with the water sample while ensuring smooth ion conduction. To compensate for its slightly lower potential stability compared to the saturated calomel reference electrode, the data processing unit 3 incorporates a real-time reference potential calibration algorithm. The calibration trigger condition is a potential drift ≥5mV, employing an iterative calibration logic every 10 minutes with a calibration range of ±10mV. This dynamically corrects potential drift, ensuring detection accuracy. The real-time reference potential calibration algorithm is a mature existing technology and is only used in this invention.

[0084] Gradient pore biofilm electrode 11: As the core detection electrode of this device, it is crucial for achieving water toxicity detection. A current collector 116 is fixedly installed at its upper end. The current collector 116 is made of copper foil with a thickness of 0.1-0.2 mm and its surface is gold-plated with a gold layer thickness of 0.5-1.0 μm. After gold plating, it undergoes passivation treatment to improve corrosion resistance. Its main function is to collect and conduct current, reduce the internal resistance of the detection system, and ensure that the electrochemical signal can be transmitted quickly and stably to the electrochemical workstation 2. At the same time, it avoids corrosion of the current collector itself and extends the service life of the electrode. The upper end of the current collector 116 is connected to the working electrode interface of the electrochemical workstation 2 through a wire to realize signal transmission.

[0085] Reference Figure 5The gradient pore biofilm electrode 11 is the core component of this device. Its unique gradient pore structure enables the layered immobilization and efficient metabolism of electroactive microorganisms 115, improving detection sensitivity and response speed. The gradient pore biofilm electrode 11 includes an electrode substrate 111, a bottom microporous region 112, a middle mesoporous region 113, a surface macroporous region 114, and electroactive microorganisms 115. The layers are stacked sequentially and tightly combined to form an integrated gradient pore structure.

[0086] Electrode substrate 111: As a support carrier for the electrode, it is made of graphite rod with a diameter of 3.0-5.0 mm and a length of 8.0-10.0 cm. It has good conductivity, mechanical strength and chemical stability, and can support the upper porous structure while conducting current. The lower end of the electrode substrate 111 is inserted into the electrode fixing seat at the bottom of the box 15 to fix the electrode. The electrode fixing seat is not shown. The upper end is fixedly connected to the current collector 116 to ensure smooth current conduction.

[0087] The bottom microporous region 112 is fixedly disposed above the electrode substrate 111, with a thickness of 100-200 μm and a pore size of 0.1-1.0 μm. It is made of activated carbon and polyvinylidene fluoride (PVDF) composite, with activated carbon content of 70-80% and PVDF as a binder content of 20-30%. The molding process is as follows: activated carbon and PVDF are mixed in a certain proportion, N-methylpyrrolidone is added as a solvent, and after stirring evenly, it is coated on the surface of the electrode substrate and dried at 80℃ for 2 hours. The molding pressure is 0.5-1.0 MPa. The microporous structure of the bottom microporous region 112 can firmly fix the electroactive microorganisms 115 and prevent the microorganisms from falling off. At the same time, the microporous structure can adsorb small molecule toxic substances in the water sample, provide a stable microenvironment for microbial metabolism, and facilitate rapid electron conduction.

[0088] The middle mesoporous region 113 is fixedly positioned above the bottom microporous region 112, with a thickness of 200-300 μm and a pore size of 2.0-50.0 nm. It is made of mesoporous silica and PVDF composite. The molding process is as follows: mesoporous silica and PVDF are mixed at a mass ratio of 8:2, ethanol is added as a dispersant, and after being ultrasonically dispersed evenly, it is coated onto the surface of the bottom microporous region and dried at 60℃ for 1.5 h. The molding pressure is 0.3-0.5 MPa. The mesoporous structure has a large specific surface area, which can support a large number of electroactive microorganisms 115, while providing sufficient space for microbial metabolism, promoting electron transfer between microorganisms, and improving the electrochemical activity of the electrode. The middle mesoporous region 113 serves to connect the bottom microporous region 112 and the surface macroporous region 114, realizing a gradient transition of pore size and avoiding uneven distribution of microorganisms caused by abrupt changes in pore size.

[0089] The surface macroporous region 114 is fixedly positioned above the mesoporous region 113, with a thickness of 140-160 μm and a pore size of 1.0-5.0 μm. It is made of a composite of nickel foam and activated carbon. The molding process is as follows: the nickel foam is cut to a size that matches the electrode substrate, immersed in an activated carbon dispersion, dried, and then pressed onto the surface of the mesoporous region. The pressing pressure is 0.2-0.3 MPa, the drying temperature is 70℃, and the time is 1.0 h. The macroporous structure allows toxic substances in the water sample to diffuse rapidly into the electrode interior and fully contact the electroactive microorganisms 115. At the same time, it facilitates the discharge of microbial metabolites, reducing the impact of product accumulation on microbial activity, thereby improving the detection response speed and sensitivity. The surface of the surface macroporous region 114 is hydrophilically treated, which can enhance the contact between the electrode and the water sample, further improving the detection performance.

[0090] Electroactive microorganisms 115: These microorganisms grow uniformly in the pore structure of the bottom microporous zone 112, the middle mesoporous zone 113, and the surface macroporous zone 114. They are domesticated electrogenic microbial communities, mainly including Shewanella and Geobacterium. These microorganisms can undergo metabolic reactions under electrochemical action, producing electron transfer. When toxic substances are present in the water sample, the toxic substances will inhibit the metabolic activity of the microorganisms, causing changes in the impedance characteristics of the electrode. Quantitative analysis of water toxicity can be achieved by detecting the impedance changes. The gradient pore structure design allows microorganisms to grow in different pore zones. The bottom microporous zone immobilizes microorganisms, the middle mesoporous zone loads microorganisms, and the surface macroporous zone facilitates contact between microorganisms and the water sample, maximizing the metabolic efficiency of microorganisms and their sensitivity to toxic substances.

[0091] The toxicity and permeation characteristics of gradient pore biomembrane electrodes are shown in Table 1 below.

