Method for detecting transmembrane transport of charges between target organism and substance interface

Abstract

The invention relates to a method for detecting transmembrane transport of charges between a target organism and a substance interface, which comprises the following steps of: providing a working electrode which is provided with a working surface provided with a functional material; adding an electrolyte containing a target bioactive substance to the working surface loaded with the functional material so as to form a micro-droplet with the volume of microliter; a reference electrode and a counter electrode are inserted into the micro-droplet to form a micro-droplet electrochemical detection loop together with the working surface; applying an excitation signal to the micro-droplet electrochemical detection loop, and collecting a corresponding response signal; and based on the response signal, determining a charge transmembrane transport characteristic between the target bioactive substance and the functionalized material interface. The micro-droplet electrochemical detection loop is constructed, a micro-droplet system and an electrochemical detection technology are successfully combined, and high-sensitivity, low-noise and quantitative detection of the charge transmembrane transport process between a target bioactive substance and a material interface is achieved.

Description

Technical Field

[0001] This invention relates to the field of bioelectrochemical detection, and in particular to a method for detecting transmembrane charge transport between a target organism and a substance. Background Technology

[0002] The process of charge transport across membranes at the microbial-material interface is a key scientific question in environmental microbiology, bioenergy, and biomedicine. Accurately elucidating this process is crucial for developing efficient microbial electrochemical sensors, optimizing the performance of microbial fuel cells, and evaluating the effectiveness of novel antimicrobial materials.

[0003] Currently, detection techniques in this field mainly rely on macroscopic electrochemical cells (such as 50 mL systems). These traditional methods have the following significant drawbacks: First, they require large sample volumes, making them unsuitable for detecting precious or low-concentration samples, and they are also costly. Second, the charge transport paths in macroscopic systems are long, and diffusion losses in the electrolyte are severe, resulting in weak intrinsic signals originating from interfacial charge transfer. Third, the random diffusion of free ions in the solution generates strong background noise, which can easily mask weak transmembrane charge transfer signals. Finally, existing technologies often only allow inference of interfacial processes indirectly from macroscopic output results (such as overall current and voltage), lacking the ability to directly and accurately quantify interfacial charge transfer kinetic parameters (such as charge transfer rate constant and charge transfer resistance), making it difficult to reveal their microscopic mechanisms.

[0004] Microdroplet technology exhibits unique advantages in biochemical analysis due to its high surface-to-volume ratio, low sample consumption, and confined reaction space. However, combining microdroplet systems with electrochemical detection techniques for the precise quantification of transmembrane charge transport processes still faces technical challenges. These include how to stably construct a three-electrode detection circuit for microdroplets, how to select functionalized materials that specifically interact with the microbial interface to amplify the signal, and how to establish a quantitative relationship between the electrical signal and the interfacial transport properties. Therefore, there is an urgent need in this field for a new method capable of highly sensitive, accurate, and quantitative detection of transmembrane charge transport processes at microbial interfaces.

[0005] It should be noted that the information disclosed in the background section above is only for understanding the background of this application, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The fundamental technical problem to be solved by this invention is: how to directly and accurately quantify the kinetic parameters of charge transmembrane transport between the target bioactive substance and the material interface; specifically, how to establish a new method that can achieve high sensitivity, low noise, and quantitative detection of charge transmembrane transport processes between the interfaces of target bioactive substances (such as microorganisms, cells, viruses, enzymes, nucleic acids, or proteins), so as to overcome the core limitations of traditional macroscopic electrochemical detection techniques, such as weak signals, high background noise, large sample consumption, and inability to directly and accurately quantify interface kinetic parameters.

[0007] The technical solution adopted in this application to solve the above-mentioned technical problems is as follows.

[0008] This application provides a method for detecting transmembrane charge transport between a target organism and a substance, comprising the following steps: S1. A working electrode is provided, the working electrode having a working surface, the working surface being provided with a functionalized material; S2. Add the electrolyte containing the target bioactive substance to the working surface loaded with functionalized material to form a microdroplet with a volume of microliters on it. S3. Insert the ends of the reference electrode and the counter electrode into the microdroplet to form a microdroplet electrochemical detection circuit together with the working surface; S4. Apply an excitation signal to the microdroplet electrochemical detection circuit and acquire the response signal from the working electrode; S5. Based on the response signal, determine the charge transmembrane transport characteristics between the target bioactive substance and the functionalized material interface.

