Anti-pollution microelectrode for identifying pseudomonas aeruginosa strain and detecting phenazine spatial distribution of pseudomonas aeruginosa strain as well as preparation method and application of anti-pollution microelectrode

Abstract

The invention belongs to the field of electrochemical sensors, and discloses an anti-pollution microelectrode for identifying pseudomonas aeruginosa strains and detecting phenazine spatial distribution of the pseudomonas aeruginosa strains as well as a preparation method and application of the anti-pollution microelectrode. The anti-pollution microelectrode comprises a carbon fiber electrode base body, a Fe-HHTP-PDA composite substrate layer and a Fe-HHTP-SBMA anti-pollution layer. The two-dimensional conductive metal organic framework material Fe-HHTP is doped in the anti-pollution layer based on the PDA substrate and the SBMA, so that the problem of signal attenuation caused by insulativity of the anti-pollution layer is successfully solved; by virtue of a two-dimensional ordered structure and a mixed valence iron node, the Fe-HHTP not only serves as a conductive bridge to maintain electron transmission, but also specifically catalyzes an oxidation-reduction reaction of phenazine substances, so that the anti-pollution function is realized, and meanwhile, the detection sensitivity is maintained and even improved. The preparation method is simple and convenient to operate and controllable in modification, and does not need a traditional high-pressure and high-temperature process.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensors, and in particular to a Fe-HHTP-doped anti-fouling microelectrode, its preparation method, and its application. Background Technology

[0002] Pseudomonas aeruginosa ( P. aeruginosa Pseudomonas aeruginosa is an opportunistic pathogen widely found in human living and medical environments. It often leads to chronic infections and persistent environmental pollution. It creates an environment conducive to biofilm formation, providing a breeding ground for the development of antibiotic resistance and tolerance. Pseudomonas aeruginosa can produce a series of redox-active secondary metabolites—phenazines, including pyocyanin (PYO), phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and 5-methylphenazine-1-carboxylic acid (5-MCA). These nitrogen-containing heterocyclic compounds play important roles in Pseudomonas aeruginosa infection and contamination, such as: (1) regulating cellular redox state; (2) mediating host tissue damage and necrosis as virulence factors; (3) regulating gene expression as quorum sensing signaling molecules; (4) affecting bacterial viability and biofilm formation; and (5) regulating antibiotic resistance and tolerance (especially in biofilms). Under the same conditions, different strains of Pseudomonas aeruginosa exhibit different phenazine secretion patterns; the same strain may also show changes in phenazine secretion patterns and content under different stress conditions (such as antibiotics). This information is correlated with the virulence and drug resistance of *Pseudomonas aeruginosa*, providing rich insights into the evolutionary direction of drug resistance under different stresses and predicting its resistance. Therefore, obtaining information on whether bacteria secrete phenazine, and the types and amounts of phenazine secreted, is not only beneficial for identifying *P. aeruginosa* and determining its strain, but also helps predict its virulence and drug resistance. Since nearly 80% of human microbial infections are caused by biofilms, and biofilms play a crucial role in the development of bacterial antibiotic resistance, identifying the presence of *P. aeruginosa* in biofilms and monitoring the phenazine secretion patterns and levels within *P. aeruginosa* biofilms are of great significance and have attracted considerable attention.