[0092]

[0093] Long-term stability experiments were conducted on n=5 batches, with three electrodes per batch. Within 30 days of continuous operation, the model prediction error was controlled within 10%, with RSD ≤ 8%. After the fifteenth day, the cumulative background impedance drift was approximately 12%, but after Kalman filtering correction, the error remained within 10%. On the 28th day, the low-frequency background impedance change rate exceeded 20% of the initial value for 2.0 mg / L Cd²⁻¹. + of The response value dropped from the initial 9.8% to 5.9%, and the sensitivity decreased by approximately 40%. The response under standard toxicity shock was below the first-level alert threshold, and the system prompted "recommend replacing the electrode". The electrode was removed and stained with live / dead bacteria, confirming that the survival rate of deep microorganisms had dropped to below 30%, which was consistent with the actual degree of electrode aging.

[0094] The comparative experimental data of gradient pore electrode and ordinary homogeneous electrode are shown in Table 2 below.

[0095]

[0096] Measurements were taken at five different locations for each electrode in n=3 batches, with data expressed as mean ± standard deviation. This data was used to calibrate the correlation between penetration depth and impedance change rate at different frequency bands, providing experimental support for establishing a layered response coupling model. It was also used to determine the warning threshold. The correlation between the model-predicted penetration depth and the measured depth indicates that the model prediction is reliable. Statistical tests showed that the gradient pore electrode exhibited significantly lower prediction errors for both scenarios compared to the ordinary homogeneous electrode. The signal separation index was improved by 3.2 times (0.99 / 0.31).

[0097] Example 1.

[0098] This embodiment 1 discloses a water toxicity detection method based on gradient pore biofilm electrode, wherein step S1 includes the following steps.

[0099] S11. Prepare an electrodeposition solution containing electroactive microorganisms and film-forming materials.

[0100] S111: Prepare the film-forming material system. Take 1.5% to 2.5% (w / v) sodium alginate as the main film-forming agent, add 2.5% to 3.5% (w / v) calcium disodium ethylenediaminetetraacetate, mix thoroughly and evenly to form the basic film-forming matrix.

[0101] S112: Add 0.15%–0.25% (w / v) graphene solution to the basic film-forming matrix. The graphene solution has a sheet diameter of 1.0–5.0 μm and a thickness of 1.0–5.0 nm. The graphene is reduced graphene with one to five layers. The dispersant is 0.05% (w / v) sodium dodecylbenzenesulfonate. Stir until uniformly dispersed to obtain a reduced graphene composite dispersion. The graphene doping amount is strictly controlled at 0.15%–0.25% (w / v) to ensure that the conductive pathway is constructed, the subsequent micropores are not blocked, and the electroactive microorganisms are not killed.

[0102] S113: Add the corresponding electroactive microbial culture solution according to the electrode type. The electrode types include anode and cathode. The anode uses *Geobacterium* ATCC 51573 culture solution, and the cathode uses *Pseudomonas* ATCC 27853 culture solution. The culture solution must meet the following requirements: OD600≈1.0, viable cell count approximately... The requirement of CFU / mL is met to form a mixed system.

[0103] S114: Add 0.05%–0.1% (w / v) L-cysteine ​​to the mixture as a microbial protectant, stir well. Its function is to inhibit damage to the microbial cell membrane by the electric field during subsequent electrodeposition, further improving the microbial survival rate without affecting the film formation effect and subsequent pore formation, thus obtaining a mixed solution. It should be noted that L-cysteine ​​itself is a known amino acid compound and belongs to existing technology. However, this invention is the first to apply it at a specific ratio of 0.05%–0.1% (w / v) in the preparation of the electrodeposition solution for a gradient pore biofilm electrode, specifically to inhibit damage to the microbial cell membrane by the electric field during electrodeposition, achieving a synergistic adaptation between microbial survival, film formation, and pore formation.

[0104] S115: After thoroughly mixing the liquid, seal and store for later use.

[0105] S12. Electrode with hydrogel coating: A graphite rod with a diameter of 3.0–5.0 mm and a length of 8.0–10.0 cm is selected as the substrate material. After the surface is polished with 800-grit sandpaper, it is ultrasonically cleaned with acetone, ethanol, and deionized water for 15 minutes each. A hydrogel coating encapsulating electroactive microorganisms is formed on the substrate surface by electrodeposition. The electrodeposition voltage is set to 5.0 V, the deposition time is at least three minutes, and the coating thickness is controlled at 480–520 μm. After polishing the electrode substrate, plasma etching is performed. The etching atmosphere is argon, the vacuum degree is ≤5.0 Pa, the etching distance is 3.0–5.0 mm, the power is 80–100 W, and the time is three to five minutes. A micro-nano-level rough structure is formed on the substrate surface, which improves the adhesion between the hydrogel coating and the substrate, avoids the coating from falling off during the detection process, and provides additional attachment sites for the colonization of underlying microorganisms, thus forming an electrode with hydrogel coating.

[0106] S13. Gradient temperature-controlled freeze-drying is performed on the electrodes with hydrogel coatings, and the temperature is controlled to be no higher than 25°C throughout the drying process. The room temperature reference is defined as 25°C to ensure that the metabolic activity of microorganisms is not destroyed. The specific process of temperature-controlled freeze-drying includes:

[0107] (1) Pre-freezing stage: Cool the sample to below -50℃ at a cooling rate of 0.5~2.0℃ / min and keep it warm for one to three hours.

[0108] (2) Ice crystal growth control stage: The sample is heated to -20.0℃ to -10.0℃ at a heating rate of 0.4 to 0.6℃ / min and kept at that temperature for two to four hours. The electrode substrate is vertically fixed on the gradient temperature control sample stage made of thermally conductive copper plate in the freeze dryer. The temperature is controlled independently by multi-point thermocouple feedback to maintain the temperature difference between the bottom and top of the electrode at 5.0 to 15.0℃. Combined with the total thickness of the electrode coating of 480 to 520μm, the temperature gradient is adjusted to 10.0 to 31.0℃ / cm. The ice crystal growth size is precisely controlled to induce the ice crystals to grow in a directional manner along the temperature gradient, matching the requirements of the three-layer pore size formation. During the ice crystal growth control stage, under the condition of vacuum degree ≤10.0Pa, inert gas nitrogen is intermittently introduced at a flow rate of 50 to 80mL / min, with an interval of 30min / time and each introduction lasting ten minutes. This inhibits the oxidation of the sample surface and avoids pore collapse during the ice crystal growth process, ensuring the integrity and connectivity of the gradient pores. It should be noted that multi-point thermocouple feedback independent temperature control is a mature existing temperature control technology, widely used in precision temperature control scenarios, and belongs to existing technology. However, this invention is the first to adapt it to the freeze-drying process of gradient pore biofilm electrodes, specifically for precisely controlling the temperature gradient in the electrode thickness direction, providing stable temperature control support for the directional growth of ice crystals and the formation of gradient pores.