[0009] In some embodiments, the excitation signal includes at least one of cyclic voltammetry (CV) scan signal, differential pulse voltammetry (DPV) scan signal, and electrochemical impedance spectroscopy (EIS) scan signal.

[0010] In some embodiments, in step S1, the functionalized material includes a two-dimensional material, which refers to a material having a nanoscale thickness and a two-dimensional planar structure.

[0011] In some embodiments, in step S2, the target bioactive substance includes microorganisms, cells, viruses, enzymes, nucleic acids, or proteins.

[0012] In some embodiments, the microorganisms include Escherichia coli, lactic acid bacteria, or Shewanella.

[0013] In some embodiments, applying the excitation signal in step S4 specifically includes the following steps: When using cyclic voltammetry, scanning is performed within a selected potential range at a selected scan rate; When using the differential pulse voltammetry method, a scan is performed within the selected potential range using selected potential steps, pulse potentials, and pulse time parameters. When using electrochemical impedance spectroscopy, a selected DC bias voltage and AC disturbance potential are superimposed on the open-circuit potential of the microdroplet electrochemical detection circuit, and scanning is performed within a selected frequency range.

[0014] In some embodiments, in step S5, the charge transmembrane transport characteristics include: interfacial charge transfer direction, charge transfer resistance, number of electrons transferred, or concentration of the target bioactive substance.

[0015] This application also provides a microbial electrochemical sensor for performing the method of this application to detect microbial concentration. The sensor includes: a housing; an electrochemical workstation disposed within the housing for generating and acquiring electrical signals; a detection cell disposed on or connected to the housing surface for accommodating microdroplet electrochemical detection circuits; and a processor electrically connected to the electrochemical workstation and configured to calculate and output microbial concentration information based on the response signals acquired by the electrochemical workstation.

[0016] This application also provides a method for evaluating the efficacy of antimicrobial materials, comprising the following steps: using the method of this application, detecting the charge transmembrane transport characteristics between the target antimicrobial material and the microorganism interface; evaluating the antimicrobial efficacy of the target antimicrobial material based on changes in the charge transmembrane transport characteristics, wherein changes in the charge transport process or different amounts of charge transfer affect the antimicrobial effect.

[0017] This application also provides a microdroplet electrochemical detection device, comprising: a working electrode having a functionalized material disposed on its working surface; a reference electrode and a counter electrode, wherein the ends of the reference electrode and the counter electrode are configured to be inserted into microdroplets formed on the working surface to jointly constitute a microdroplet electrochemical detection circuit.

[0018] The present invention has the following beneficial effects: This application successfully combines a microdroplet system with electrochemical detection technology by constructing a "microdroplet electrochemical detection loop," achieving direct and precise quantitative analysis of the charge transmembrane transport dynamics process between the target bioactive substance and the material interface. Specifically, this application utilizes the high surface-to-volume ratio and confined reaction space unique to microdroplets to significantly shorten the charge transmembrane transport path, reduce diffusion interference and background noise of free ions in solution, and greatly reduce sample consumption. By loading functionalized materials onto the working surface of the working electrode and forming a tight interface with the target bioactive substance within the microdroplet, the intrinsic electrochemical signal originating from charge transmembrane transport can be efficiently captured and amplified. Combined with specific electrochemical excitation signals and response analysis, this ultimately achieves highly sensitive, low-noise, and quantitative detection of the charge transmembrane transport process between the target bioactive substance and the material interface, overcoming the core limitations of traditional macroscopic electrochemical detection technologies.

[0019] Furthermore, this application significantly reduces the consumption of biological samples by employing micro-upgraded microdroplets, which is crucial for the detection of precious, rare, or difficult-to-cultivate biological samples, resulting in substantial cost savings. In addition, the high surface-to-volume ratio of microdroplets greatly enhances the probability and tightness of interfacial contact between the target bioactive substance (such as microorganisms) and the working surface of the functionalized material, effectively increasing the reaction sites for charge transmembrane transport and thus amplifying the intensity of the response signal. Moreover, the confined droplet space restricts the long-range disordered diffusion of ions, effectively reducing the noise generated by the background electrolyte, allowing the weak interfacial charge transmembrane signal to be clearly displayed, thereby improving the signal-to-noise ratio and detection sensitivity.