[0003] Since the discovery of phenazine metabolites, various detection methods have been developed. Traditional methods mainly rely on techniques such as chromatography or spectrophotometry. These methods have good quantitative results, but require extraction of phenazine substances from P. aeruginosa cultures before measurement, which is tedious, time-consuming, and cannot achieve in-situ analysis. As an alternative, people have developed various electrochemical sensing methods based on the redox activity of phenazine substances for rapid, direct, and in-situ detection of P. aeruginosa liquid cultures and biofilms. Most of these methods are based on conventional macroelectrodes and only measure the total concentration of one or a few phenazine substances in the sample. Notably, biofilms have structural and metabolic heterogeneity, making phenazine substances unevenly distributed within them. This heterogeneity can lead to differences in antibiotic sensitivity of bacteria at different parts of the biofilm, increasing the difficulty of treating biofilm infections. Therefore, the spatiotemporal distribution of phenazine substances in biofilms may provide more valuable information than their total concentration. In order to thoroughly elucidate their potential patterns and mechanisms, it is urgent to develop methods with sufficient spatial resolution for in-situ detection of phenazines in biofilms. Given the advantages of microelectrodes in spatial resolution, Belilin et al. developed a microelectrode array based on integrated circuits, which first achieved the detection of the spatial distribution of multiple phenazine substances in biofilms, thus opening up avenues for in-depth exploration of the potential functional roles of phenazine substances. However, this method has some limitations. It is costly and complex to prepare, which hinders its widespread application. Since microelectrodes are more susceptible to contamination than conventional electrodes, a layer of agar is placed between the biofilm and the microelectrode array chip to prevent signal attenuation caused by biofilm contamination. Similar methods of isolating contamination through agar have also been used in other methods developed based on conventional electrodes. For microelectrodes, this isolation method will cause a decrease in measurement resolution due to the diffusion of analytes in agar, inevitably causing differences between measured data and the actual situation inside the biofilm. A potential solution to these problems is to develop easy-to-prepare anti-pollution microelectrodes and combine them with a scanning electrochemical microscope (SECM) with precise positioning capabilities, which can directly perform in-situ quantitative analysis of phenazines in biofilms. Currently, SECM has been used to characterize various metabolites (including pyocyanin, a phenazine) within biofilms. Typically, biofilms are immersed in an electrolyte solution, and microelectrodes are placed in the diffusion layer above the biofilm for imaging or monitoring, rather than being directly inserted into the biofilm. Direct insertion into the biofilm would cause severe signal attenuation due to microelectrode contamination, affecting the reliability of measurement results in continuous measurements.

[0004] Electrode fouling caused by biofilms is mainly due to the adsorption of biomolecules, microbial cells, and redox byproducts onto the electrode surface through hydrophobic and electrostatic interactions. Accordingly, modifying microelectrode surfaces with hydrophilic and electrically neutral coatings is a reasonable choice for antifouling. Zwitterions, electrically neutral compounds possessing both positive and negative charge groups within the same molecule, are ideal candidates for constructing such coatings. Due to their excellent hydrophilicity and ability to form stable hydrated layers, they exhibit good resistance to protein adsorption and bacterial adhesion, as well as excellent biocompatibility. As highly superhydrophilic compounds, zwitterions require a strong binder to be fixed onto the surface to be modified. Polydopamine (PDA), synthesized from dopamine through spontaneous oxidative polymerization, is such a powerful "bioglue." As an intermediate layer, it can fix zwitterions onto the surfaces of various materials through chemical reactions of thiols and amines. In recent years, considerable progress has been made in the development of antifouling interfaces based on PDA-zwitterion conjugation, but many problems still remain. Specifically, the main challenge in anti-fouling modification of microelectrodes lies in the difficulty of controllable modification of the micro-nano scale interface, and the poor conductivity of the PDA-zwitterion layer leads to a decrease in electrode sensitivity due to its modification.

[0005] Currently, the only electrochemical method capable of detecting the spatial distribution of multiple phenazines in biofilms is the microelectrode array based on integrated circuits developed by Belilin et al. This method relies on complex photolithography technology and requires matching signal collection and conversion interfaces, resulting in high detection costs. Furthermore, to avoid contaminating the electrode array and causing signal attenuation, an agar layer needs to be placed between the electrode and the biofilm, which introduces diffusion interference and causes the measured signal to deviate from the in-situ information. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the signal attenuation problem caused by the anti-fouling layer, and to provide an anti-fouling microelectrode for the identification of Pseudomonas aeruginosa strains and the detection of its phenazine spatial distribution, as well as the application of this microelectrode in detecting phenazine substances and their spatial distribution in bacterial biofilms and identifying Pseudomonas aeruginosa. P. aeruginosa Applications of strains and their types.