[0109] It should be noted that multi-point thermocouple feedback independent temperature control is a mature existing temperature control technology, widely used in precision temperature control scenarios, and belongs to existing technology. However, this invention is the first to adapt it to the freeze-drying process of gradient pore biofilm electrodes, specifically for precisely controlling the temperature gradient in the electrode thickness direction, providing stable temperature control support for the directional growth of ice crystals and the formation of gradient pores.

[0110] (3) First drying stage: vacuum degree is lower than 10.0 Pa, sample temperature is controlled at -30.0℃ to -20.0℃, and drying is carried out for ten to fifteen hours.

[0111] (4) Secondary drying stage: Heat to 20.0℃~30.0℃ and dry for five to ten hours to obtain freeze-dried electrode.

[0112] By inducing the directional growth of ice crystals through temperature gradients, a pore gradient structure with a continuous and gradual change along the thickness direction is formed inside the electrode. Simultaneously, the thickness ratio of the three pore layers is precisely controlled: the surface macroporous layer is 140–160 μm thick, the middle mesoporous layer is 190–210 μm thick, and the bottom microporous layer is 140–160 μm thick. The porosity decreases progressively from the surface to the interior: 65%–70% for the surface layer, 50%–55% for the middle layer, and 28%–35% for the bottom layer. This adapts to the mechanism of toxicity permeating from the surface inwards and the multi-band impedance stratification response. The coefficient of variation of the pore size distribution of the three pore layers is controlled within 15%–20%, ensuring uniform pore size within the same pore layer and avoiding frequency response interference caused by local pore size anomalies, further improving the stability of signal separation. The pore size of the surface macroporous region is 1.0–5.0 μm, the middle mesoporous region is 2.0–50.0 nm, and the bottom microporous region is 0.1–1.0 μm.

[0113] Figure 2 The SEM cross-sectional image of the gradient pore electrode shows a clear gradient distribution: the surface macropore region (1.0–5.0 μm), the middle mesopore region (2.0–50.0 nm), and the bottom micropore region (0.1–1.0 μm). The three-layer structure is tightly bonded, with no pore collapse and uniform pore size, perfectly matching the preset gradient pore parameters. This visually demonstrates the forming effect of the gradient pore structure of the present invention.

[0114] Figure 2 In the figure, 'a' represents the pore diameter distribution of the macroporous region on the surface: corresponding to the outermost layer of the electrode, the photo clearly shows that the pore diameter is distributed in the range of 30~45μm, presenting a loose and porous sponge-like structure, providing ample space for the initial attachment of microorganisms and the exchange of substances.

[0115] Figure 2 In the diagram, b represents the pore diameter distribution of the middle mesoporous region: corresponding to the intermediate transition layer of the electrode, the pore diameter is between 8 and 15 μm, the structure tends to be dense, connecting the surface layer and the bottom layer, and regulating the material transport rate.

[0116] Figure 2 In the diagram, 'c' represents the pore diameter distribution of the bottom microporous region: the inner layer of the electrode, which is close to the substrate, has a pore diameter distribution in the range of 2~4μm. This region has the densest structure and provides stable attachment points and a protective microenvironment for the inner microorganisms.

[0117] S14. Place the freeze-dried electrode in a sterile, pre-cooled 1% (w / v) calcium chloride solution for mild cross-linking enhancement treatment at a temperature of 3.0–5.0℃ for 14–16 minutes to obtain a cross-linked electrode. A low-temperature, short-time cross-linking method is used to lock the pore structure without damaging microbial activity. After cross-linking, wash three times with sterile deionized water. After washing, detect the residual calcium ion concentration on the electrode surface to ensure the residual concentration is ≤10%. -6The concentration of mannitol was controlled at mol / L, while the gel swelling rate was kept within the range of 5% to 10%. 0.1% to 0.2% (w / v) of mannitol was added to the crosslinking solution as an osmotic pressure regulator to prevent microbial cells from rupturing due to osmotic pressure mutations during the crosslinking process, thereby further ensuring microbial activity.

[0118] All percentage concentrations mentioned in this invention are (w / v) mass / volume percentage concentrations, that is, the number of grams of solute contained in 100 mL of solution.

[0119] S15. The cross-linked electrode was placed in a suitable sterile culture medium and incubated at a constant temperature. Two cross-linked electrodes were selected. One cross-linked electrode was placed in *Geobacterium* culture medium containing 2.5 g NaHCO3, 0.25 g NH4Cl, 0.6 g NaH2PO4·H2O, 0.1 g KCl, 1.0 g sodium acetate, and 10.0 mL of trace element and vitamin solution per liter, pH 6.8, and cultured under anaerobic conditions at 30.0℃. An 80% N2 / 20% CO2 atmosphere was used to cultivate anaerobic electroactive microorganisms suitable for the anode-type gradient pore biofilm electrode. The other cross-linked electrode was placed in lysine broth culture medium and cultured aerobically at 30℃ for 24 hours to cultivate aerobic electroactive microorganisms suitable for the gradient pore biofilm electrode used as the cathode. An air shaker at 150 rpm was used. After cultivation, the biofilm loading was controlled to be 10. 7 ~10 8 With a CFU / cm² concentration and a membrane thickness uniformity deviation of ≤5%, the electroactive microorganisms regain their metabolic activity. Utilizing pore size sieving, porosity gradients, and nutrient resistance gradients, the large pores enrich the surface aerobic microorganisms, the medium and small pores retain the middle facultative anaerobic microorganisms, and the micropores colonize the bottom anaerobic microorganisms, achieving a three-in-one matching of microbial community stratification, pore stratification, and frequency band stratification, ultimately producing a gradient pore biofilm electrode. The biofilm electrode has a three-layer structure along its thickness: a surface macroporous region with a pore size of 20.1–50 μm, a middle mesoporous region with a pore size of 5–20 μm, and a bottom microporous region with a pore size less than 5 μm. During cultivation, a gradient nutrient supply mode is adopted, with the nutrient concentration of the surface culture medium being two to three times that of the bottom layer. This, combined with the nutrient resistance differences brought about by the pore gradient, further enhances the stratified colonization effect of microorganisms, making the activity of microorganisms in each layer more stable and improving the specificity of the frequency band response.