[0020] Furthermore, this application loads specific functionalized materials onto the working surface of the working electrode. These functionalized materials include two-dimensional materials (such as hexagonal boron nitride (h-BN)). These materials have nanoscale thickness (0.333-100 nm), large specific surface area, high conductivity, and good biocompatibility. They can provide an excellent adhesion interface for the target bioactive substance and act as a highly efficient electronic mediator or conductor to promote the transfer of negative charges around the target bioactive substance to the electrode surface, thereby specifically enhancing the interfacial charge transport signal and laying the foundation for subsequent precise quantification.

[0021] Furthermore, this application employs multiple electrochemical excitation and scanning techniques, including cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS), and optimizes their parameters (such as scan potential range, scan rate, pulse parameters, and frequency). CV scanning can be used to rapidly assess the reversibility and activity of interfacial reactions and preliminarily determine charge transmembrane transport efficiency. DPV scanning, with its high sensitivity and ability to effectively eliminate background capacitive current, can accurately distinguish and quantify the minute Faraday currents generated during charge transmembrane transport, making it particularly suitable for detecting low-concentration target bioactive substances. EIS scanning allows for in-depth analysis of the kinetic processes of charge transmembrane transport; by fitting equivalent circuits, key kinetic parameters such as charge transmembrane transport resistance can be directly and accurately quantified, achieving a leap from macroscopic signal analysis to microscopic mechanism analysis. The combined use of multiple techniques provides multi-dimensional verification and complementary information, ensuring the comprehensiveness and reliability of the detection results.

[0022] In summary, the various technical features of this application, through mutual cooperation and synergy, constitute a complete and efficient technical solution: the microdroplet electrochemical detection platform solves the problems of sample quantity and noise; the functionalized material interface solves the problems of signal specificity and amplification; and the multi-mode electrochemical detection technology solves the problems of signal acquisition and quantitative analysis. This design enables the present invention to successfully establish a new method for highly sensitive, low-noise, and quantitative detection of charge transmembrane transport processes between interfaces of trace biological samples, providing a powerful tool for related basic research and application development.

[0023] Other beneficial effects of the present invention will be further described below. Attached Figure Description

[0024] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 This is a schematic diagram of the transmembrane charge transport process at the detection interface of the microdroplet electrochemical detection platform. Figure 2 The images show the electrochemical test patterns of the excitation signal, where a is the CV electrochemical test pattern of hexagonal boron nitride (h-BN) and E. coli, b is the DPV electrochemical test pattern of hexagonal boron nitride (h-BN) and E. coli, and c is the EIS electrochemical test pattern of hexagonal boron nitride (h-BN) and E. coli. Figure 3 This is a flowchart of one method of this application. Detailed Implementation

[0025] The following provides a detailed description of the embodiments of this application. It should be emphasized that the following description is merely exemplary and is not intended to limit the scope and application of this application.

[0026] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the embodiments of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0027] To address the core issues in existing technologies regarding the weak and difficult-to-directly capture signals of charge transmembrane transport processes between target bioactive substances (such as microorganisms, cells, viruses, enzymes, nucleic acids, or proteins) and materials, and the reliance on indirect inference from macroscopic results for mechanism analysis, which lacks precise quantitative tools, this application proposes a unique technical solution for constructing a "microdroplet electrochemical detection platform." This solution leverages the high surface area to volume ratio and dynamic osmotic pressure of microdroplet systems to significantly amplify charge transmembrane transport signals, and the ability of functionalized two-dimensional materials to significantly enhance charge transport efficiency with target bioactive substances.

[0028] In a macroscopic system (50 mL electrolytic cell), the probability of interfacial contact between the target bioactive substance and the two-dimensional material is low, and the charge diffusion distance in the solution is long and severely depleted, resulting in a weak signal. However, in the microdroplet electrochemical detection platform, the high surface area to volume ratio significantly compresses the charge transmembrane transport path, reducing electron diffusion loss in the solution. Simultaneously, the high surface tension allows the target bioactive substance to form a tightly adhered interfacial interface with the two-dimensional material surface, increasing the effective contact area for charge transmembrane transport and significantly increasing the amount of charge transported per unit time, amplifying the signal intensity by tens of times. Furthermore, in a macroscopic solution system, the random diffusion of free ions and the migration of stray charges generate strong background noise, masking the weak charge transmembrane transport signal. Microdroplets, by confining the reaction space, reduce the diffusion range of bacteria and shorten the distance between the three electrodes, reducing interference from stray charges.