[0007] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: An antifouling microelectrode for identifying Pseudomonas aeruginosa strains and detecting the spatial distribution of its phenazine residues includes a carbon fiber electrode substrate, an Fe-HHTP-PDA composite substrate layer, and a Fe-HHTP-SBMA antifouling layer. The Fe-HHTP-PDA composite substrate layer is modified onto the surface of the carbon fiber electrode substrate by electrodeposition, and the Fe-HHTP-SBMA antifouling layer is modified onto the surface of the Fe-HHTP-PDA composite substrate layer by chemical grafting. The Fe-HHTP-SBMA antifouling layer includes an SBMA antifouling layer and Fe-HHTP doped on the SBMA antifouling layer. The Fe-HHTP-PDA composite substrate layer includes a polydopamine substrate and Fe-HHTP doped on the polydopamine substrate. The Fe-HHTP on the Fe-HHTP-PDA composite substrate layer and the Fe-HHTP on the Fe-HHTP-SBMA antifouling layer are in contact to form a conductive pathway. The Fe-HHTP is a two-dimensional conductive metal-organic framework material containing mixed-valence Fe metal nodes.

[0008] Furthermore, in the aforementioned anti-fouling microelectrode, the diameter of the carbon fiber electrode substrate is 7~30μm, and its length exposed outside the insulating glass layer is 50~150μm.

[0009] Furthermore, the Fe-HHTP is obtained by reacting HHTP with a ferrous salt, wherein the ferrous salt is selected from at least one of ferrous acetate, ferrous chloride, ferrous sulfate, and ferrous nitrate; the Fe in the mixed valence state Fe metal node 2+ and Fe 3+ The ratio of the number of atoms is 38-40:60-62.

[0010] The antifouling microelectrode of this invention, based on a 2D-cMOF-doped composite antifouling layer, allows for controllable modification of the microelectrode for sensitive, continuous, and signal-attenuation-free detection of redox-active metabolites (represented by phenazine compounds) in bacterial biofilms. The composite antifouling layer consists of Fe-HHTP, PDA, and 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt (i.e., sulfobetaine methacrylate, SBMA). The microelectrode is controllably functionalized through a two-step modification method involving electrodeposition and post-modification. PDA acts as a "strong binder" to fix Fe-HHTP onto the carbon fiber surface and as an anchoring group to graft SBMA onto the substrate, achieving the antifouling purpose. Fe-HHTP acts as a conductive bridge, maintaining electron transfer between the underlying carbon electrode and the surface analytes. Simultaneously, its mixed-valence Fe metal nodes act as catalysts for the electrochemical redox reaction of phenazines, further improving electron transfer efficiency. The synergistic effect of these two mechanisms enhances the analytical performance of the prepared microelectrode, compensating for signal attenuation caused by the PDA-SBMA coating. Using the developed electrode as a SECM probe, precise positioning allows for continuous insertion into a series of locations of interest within the *Pseudomonas aeruginosa* biofilm. Continuous detection using differential pulse voltammetry (DPV) yields spatial distribution information for various phenazines within the biofilm. Alternatively, the electrode can be positioned to a series of single colonies isolated from mixed bacteria, allowing for rapid determination of the presence of *Pseudomonas aeruginosa* and preliminary identification of the strain type by measuring DPV patterns.

[0011] This invention involves doping a two-dimensional conductive metal-organic framework material (Fe-HHTP) containing mixed-valence Fe metal nodes into an antifouling layer composed of polydopamine (PDA) and sulfobetaine (SBMA), which is then modified onto the surface of carbon fibers to fabricate a microelectrode with antifouling capabilities and the ability to distinguish multiple phenazines. This microelectrode is then used as a probe in a scanning electrochemical microscope to locate specific bacterial biofilms or specific sites within them. Differential pulse voltammetry is used to simultaneously detect the content of multiple electrochemically active metabolites (such as phenazines) at these specific sites. By measuring multiple sites consecutively within a single biofilm, the spatial distribution information of various electrochemically active metabolites can be obtained. Furthermore, by acquiring the spectral data of phenazines in a specific biofilm, the presence and strain type of *Pseudomonas aeruginosa* can be determined. It can be applied to the following fields: in the field of medical testing, it is used to identify infection types and analyze and predict drug resistance and virulence; in the field of public health, it is used to study the relationship between the evolution of bacterial biofilm resistance and the spatiotemporal changes of metabolites under the pressure of antibiotics and disinfectants; in the field of environmental monitoring and pollution control, it is used to identify polluting strains, monitor the status of biofilm control, and predict the difficulty of control.