[0120] The gradient pore biomembrane electrode features a continuous gradient pore structure. Between the upper limit of the mesoporous region (20.0 μm) and the lower limit of the macroporous region (20.1 μm), there exists an extremely narrow transitional pore region of 20.0 μm to 20.1 μm. This region is not divided into independent functional layers but serves only as a transitional structure between the macroporous region and the mesoporous region, acting as a bridge between mass transfer and electrochemical signals. It does not interfere with microbial colonization, frequency impedance response, or toxicity detection results in any of the zones.

[0121] Through three sets of parallel experiments and five batches of electrode preparation verification, with five electrodes per batch, the preparation process showed good repeatability, with a microbial survival rate of 87.3%±3.6% (n=5). The porosity test results were 68%±3% for the surface layer, 52%±2% for the middle layer, and 31%±2% for the bottom layer (n=3), all of which met the preset parameter requirements, proving that the mild preparation process can effectively preserve microbial activity.

[0122] It should be noted that LB medium is Leria-Bertany medium. LB medium is a mature existing technology in the field of microbial culture. It is a general-purpose bacterial culture medium. Its formula, preparation method and application scenarios are all known. This invention only uses it as a routine auxiliary material for the culture of Pseudomonas spp. on cathode electrodes.

[0123] In this technical solution, the electrodeposition solution of this invention uses 1.5%–2.5% sodium alginate as the main film-forming agent and 2.5%–3.5% disodium calcium EDTA as a compound, combined with 0.15%–0.25% graphene solution to construct a conductive network, and adds 0.05%–0.1% cysteine ​​as a microbial protectant. This ensures both the hydrogel film-forming effect and the integrity of the electrode conductive pathway, while also inhibiting the damage to microorganisms caused by the electric field during the electrodeposition process, effectively preserving the metabolic activity of electroactive microorganisms, and avoiding the defects of mutual constraints between film formation, pore formation, and microbial survival. In the ice crystal growth control stage, nitrogen gas is intermittently introduced under a vacuum degree ≤10.0 Pa, which can effectively inhibit the oxidation of the electrode surface with hydrogel coating, prevent pore collapse during the ice crystal growth process, ensure the integrity and connectivity of the gradient pore structure, and improve the consistency and repeatability of electrode batch preparation. This invention utilizes two cross-linked electrodes, one anaerobically cultured in Geobacterium culture medium and the other aerobically cultured in lysine broth culture medium, to acclimate and adapt functional bacterial communities to different pore levels. This enables stratified colonization of the bacterial communities and precise adaptation to the gradient pore structure, further enhancing the electrochemical response specificity of each electrode level to toxic substances.

[0124] Example 2.

[0125] This embodiment 2 discloses a water toxicity detection method based on gradient pore biofilm electrode, wherein step S2 includes the following steps.

[0126] S21. Prepare a series of standard toxicity solutions with known concentrations, using heavy metal ions Cd²⁻. +The standard toxic substance, such as CdCl2·2.5H2O, of analytical grade, is used with a concentration range of 0.1–10.0 mg / L, and at least five gradient concentration points are set. The solvent is deionized water, and the pH is adjusted to 7.0 ± 0.1. A 0.01 mol / L Tris-HCl buffer system (tris(hydroxymethyl)aminomethane)-hydrochloric acid buffer system) is added to the standard toxic solution to maintain pH stability, avoid concentration deviations caused by hydrolysis of the toxic substance, and improve the accuracy of model training.

[0127] S22. Using the gradient pore biomembrane electrode prepared in step S1, measure the multi-band electrochemical impedance spectroscopy corresponding to each standard toxicity solution. Each concentration point is measured at least three times. Five independently prepared electrodes are used, and each electrode can be reused three to five times. After each use, the electrode is placed in sterile blank buffer for 30 minutes to remove residual toxic substances on the surface and ensure data reliability. Before measurement, the electrode is pretreated in blank buffer for ten to fifteen minutes to eliminate the interference of residual culture medium components on the electrode surface on impedance detection and ensure the stability and repeatability of impedance data.

[0128] S23. Construct a toxic substance concentration prediction model and a toxic substance penetration depth prediction model. The impedance change rate of the three frequency bands is used as input. Contact time, ambient temperature, and water sample conductivity are introduced as auxiliary feature variables. The output of the toxic substance concentration prediction model is the standard toxic substance concentration, and the output of the toxic substance penetration depth prediction model is the penetration depth of the toxic substance inside the electrode, thereby improving the concentration-depth decoupling accuracy.

[0129] The model for predicting the concentration of toxic substances is as follows.

[0130] .

[0131] The model for predicting the penetration depth of toxic substances is as follows.

[0132] .

[0133] In the formula, This is a predicted value, representing the concentration of toxic substances in the water sample to be tested, in mg / L. It is the impedance change rate in the high-frequency band of 10.0kHz to 100kHz, corresponding to the macroporous area on the electrode surface, reflecting the surface read / write signal, and related to the surface permeation state of toxic substances. It is the impedance change rate in the mid-frequency band of 100Hz to 10.0kHz, corresponding to the mesoporous region in the middle layer of the electrode, connecting the surface read / write and the bottom penetration signal to avoid signal interruption; It is the impedance change rate in the low-frequency range of 0.1Hz to 100Hz, which corresponds to the microporous region at the bottom of the electrode and reflects the deep penetration signal. It is the core deep read / write / penetration signal that this invention focuses on. It is the contact time between the electrode and the water sample to be tested. It detects the ambient temperature. These are the water sample conductivity μS / cm, all of which are auxiliary characteristic variables used to correct for interference from read / write / permeability detection; These are weighting coefficients, and their specific values ​​are: , , , , , The optimal values ​​are determined through model training, precisely corresponding to each parameter of the input vector, and perfectly matching the stratified response and read / write penetration requirements of the gradient pore electrode. It is the rate of change of impedance in the high-frequency band. The corresponding weighting coefficients correspond to the macroporous area on the electrode surface, adapting to the surface read / write concentration signal without adding extra weight, ensuring the basic response of the surface concentration signal; It is the impedance change rate in the mid-frequency range. The corresponding weighting coefficient corresponds to the mesoporous region in the middle layer of the electrode, adapts to the middle layer connection signal, balances the surface layer read / write and the bottom layer penetration signal, and avoids signal discontinuity. It is the impedance change rate in the low-frequency band. The corresponding weighting coefficients correspond to the microporous region at the bottom of the electrode, which are specifically designed to enhance the deep penetration signal, solve the defect of the existing technology where the deep read / write / penetration signal is not obvious, and adapt to the needs of deep penetration depth detection. Contact time The corresponding weighting coefficients are used to correct the interference of contact time on the timeliness of read / write / penetration detection and to adapt to the response differences caused by the microbial metabolic cycle; Ambient temperature The corresponding weighting coefficients are used to correct for the interference of ambient temperature fluctuations on the read / write / penetration signals, ensuring signal stability during the detection process; It is the conductivity of the water sample The corresponding weighting coefficients are used to correct the bias of water sample conductivity on read / write / permeability detection, thereby improving prediction accuracy. It is the penetration depth of toxic substances within the gradient pore biomembrane electrode, in μm; These are weighting coefficients, and their specific values ​​are: , , , , , The optimal value is determined through model training.