[0029] This application utilizes microscale measurements (e.g., 50 μL microdroplets as reaction vessels) to integrate multi-dimensional detection technologies such as cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy to target and capture specific charge transmembrane transport signals at the interface between target bioactive substances and materials. Ultimately, it achieves "visualization + quantification" analysis of this microscopic process, not only accurately revealing the charge transmembrane transport pathway and mechanism but also providing an efficient detection tool for antibacterial material development, microbial energy production, and bioelectrochemical device optimization. It achieves excellent results in improved signal sensitivity, shortened detection cycle, and reduced sample consumption, filling the technological gap in the accurate detection of charge transmembrane transport in low-conductivity microorganisms.

[0030] In summary, the technical solution of this application mainly improves upon the existing technology in three aspects, including: a new mechanism of action: by studying the interaction mechanism between the target bioactive substance and the two-dimensional material, a charge transmembrane transport interface for the target bioactive substance is constructed, providing a foundation for elucidating the transmembrane transport process of the target bioactive substance interface; a new method: in detecting the transmembrane transport process of the target bioactive substance interface, an electrochemical detection method based on microdroplets is proposed, enabling real-time, rapid, and accurate detection of the charge transmembrane transport process of the target bioactive substance; and new characteristics: in the microdroplet-based electrochemical detection process, the detection sensitivity is significantly improved by utilizing micro-level droplets, enabling the detection of target bioactive substances with poor conductivity and rare samples.

[0031] In some embodiments, this application uses hexagonal boron nitride (BN1) and modified h-BN (BN2, BN3, BN4) two-dimensional materials and Escherichia coli (E. coli). E. coli Taking a sample as an example, an electrochemical test was conducted on the interaction between BN series materials and Escherichia coli using a PalmSens4 potentiostat, PSTrace5 software, and a three-electrode system.

[0032] refer to Figures 1 to 3 The method for detecting transmembrane charge transport between the target biological and material interfaces in this application specifically includes the following steps: Cultivation and treatment of the bacterial strain: Instruments and solutions used for antimicrobial treatment were autoclaved at 120°C for at least 30 minutes. The entire antimicrobial operation was performed in a biosafety cabinet. *Escherichia coli* (ATCC11775 strain) was selected as the model strain and inoculated into LB (Luria-Bertani) medium. The strain was incubated at 37°C and 220 rpm for 12 hours to resuscitate it. The resuscitated *E. coli* was diluted in LB medium and incubated for another 3 hours to allow it to enter the logarithmic growth phase. Subsequently, the bacterial suspension was centrifuged at 12,000 rpm for 3 minutes to separate the bacterial precipitate. The precipitate was washed with 0.9% sodium chloride solution. This centrifugation-washing step was repeated three times to obtain the final bacterial suspension required for the experiment.

[0033] Electrode preparation: (1) First, prepare the electrode slurry: weigh 5 mg of the target two-dimensional material and 5 mg of conductive carbon (C) material, add them to 2000 μL of water-isopropanol mixed solution and disperse them fully; then add 120 μL of Nafion solution as a binder and continue stirring until the system forms a uniform and stable electrode slurry. (2) Test electrode preparation: coat the electrode slurry uniformly on the surface of the glass carbon electrode sheet, and complete the electrical conduction between the working electrode and the PalmSens4 potentiostat by using copper tape; then use polyimide (PI) tape to encapsulate and isolate the non-test area of ​​the electrode, leaving only the designated exposed area as the effective working interface.

[0034] Microdroplet formation and testing: A platinum (Pt) needle was used as the counter electrode (CE), and a silver / silver chloride electrode (Ag / AgCl) was used as the reference electrode (RE). The target material was uniformly coated on the surface of a platinum carbon sheet as the working electrode (WE). Copper tape was used to electrically connect the working electrode to a PalmSens4 potentiostat. Subsequently, PI tape was used to isolate the non-test areas of the electrode, leaving only the exposed working interface. Finally, bacterial solution was dropped onto the working interface to test the electrochemical reaction between the bacterial solution and the material.