[0012] Based on a general inventive concept, the present invention also provides a method for preparing an anti-fouling microelectrode, comprising the following steps: (1) HHTP and ferrous salt were dissolved in N,N-dimethylformamide, mixed and sonicated, stirred and reacted, and then Fe-HHTP powder was obtained after washing, drying, grinding and sieving. (2) The carbon fiber is fixed in a glass capillary tube, drawn in two steps by a vertical needle drawing instrument, filled with carbon powder and inserted with copper wire, and sealed and fixed with epoxy resin to obtain the carbon fiber electrode matrix. (3) After preparing Fe-HHTP powder into Fe-HHTP dispersion, it is mixed with hydrochloric acid dopamine solution. Under nitrogen protection, using carbon fiber electrode substrate as working electrode, Fe-HHTP-PDA composite substrate layer is formed on the surface of carbon fiber electrode substrate by cyclic voltammetry electrodeposition. (4) The carbon fiber electrode modified with Fe-HHTP-PDA composite substrate is placed in SBMA solution containing Fe-HHTP for grafting reaction. After ultrasonic cleaning and nitrogen drying, the anti-pollution microelectrode with good conductivity is obtained.

[0013] In the above preparation method, further, in step (1), the stirring reaction temperature is 80~90℃ and the time is 12~24h; the cleaning is carried out by N,N-dimethylformamide, anhydrous ethanol and water in sequence; the drying is carried out by vacuum drying at 50~80℃ for 12~24h; and the carbon fiber is passed through a 200~1000 mesh sieve; in step (2), the diameter of the carbon fiber is 7~30 μm, the two heating temperature levels of the two-step vertical needle pulling method are 70~80% and 75~85% in sequence, and the positions of the upper and lower sliding rods are 3~5mm and 4~6mm, respectively.

[0014] Further, in step (3), the concentration of the Fe-HHTP dispersion is 2~20 mg / mL, and ultrasonic dispersion is performed for 1~2 h; the dopamine hydrochloride solution is prepared using 50 mM Tris-HCl buffer as solvent, with a concentration of 2~20 mg / mL and pH adjusted to 7.2~7.6; the reference electrode is an Ag / AgCl electrode containing 3 M KCl, and the counter electrode is a platinum wire; the potential window for the cyclic voltammetry electrodeposition is 0~0.8 V, the scan rate is 50~200 mV / s, and the number of scans is 15~30; after deposition, the carbon fiber electrode is dried in an oven at 50~60℃ for 1~2 h to obtain a carbon fiber electrode modified with a Fe-HHTP-PDA composite substrate layer.

[0015] Furthermore, in step (4), the SBMA solution containing Fe-HHTP contains SBMA at a concentration of 30~150 mg / mL, Fe-HHTP at a concentration of 0.05~0.5 mg / mL, and the concentration ratio of Fe-HHTP to SBMA is 1:100~1000; the grafting reaction is carried out by stirring at 35~60℃ for 12~24 h.

[0016] Based on a general inventive concept, this invention also provides an application of an antifouling microelectrode in detecting phenazine substances and their spatial distribution in bacterial biofilms. The method of application includes the following steps: using the antifouling microelectrode as the working electrode, a silver wire coated with AgCl as the quasi-reference electrode, and a platinum wire as the counter electrode, the working electrode is positioned at multiple test sites on the surface of the bacterial biofilm using a scanning electrochemical microscope; differential pulse voltammetry is used to detect at each test site to obtain a voltammetric spectrum containing redox current signals of various phenazine substances; based on the voltammetric spectrum, the content of various phenazine substances is quantified, and combined with the spatial coordinates of each site, the spatial distribution information of various phenazine substances in the bacterial biofilm is obtained.

[0017] In the above application, the parameters of the differential pulse voltammetry method are as follows: initial potential 0~0.2V, final potential -0.6~-0.8V, potential increment 0.005~0.02V, amplitude 0.01~0.05V, pulse width 0.01~0.04s, sampling width 0.01~0.1s, and pulse period 0.05~0.2s; The parameters for positioning using the scanning electrochemical microscope are: potential bias -0.1~0.1V, z-axis approach velocity 5~20μm / s, and stop current 1×10⁻⁶. -10 ~1×10 -9 A, after approaching the surface of the biofilm, continues to advance for 50~150μm; The bacteria is Pseudomonas aeruginosa (Pseudomonas aeruginosa) P. aeruginosa The phenazines include at least one of pyocyanin (PYO), phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and 5-methylphenazine-1-carboxylic acid (5-MCA).