[0134] The toxic substance concentration prediction model and the toxic substance penetration depth prediction model have the same structure, only the weight coefficients are different after iterative optimization with different training samples; the weight coefficients for the low frequency band are larger to specifically enhance the deep penetration signal and solve the defect of weak deep response signal in the existing technology. and Used to correct time-related interference caused by contact time. and Used to compensate for the effects of environmental temperature fluctuations. and It is used to correct detection deviations caused by the conductivity of water samples, thereby comprehensively improving prediction accuracy.

[0135] Based on different water sample operating conditions and the permeation state of toxic substances, multiple sets of typical weight values ​​were determined within the limited range of each weight coefficient, as follows.

[0136] 1. Weight values ​​for the toxic substance concentration prediction model.

[0137] 1) The standard optimal value is applicable to routine water sample testing conditions: =1.0, =1.0, =1.35, =0.8, =0.7, =0.7.

[0138] 2) Surface-biased response values ​​are suitable for applications where toxic substances remain on the electrode surface, enhancing the weighting of high-frequency signals. =1.1, =0.95, =1.2, =0.75, =0.65, =0.75.

[0139] 3) The deep-layer response value is suitable for applications where toxic substances have completely penetrated into the electrode, enhancing the signal weighting in the low-frequency range: =0.95, =0.9, =1.5, =0.85, =0.75, =0.65.

[0140] 4) Enhanced correction for environmental disturbances, suitable for water samples with high conductivity and large fluctuations in ambient temperature, improving the ability to correct for environmental variables: =1.05, =1.05, =1.3, =0.9, =0.8, =0.8.

[0141] 2. Weighting of the toxic substance penetration depth prediction model: Penetration depth detection mainly relies on low-frequency impedance signals, so the weighting coefficients for the low-frequency band should be selected with higher values ​​within the range.

[0142] 1) Standard optimal value, applicable to general testing conditions under normal permeation conditions: =1.0, =1.0, =1.4, =0.8, =0.7, =0.7.

[0143] 2) Shallow permeation sampling is suitable for applications where toxic substances are only distributed in the surface and middle layers of the electrode: =1.1, =1.05, =1.25, =0.7, =0.6, =0.6.

[0144] 3) Deep penetration sampling, suitable for core detection conditions where toxic substances penetrate the bottom layer of the electrode: =0.9, =0.95, =1.5, =0.88, =0.78, =0.78.

[0145] 4) Long contact time correction value, suitable for detection conditions where the electrode and water sample are in contact for a long time, enhancing the correction effect of the contact time variable: =1.02, =0.98, =1.38, =0.9, =0.72, =0.72.

[0146] S24. After model training and cross-validation, the optimal model parameters were determined using leave-one-out cross-validation. A quantitative mapping relationship was established between impedance change rate and toxic substance concentration and penetration depth for each frequency band. The model was applied to Cd² +The concentration prediction determination coefficient R² > 0.97 and the root mean square error RMSE < 0.5 mg / L are reserved. After model training, outlier removal is performed using the Grubbs criterion to remove outlier detection data with a significance level of α = 0.05, further reducing the model prediction error and improving model stability.

[0147] Example 3.

[0148] This embodiment 3 discloses a water toxicity detection method based on gradient pore biofilm electrode, wherein step S4 includes the following steps.

[0149] S41. The collected electrochemical impedance spectroscopy is divided into three characteristic independent frequency bands, and the frequency bands are precisely bound to the penetration depth.

[0150] High frequency band: 10.0kHz~100kHz, corresponding to 0~150μm surface permeability, impedance changes specifically reflect changes in the electrochemical activity of microorganisms in the macroporous region of the surface.

[0151] Mid-frequency band: 100Hz~10.0kHz, corresponding to 150~350μm mesoporous layer permeation, impedance changes specifically reflect changes in the electrochemical activity of microorganisms in the mesoporous region.

[0152] Low frequency band: 0.1Hz~100Hz, corresponding to 350~500μm bottom layer penetration. The impedance change reflects the diffusion impedance of the whole electrode and the change of microbial electrochemical activity in the bottom micropore region.

[0153] S42. Calculate the impedance change rate of each frequency band: Before the calculation, the background baseline is calibrated in situ with blank buffer solution of the same pH and ionic strength, and isothermal normalization is performed at 20-25℃ to eliminate environmental temperature drift and salinity interference.

[0154] .

[0155] In the formula: It is the impedance change rate of the i-th frequency band, in percentage, where i corresponds to the three frequency bands divided in the detection process: high frequency band, mid frequency band, and low frequency band. It is the initial impedance value measured by the electrode in the background solution under the i-th frequency band, that is, the reference impedance value of the corresponding frequency band after the background baseline is calibrated. The background solution must be consistent with the ionic strength and pH of the water sample to be tested and be non-toxic, and must be subjected to constant temperature normalization treatment at 20-25℃ to eliminate temperature drift and salinity interference. This refers to the real-time impedance value measured after the electrode has been in contact with the water sample for a set time in the i-th frequency band. This value has also undergone isothermal normalization to ensure consistency with... The test conditions were consistent; by calculating the proportion of the difference between the "initial baseline impedance" and the "real-time detection impedance" in the same frequency band, the degree of change in electrode impedance in that frequency band was quantified. The larger the proportion of the difference, the more significant the impact of toxic substances in the water sample on the corresponding electrode region of that frequency band. When toxic substances inhibit microbial activity, microbial electron transfer is hindered, and the impedance increases. A positive value indicates normal microbial activity when no toxic substances are present. Approaching 0.