[0035] Experimental setup: 50 μL of the test solution (an electrolyte system containing E. coli) was precisely dropped onto the surface of the working electrode, forming a closed microdroplet electrochemical reaction region together with the reference electrode. The working electrode was connected to copper glue via a Pt needle to achieve effective conduction and acquisition of electrochemical signals. A Pt needle was used as the counter electrode, precisely inserted into the microdroplet between the reference and working electrodes, forming a complete three-electrode detection circuit to ensure stable conduction of charge transmembrane transport reactions and signal detection. When bacteria cover the electrode surface, a microbial interface is formed, allowing for further testing of the microdroplet electrochemical interface transmembrane transport process. The electrochemical test parameters described in this method are based on specific microorganisms (…). E. coli These are exemplary parameters optimized based on the typical redox properties between H-BN and H-BN. For different microorganism-material combinations, those skilled in the art can adaptively adjust the above parameters according to their specific redox potentials through routine preliminary experiments (such as performing a rapid CV scan to determine the reaction range).

[0036] By integrating the area of ​​the CV curve, it can be shown that a large number of electrons are transferred from bacteria to the material, and the charge transmembrane transport efficiency of different BN series materials can be compared based on the area size (reference). Figure 2 (a) Simultaneously, CV tests were performed using a pure carrier electrode sheet (glassy carbon electrode sheet). The experimental results showed that the current response between the glassy carbon electrode sheet and bacteria was extremely weak, and no significant redox reaction occurred. (Reference) Figure 2 In the figure, b represents the DPV curves of different materials. The DPV curves show a clear reduction peak, indicating that BN can accept electrons generated by bacteria in the respiratory chain, completing the transmembrane charge transport process. (Reference) Figure 2 In the figure, c represents the EIS curves of different materials, which can be used to compare the resistance to transmembrane charge transport in different materials.

[0037] It should be noted that this application also has the following alternatives: using transition metal sulfides (molybdenum disulfide (MoS2), etc.), graphene (C), black phosphorus (BP), or titanium carbide MXene (Ti3C2T). XOther two-dimensional materials, such as hexagonal boron nitride (h-BN), can be used instead of boron nitride (h-BN); microorganisms such as Shewanella oneidensis MR-1, lactic acid bacteria, or mixed microbial communities derived from activated sludge can be used instead of Escherichia coli. In addition, this application can also be applied to bioactive substances such as cells, viruses, enzymes, nucleic acids, or proteins other than microorganisms; different modification methods (such as noble metal nanoparticle modification, fluorine doping, or surface functionalization treatment) can be used to modify two-dimensional materials to enhance the interfacial interaction between microorganisms.

[0038] In some embodiments, this application also provides a microbial electrochemical sensor for performing the method of this application to detect microbial concentration. The sensor includes: a housing; an electrochemical workstation disposed within the housing for generating and acquiring electrical signals; a detection cell disposed on or connected to the housing surface for accommodating microdroplet electrochemical detection circuits; and a processor electrically connected to the electrochemical workstation and configured to calculate and output microbial concentration information based on the response signals acquired by the electrochemical workstation.

[0039] In some embodiments, this application also provides a method for evaluating the performance of a microbial fuel cell, comprising the following steps: using the method of this application to detect the charge transmembrane transport characteristics between different electrode materials and microorganisms; comparing the charge transmembrane transport characteristics, and screening out electrode materials with high charge transport efficiency for constructing a microbial fuel cell.

[0040] In some embodiments, this application also provides a method for evaluating the efficacy of antimicrobial materials, comprising the following steps: using the method of this application, detecting the charge transmembrane transport characteristics between the target antimicrobial material and the microorganism interface; evaluating the antimicrobial efficacy of the target antimicrobial material based on changes in the charge transmembrane transport characteristics, wherein obstructed charge transport or increased charge transfer resistance indicates enhanced antimicrobial efficacy.

[0041] In some embodiments, this application also provides a microdroplet electrochemical detection device, comprising: a working electrode having a functionalized material disposed on its working surface; a reference electrode and a counter electrode, wherein the ends of the reference electrode and the counter electrode are configured to be inserted into microdroplets formed on the working surface to jointly constitute a microdroplet electrochemical detection circuit.