[0018] Based on a general inventive concept, the present invention also provides an anti-fouling microelectrode for identifying Pseudomonas aeruginosa (… P. aeruginosaThe application of the anti-fouling microelectrode and / or its strain type includes the following steps: using the anti-fouling microelectrode as the working electrode, a silver wire coated with AgCl as the quasi-reference electrode, and a platinum wire as the counter electrode, the working electrode is positioned in the bacterial colony or biofilm to be tested using a scanning electrochemical microscope; differential pulse voltammetry (DPV) is used for detection to obtain a DPV spectrum; based on whether the characteristic redox peaks of phenazine substances appear in the DPV spectrum, it is determined whether the bacterial colony is Pseudomonas aeruginosa; if it is Pseudomonas aeruginosa, its strain type is preliminarily determined based on the characteristic current combinations of different phenazine substances in the DPV spectrum.

[0019] The above applications, further, whether in detecting phenazine substances and their spatial distribution in bacterial biofilms, or in identifying Pseudomonas aeruginosa (…), P. aeruginosa The application of this strain and its strain type does not involve the addition of any electrolyte solution.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) By doping a two-dimensional conductive metal-organic framework material Fe-HHTP into an antifouling layer based on polydopamine (PDA) substrate and sulfobetaine methacrylate (SBMA), the problem of signal attenuation caused by the insulation of the antifouling layer was successfully solved. Fe-HHTP, with its two-dimensional ordered structure and mixed valence iron nodes, not only acts as a "conductive bridge" to maintain electron transport, but also specifically catalyzes the redox reaction of phenazine substances, thus maintaining or even improving the detection sensitivity while achieving the antifouling function.

[0021] (2) This method is simple to prepare and the modification is controllable. Fe-HHTP can be synthesized in a water bath at 80~90℃ without the need for traditional high pressure and high temperature processes. The cyclic voltammetric electrodeposition method can achieve synchronous and stable modification of PDA and Fe-HHTP within 3~4 minutes, which overcomes the disadvantages of unevenness and weak bonding of the drop coating method and ensures the consistency of batch preparation.

[0022] (3) The doping of Fe-HHTP also significantly improved the peak separation of various phenazine substances in the DPV spectrum, solved the problem of difficult analysis of overlapping peaks, and ensured quantitative accuracy.

[0023] (4) Compared with microelectrode array chips, this solution has significantly reduced costs and does not require an agar isolation layer, thus avoiding diffusion interference. It can be directly inserted into the biofilm to obtain real in-situ information. As a scanning electrochemical microscope probe, its clever positioning capability supports on-demand spatial distribution detection, making it more convenient to use. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the material synthesis, electrode modification, and anti-fouling detection of phenazine in biofilm in the embodiments of the present invention, wherein: (A) schematic diagram of the synthesis principle of Fe-HHTP; (B) schematic diagram of the principle of Fe-HHTP / PDA / SBMA composite coating modified by carbon fiber microelectrode (CFE); (C) schematic diagram of the electrode used to detect phenazine substances in biofilm.

[0026] Figure 2 These are the (A) scanning electron microscope image and (BD) elemental distribution map of Fe-HHTP in this embodiment of the invention.

[0027] Figure 3 This is an X-ray photoelectron spectrum of the valence state composition of iron in Fe-HHTP in this embodiment of the invention.

[0028] Figure 4 These are low-magnification (B) and high-magnification (C) scanning electron microscope images of the carbon fiber electrode before modification (A) and after modification with Fe-HHTP / PDA / SBMA in this embodiment of the invention. (DH) is an elemental distribution mapping map on the carbon fiber electrode after modification with Fe-HHTP / PDA / SBMA.

[0029] Figure 5 This is a comparison of the reduction current signal DPV spectra (n=3) measured in PBS solution containing 10 μM PYO before and after different modified electrodes (bare carbon fiber, Fe-HHTP / PDA and Fe-HHTP / PDA / SBMA modified electrodes) were immersed in 1% bovine serum albumin (BSA) for 1 h for contamination.

[0030] Figure 6 The images show the DPV patterns of the Fe-HHTP / PDA / SBMA modified electrode and the bare electrode in a mixed PBS solution containing 10 μM PYO, 10 μM PCA and 10 μM PCN in this embodiment of the invention.