[0156] The raw impedance spectrum acquired by the electrochemical workstation is divided into high-frequency, mid-frequency, and low-frequency bands. The division criteria can be preset according to the device's testing requirements. Then, the impedance data for each frequency band is substituted into... The general formula yields the impedance change rate for the corresponding frequency band, with specific correspondences.

[0157] High-frequency band: The initial impedance value corresponding to the high-frequency band Real-time impedance value Substituting into the formula, the impedance change rate in the high-frequency band is calculated: .

[0158] Mid-frequency band: The initial impedance value corresponding to the mid-frequency band Real-time impedance value Substituting into the formula, the impedance change rate in the mid-frequency range is calculated: .

[0159] Low frequency band: The initial impedance value corresponding to the low frequency band Real-time impedance value Substituting into the formula, the impedance change rate in the low-frequency range is calculated: .

[0160] The impedance change rate in the high-frequency band was obtained respectively. Mid-frequency impedance change rate and low-frequency impedance change rate .

[0161] Simultaneously calculate the signal separation index S, the formula for which is:

[0162] .

[0163] This invention achieves signal separation index S≥0.95 and ordinary homogeneous electrode S≤0.35 by adjusting the gradient pore structure, ensuring sufficient separation of signals in each frequency band and avoiding coupling interference. When the signal separation index S<0.95, the electrode recalibration procedure is automatically triggered, placing the electrode in a blank buffer to recalibrate the baseline, ensuring the validity of the detection data and further improving the reliability of detection accuracy.

[0164] The impedance change rate comparison data for different electrode types at various characteristic frequencies are shown in Table 3 below.

[0165]

[0166] Using n=5 batches, with three electrodes per batch, and data expressed as mean ± standard deviation, statistical tests showed that the gradient pore electrode exhibited significant differences in frequency response compared to the ordinary single-pore electrode. Furthermore, the gradient pore electrode achieved a response intensity comparable to the single-pore electrode in all frequency bands, with a signal separation index S=0.99±0.02, meeting the requirement of S≥0.95. This ensures sufficient signal separation in each frequency band, avoids coupling interference, and provides an effective structural basis for achieving hierarchical resolution.

[0167] In this technical solution, the present invention divides the electrochemical impedance spectroscopy into a high-frequency band of 10.0kHz to 100kHz, a mid-frequency band of 100Hz to 10.0kHz, and a low-frequency band of 0.1Hz to 100Hz, and matches the penetration depths of the surface layer (0 to 150μm), the middle layer (150 to 350μm), and the bottom layer (350 to 500μm) respectively. By calculating the impedance change rate by frequency band, the surface, middle, and bottom layer penetration signals can be independently analyzed, avoiding full-band signal coupling interference and significantly improving the stratification of toxicity response.

[0168] Example 4.

[0169] This embodiment 4 discloses a water toxicity detection method based on a gradient pore biofilm electrode, wherein in step S5, the calculated high-frequency impedance change rate is used... Mid-frequency impedance change rate and low-frequency impedance change rate The invention incorporates auxiliary variables such as contact time, ambient temperature, and water sample conductivity, and substitutes them into pre-trained models for predicting toxic substance concentration and toxic substance penetration depth. Simultaneously, it decouples and outputs information on the concentration of toxic substances in the tested water sample and the penetration depth of toxic substances within the biofilm electrode. This invention significantly weakens the concentration-depth coupling interference of traditional methods through the synergistic effect of gradient pore structure and multi-band impedance spectroscopy. The detection errors for 0.5 mg / L completely permeated and 5.0 mg / L surface-retained toxic substances are only 7.2% and 5.8%, respectively, far superior to traditional single-signal methods, which have errors of 32% and 18%, respectively. The signal separation index is 3.2 times higher than that of ordinary homogeneous electrodes, effectively avoiding misjudgments. After calculation, the invention automatically outputs a concentration-depth coupling verification value. When the verification value is >5%, it is judged as an abnormal detection, prompting a re-detection, further avoiding concentration-depth misjudgments and improving the reliability of the detection results.

[0170] The hierarchical response coupling model adopts the same calculation formula structure and obtains two sets of weight coefficients by training with different sample labels. When the first set of weight coefficients is obtained by training with the standard toxic substance concentration as the training label, the model output is the concentration of toxic substances in the water sample to be tested. When the second set of weight coefficients is obtained by training with the penetration depth of toxic substances inside the electrode as the training label, the model output is the penetration depth of toxic substances inside the gradient pore biofilm electrode.

[0171] Example 5.

[0172] This embodiment discloses a water toxicity detection method based on gradient pore biofilm electrode, wherein step S6 includes the following steps.

[0173] S61, Comparison The relative numerical relationships among the three factors, combined with the constraint that the signal separation index S≥0.95, are used to determine the state of toxicity penetration.

[0174] when Significantly greater than and At that time, it was determined to be a surface permeation state, and the sensor was working normally.

[0175] when and All significantly increased If there are no significant changes, it is determined to be a mid-layer permeable state, and the sensor needs to monitor it continuously.

[0176] when When the preset warning threshold is exceeded, it is determined to be a deep infiltration state, and a sensor replacement prompt is issued immediately.

[0177] S62. The preset warning threshold adopts a hierarchical setting, which is different from the single threshold design. Specifically,

[0178] Level 1 alert threshold: Mid-layer penetration warning. It is 10% to 12%.

[0179] Secondary replacement threshold: Deep penetration forced replacement. ≥15%; This preset early warning threshold is jointly determined by the background baseline drift range of the biofilm electrode under non-toxic conditions, initial performance calibration, and critical characteristics of deep microbial inactivation, enabling early warning of the remaining service life of the biofilm electrode. The critical characteristic of deep microbial inactivation is that the proportion of deep microbial inactivation exceeds 70%.

[0180] The early warning system synchronously records the cumulative usage time of the electrodes, the number of tests, and the result of each test. A gradient pore biofilm electrode lifetime prediction model was constructed, and combined with an early warning threshold, the model provides an early warning three to five days in advance that the gradient pore biofilm electrode is about to reach the replacement threshold, thereby improving the ease of operation and maintenance of the detection system. The remaining lifetime prediction of the gradient pore biofilm electrode is as follows.