[0042] The beneficial effects of this application are as follows: This application combines two-dimensional materials with microdroplet electrochemical technology to optimize the charge transmembrane transport efficiency at microbial interfaces. Utilizing interfacial and confinement effects at the microscale, it significantly amplifies the charge transmembrane transport signal. Simultaneously, it enables the analysis of factors influencing the transmembrane charge transport efficiency at microbial interfaces. Furthermore, it accurately captures the reduction peaks of electron transfer from bacterial and cell membranes to the surface of two-dimensional materials, demonstrating that two-dimensional materials can influence the transmembrane transport process of bacterial extracellular charges (specifically, based on the reduction peaks in DPV testing representing the direction of interfacial charge transfer). This invention advances fundamental research on the application of microdroplets in biosensing and provides technical support for the development of environmental monitoring, food safety, and digital healthcare.

[0043] The background section of this invention may include background information about the problems or environment in which the invention is being developed, and is not necessarily a description of prior art. Therefore, the content included in the background section does not constitute an admission of prior art by the applicant.

[0044] The above description provides a further detailed explanation of the present invention in conjunction with specific / preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the concept of the present invention, and all such substitutions or modifications should be considered within the scope of protection of the present invention. In the description of this specification, the reference to terms such as "an embodiment," "some embodiments," "preferred embodiment," "example," "specific example," or "some examples," etc., indicates that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more embodiments or examples. Without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. A method for detecting transmembrane charge transport between a target organism and a substance interface, characterized in that, Includes the following steps: S1. A working electrode is provided, the working electrode having a working surface, the working surface being provided with a functionalized material; S2. An electrolyte containing the target bioactive substance is added to the working surface loaded with the functionalized material to form a microdroplet with a volume of microliters on it. S3. Insert the ends of the reference electrode and the counter electrode into the microdroplet, which together with the working surface forms a microdroplet electrochemical detection circuit; S4. Apply an excitation signal to the microdroplet electrochemical detection circuit and acquire the response signal from the working electrode; S5. Based on the response signal, determine the charge transmembrane transport characteristics between the target bioactive substance and the functionalized material interface.

2. The method according to claim 1, characterized in that, The excitation signal includes at least one of cyclic voltammetry (CV) scan signal, differential pulse voltammetry (DPV) scan signal, and electrochemical impedance spectroscopy (EIS) scan signal.

3. The method according to claim 1, characterized in that, In step S1, the functionalized material includes two-dimensional materials, which refer to materials with nanoscale thickness and two-dimensional planar structure.

4. The method according to claim 1, characterized in that, In step S2, the target bioactive substance includes microorganisms, cells, viruses, enzymes, nucleic acids, or proteins.

5. The method according to claim 4, characterized in that, The microorganisms include Escherichia coli, lactic acid bacteria, or Shewanella.

6. The method according to claim 2, characterized in that, Applying the excitation signal in step S4 includes the following steps: When using cyclic voltammetry, scanning is performed within a selected potential range at a selected scan rate; When using the differential pulse voltammetry method, a scan is performed within the selected potential range using selected potential steps, pulse potentials, and pulse time parameters. When using electrochemical impedance spectroscopy, a selected DC bias voltage and AC disturbance potential are superimposed on the open-circuit potential of the microdroplet electrochemical detection circuit, and scanning is performed within a selected frequency range.

7. The method according to claim 1, characterized in that, In step S5, the charge transmembrane transport characteristics include: the direction of interfacial charge transfer, charge transfer resistance, number of electrons transferred, or concentration of the target bioactive substance.

8. A microbial electrochemical sensor, characterized in that, The sensor is used to perform the method of any one of claims 1-9 to detect microbial concentration, the sensor comprising: a housing; an electrochemical workstation disposed within the housing for generating and acquiring electrical signals; a detection cell disposed on or connected to the housing surface, the detection cell for accommodating a microdroplet electrochemical detection circuit; and a processor electrically connected to the electrochemical workstation and configured to calculate and output the concentration information of the microorganisms based on the response signals acquired by the electrochemical workstation.

9. A method for evaluating the efficacy of antibacterial materials, characterized in that, The method includes the following steps: using the method of any one of claims 1-9, detecting the charge transmembrane transport characteristics between the target antimicrobial material and the microorganism interface; and evaluating the antimicrobial efficacy of the target antimicrobial material based on the changes in the charge transmembrane transport characteristics, wherein changes in the charge transport process or different amounts of charge transfer affect the antimicrobial effect.

10. A microdroplet electrochemical detection device, comprising: The working electrode has a functionalized material on its working surface; A reference electrode and a counter electrode, the ends of which are configured to be inserted into micro-droplets formed on the working surface, together forming a microdroplet electrochemical detection circuit.

Images