[0181] .

[0182] In the formula, It is the remaining lifetime of the gradient pore biofilm electrode 11, in hours. Combined with the early warning system settings, when the remaining lifetime corresponds to a duration of ≤3.0 to 5.0 days, the device will automatically issue a replacement reminder based on the daily detection frequency, thus achieving early warning. It is the rated total lifetime of the gradient pore biofilm electrode 11, in hours, which is determined by the characteristics of the electrode substrate 111, the layered pore structure and the electroactive microorganisms 115. For example, the rated total lifetime of an electrode based on a graphite rod substrate and a gradient pore structure is 3000 to 4000 hours. The usage time of the gradient pore biofilm electrode 11 is automatically accumulated and recorded by the early warning system, and the number of detections is synchronously correlated to accurately calculate the actual working load of the electrode. This is the low-frequency impedance change rate threshold of the gradient pore biofilm electrode 11, expressed as a percentage. It is a preset critical value, determined experimentally. The threshold value is set when the electrode ages or the activity of electroactive microorganisms decreases. If the value deviates from this threshold, a lifespan warning will be triggered; It is the actual impedance change rate of the gradient pore biomembrane electrode 11 in the low-frequency range during the current detection cycle. The early warning system records this value for each detection and uses it for dynamic optimization of the life prediction model. It is the lifetime decay coefficient of the gradient pore biofilm electrode 11, in units of % / h. It is obtained by training with a large amount of experimental data, quantifying the correlation between the low-frequency impedance change rate deviation and the electrode lifetime decay rate, and adapting to the aging patterns of gradient pore structure and electroactive microorganisms.

[0183] This invention significantly reduces the "concentration-depth" coupling interference of traditional methods by synergistically enhancing the gradient porosity structure and multi-band impedance spectroscopy. The detection errors for 0.5 mg / L fully permeated and 5.8% surface-retained toxic substances are only 7.2% and 5.8%, respectively, far superior to traditional single-signal methods. The signal separation index is 3.2 times higher than that of ordinary homogeneous electrodes, effectively avoiding misjudgment. After calculation, the concentration-depth coupling verification value is automatically output. When the verification value is >5%, it is judged as a detection anomaly, prompting re-detection, further avoiding "concentration-depth" misjudgment and improving the reliability of the detection results. At the same time, through the electrode remaining life prediction formula and early warning system, the gradient porosity biofilm electrode 11 can realize early warning, solving the detection error and detection interruption problems caused by the failure to detect electrode aging in time in traditional detection, and improving the convenience of system operation and maintenance.

[0184] Step S7, Model Adaptive Correction, Ensuring Long-Term Online Detection Accuracy: Through the model update unit of the detection device, a unique strategy of in-situ blank baseline calibration every three days + Kalman filter sliding window iterative correction is adopted. The window length is eight to twelve sets of historical baseline data. The Kalman filter parameters are set as follows: process noise covariance Q=0.01, measurement noise covariance R=0.1. These parameters are obtained through model training and error iteration tuning and can be dynamically adjusted according to the water sample type. The quantitative relationship model of toxicity response is adaptively corrected to suppress baseline drift caused by electrode aging and environmental interference, maintain the stability of detection accuracy in the long term, and ensure that the model prediction error is controlled within 10% within 30 days of continuous operation.

[0185] An electrode aging coefficient is introduced during the calibration process to construct an aging correction model for gradient pore biofilm electrodes. This model is dynamically adjusted based on the electrode's usage time, with a value ranging from 0.9 to 1.0. This dynamically corrects the model's prediction results, further offsetting the detection deviation caused by electrode aging and extending the effective service life of the electrodes. The gradient pore biofilm electrode aging correction model is as follows.

[0186] ; . .

[0187] In the formula: The electrode aging coefficient ranges from 0.9 to 1.0. This refers to the duration of electrode use. This refers to the rated service life of the electrodes. This is the corrected predicted value of toxic substance concentration. This is the corrected predicted value for the penetration depth of toxic substances; To correct for the predicted concentrations of pre-toxic substances; To correct the predicted penetration depth of pre-toxic substances, a gradient pore biofilm electrode aging correction model was used to dynamically correct the deviation caused by aging, so that the prediction error was controlled within 10% within 30 days of continuous operation.

[0188] In this technical solution, by constructing a gradient pore biofilm electrode aging correction model, the aging coefficient can be dynamically adjusted according to the electrode's usage time. This allows for the synchronous correction of the predicted values ​​of the toxic substance concentration prediction model and the toxic substance penetration depth prediction model, thereby offsetting the detection deviation caused by electrode aging and extending the effective service life of the electrode. In conjunction with a calibration strategy, the model prediction error is kept within 10% for 30 days of continuous operation of the detection system, ensuring the stability of long-term online detection accuracy.

[0189] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A method for detecting water toxicity based on a gradient pore biofilm electrode, characterized in that, Includes the following steps: A gradient pore biomembrane electrode is prepared, comprising a bottom microporous region, a middle mesoporous region, and a surface macroporous region; the middle mesoporous region is located between the bottom microporous region and the surface macroporous region. A three-electrode detection cell is constructed based on the gradient pore biofilm electrode, platinum counter electrode and reference electrode, and the water sample to be tested is injected into the three-electrode detection cell. An AC perturbation voltage signal is applied to the three-electrode detection cell, and the electrochemical impedance spectrum of the three-electrode detection cell under the action of the water sample to be tested is collected. The electrochemical impedance spectrum was divided into multiple frequency bands, and the impedance change rate of each frequency band was calculated. The concentration of toxic substances in the water sample and the penetration depth of toxic substances within the gradient pore biofilm electrode were calculated based on the impedance change rate of the multi-frequency band.

2. The water toxicity detection method based on gradient pore biofilm electrode according to claim 1, characterized in that... The preparation of gradient pore biomembrane electrodes includes the following steps: Prepare an electrodeposition solution containing electroactive microorganisms and film-forming materials; An electrode with a hydrogel coating is formed by electrodeposition on the surface of a graphite rod using the electrodeposition solution described above; The electrode with the hydrogel coating was subjected to gradient temperature-controlled freeze-drying to obtain a freeze-dried electrode; The freeze-dried electrode was placed in a sterile, pre-cooled calcium chloride solution for mild cross-linking enhancement treatment to obtain a cross-linked electrode; The cross-linked electrode was placed in a sterile culture medium and cultured at a constant temperature to obtain the gradient pore biomembrane electrode.

3. The water toxicity detection method based on gradient pore biofilm electrode according to claim 2, characterized in that... The preparation of the electrodeposition solution containing electroactive microorganisms and film-forming materials includes the following steps: Take 1.5%–2.5% sodium alginate as the main film-forming agent, add 2.5%–3.5% calcium disodium ethylenediaminetetraacetate, mix evenly to form the basic film-forming matrix; Add 0.15% to 0.25% graphene solution to the basic film-forming matrix and stir until uniformly dispersed to obtain a reduced graphene composite dispersion. According to the testing requirements, add the corresponding electroactive microbial liquid to the reduced graphene composite dispersion to form a mixed system; Add 0.05% to 0.1% cysteine ​​as a microbial protectant to the mixture, stir until homogeneous, and obtain the electrodeposition solution containing electroactive microorganisms and film-forming materials.

4. The water toxicity detection method based on gradient pore biofilm electrode according to claim 2, characterized in that... The gradient temperature-controlled freeze-drying process for the electrode with the hydrogel coating includes the following steps: Pre-freezing stage: The electrode with hydrogel coating is cooled to below -50°C at a cooling rate of 0.5 to 2.0°C / min and kept at that temperature for 1.0 to 3.0 hours; Ice crystal growth control stage: The electrode with hydrogel coating is heated to -20.0℃ to -10.0℃ at a heating rate of 0.4 to 0.6℃ / min and kept at that temperature for two to four hours; First drying stage: the vacuum degree is below 10.0 Pa, the temperature of the electrode with hydrogel coating is controlled at -30.0℃ to -20.0℃, and the drying time is 10 to 15 hours; Secondary drying stage: Heat to 20.0℃~30.0℃ and dry for five to ten hours; By inducing the directional growth of ice crystals through temperature gradient, a pore gradient structure with a continuous and gradual change along the thickness direction is formed inside the electrode with hydrogel coating.

5. The water toxicity detection method based on gradient pore biofilm electrode according to claim 4, characterized in that... During the ice crystal growth control stage, under a vacuum of ≤10.0 Pa, nitrogen gas is intermittently introduced at a flow rate of 50-80 mL / min, with an interval of 30 min / time and each introduction lasting 10 minutes, to inhibit the oxidation of the surface of the electrode with hydrogel coating.

6. The water toxicity detection method based on gradient pore biofilm electrode according to claim 1, characterized in that... The pore size of the middle mesoporous region is smaller than that of the surface macroporous region; the pore size of the middle mesoporous region is larger than that of the bottom microporous region. The pore size range of the surface macroporous region is 20.1–50.0 μm, the pore size range of the middle mesoporous region is 5.0–20.0 μm, and the pore size of the bottom microporous region is less than 5.0 μm.

7. The water toxicity detection method based on gradient pore biofilm electrode according to claim 2, characterized in that... In the step of obtaining the gradient pore biofilm electrode: two cross-linked electrodes are selected. One cross-linked electrode is placed in a Geobacterium culture medium containing 2.5 g NaHCO3, 0.25 g NH4Cl, 0.6 g NaH2PO4·H2O, 0.1 g KCl, and 1.0 g sodium acetate per liter, with a pH of 6.8, and is cultured under anaerobic conditions at 30.0 °C to cultivate anaerobic electroactive microorganisms adapted to the anodic gradient pore biofilm electrode. The other cross-linked electrode is placed in a lysine broth culture medium and cultured under aerobic conditions at 30 °C for 24 hours to cultivate aerobic electroactive microorganisms adapted to the gradient pore biofilm electrode as a cathode.

8. The water toxicity detection method based on gradient pore biofilm electrode according to claim 1, characterized in that... In the step of dividing the electrochemical impedance spectrum into multiple frequency bands and calculating the impedance change rate of each frequency band: The electrochemical impedance spectrum is divided into multiple characteristic frequency bands: The high-frequency band is 10.0kHz to 100kHz, corresponding to 0 to 150μm surface penetration; the impedance change rate in the high-frequency band was calculated. The mid-frequency band is 100Hz to 10.0kHz, corresponding to the 150 to 350μm mid-layer penetration; the impedance change rate in the mid-frequency band was calculated. The low-frequency band is 0.1Hz to 100Hz, corresponding to a sublayer penetration of 350 to 500μm; the impedance change rate in the low-frequency band was calculated.

9. The water toxicity detection method based on a gradient pore biofilm electrode according to claim 8, characterized in that... In the process of calculating the concentration of toxic substances in the water sample to be tested and the penetration depth of toxic substances in the gradient pore biofilm electrode based on the impedance change rate, a toxic substance concentration prediction model is constructed to obtain the concentration of toxic substances in the water sample to be tested, and a toxic substance penetration depth prediction model is constructed to obtain the penetration depth of toxic substances in the water sample to be tested in the gradient pore biofilm electrode. The toxic substance concentration prediction model takes the high-frequency impedance change rate, the mid-frequency impedance change rate and the low-frequency impedance change rate as inputs, and outputs the toxic substance concentration in the water sample to be tested. The toxic substance penetration depth prediction model takes high-frequency impedance change rate, mid-frequency impedance change rate and low-frequency impedance change rate as inputs, and outputs the penetration depth of toxic substances in the water sample to be tested within the gradient pore biofilm electrode.

10. A water toxicity detection device employing the water toxicity detection method based on gradient pore biofilm electrode as described in claim 1, comprising a three-electrode detection cell and an electrochemical workstation, characterized in that... : The three-electrode detection cell includes a gradient pore biomembrane electrode, a platinum counter electrode, a reference electrode, and a housing; The gradient pore biomembrane electrode comprises a bottom microporous region, a middle mesoporous region, and a surface macroporous region; the middle mesoporous region is located between the bottom microporous region and the surface macroporous region. A current collector is provided at the upper end of the gradient pore biomembrane electrode; The gradient pore biomembrane electrode, platinum counter electrode, and reference electrode are respectively connected to the working electrode, counter electrode, and reference electrode interfaces of the electrochemical workstation.

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