[0031] Figure 7 The calibration curves are obtained by measuring the Fe-HHTP / PDA / SBMA modified electrode prepared in the embodiments of the present invention in a series of PBS solutions of (A) PYO, (B) PCA and (C) PCN.

[0032] Figure 8 In this embodiment of the invention, the DPV fingerprints of six bacteria were determined, including four Pseudomonas aeruginosa bacteria: PAO1, PA14, PA, and ΔPYO, and two non-Pseudomonas aeruginosa bacteria: SA (Staphylococcus aureus) and EC (Escherichia coli).

[0033] Figure 9 This is a spatial distribution diagram of four phenazines (PYO, PCN, PCA and 5-MCA) in a Pseudomonas aeruginosa PAO1 biofilm cultured for 24 hours, as shown in the embodiment of the invention. Detailed Implementation

[0034] This invention first synthesizes 2D-cMOF Fe-HHTP containing mixed valence state Fe metal nodes, mixes it with dopamine, and then electrodeposits it onto the prepared carbon fiber microelectrode by cyclic voltammetry. The electrode is then immersed in a mixed solution of SBMA and Fe-HHTP, and SBMA is grafted onto a PDA substrate doped with Fe-HHTP by a chemical reaction to form an anti-fouling layer with good conductivity. Using the prepared electrode as a probe for SECM, biofilms can be detected without interference from biological contaminants, achieving the following functions: (1) Precisely locating a series of sites in the Pseudomonas aeruginosa biofilm and performing differential pulse voltammetry (DPV) measurement. The obtained spectrum contains redox currents of multiple phenazines, which can be used to quantify the corresponding phenazine content levels, thereby obtaining spatial quantitative distribution information of multiple phenazines; (2) Locating the probe to a single colony isolated from mixed bacteria on a solid culture medium and performing DPV measurement. The presence of Pseudomonas aeruginosa can be determined based on the presence of redox currents of phenazines in the spectrum, and the strain type of Pseudomonas aeruginosa can be preliminarily determined through the obtained phenazine DPV spectrum information. The specific technical solution is as follows: (1) Synthesis of Fe-HHTP Dissolve 161.33 mg HHTP and 198.8 mg ferrous chloride tetrahydrate in 20 mL of N,N-dimethylformamide (DMF), respectively. Pour the two solutions into a glass bottle, mix thoroughly, and sonicate for 5 min. Place the glass bottle containing the mixture on a constant temperature magnetic stirrer and stir at 85 ℃ for 24 h. After the synthesis is complete, cool the solution to room temperature for 2 h, centrifuge and discard the supernatant. Wash the solution three times each with DMF, anhydrous ethanol, and water, and then dry it in a vacuum drying oven at 60 ℃ for 12 h. Grind the dried material in an agate mortar and pestle, and then pass it through a 200-mesh sieve for later use.

[0035] (2) Fabrication of carbon microelectrodes (CFE) Take a carbon fiber approximately 1.5 cm long (30 μm or 7 μm in diameter) and place it inside a glass capillary tube (outer diameter 1.2 mm, inner diameter 0.69 mm). Use a copper wire to move the carbon wire to the middle of the tube. Insert a 0.5 mm diameter copper wire into the tube to block one end of the glass tube opening, securing the carbon wire in the middle of the tube to prevent displacement or detachment. Mount the glass tube on a vertical needle drawing apparatus (brand: Narisuge, model: PC-100), with the copper wire end facing down. Perform a two-step drawing process using four counterweights. The heating temperatures for the two steps are 75% and 80% respectively, with the upper and lower slide positions at 4 mm and 5 mm respectively. After drawing, trim the exposed carbon fiber tip to approximately 100 μm (or to other lengths depending on the research purpose) under a microscope. Further fine-tune the exposed carbon fiber length and tip flatness using a needle grinding instrument. Finally, fill the tube with carbon powder to one-third to one-half of its volume, and then insert a 400 mm diameter needle. A copper wire of μm was used, and the open end of the glass tube was sealed with epoxy resin to fix the position of the copper wire.

[0036] (3) Electrodeposition modification of Fe-HHTP-PDA composite substrate A 20 mg / mL Fe-HHTP dispersion was prepared using ultrapure water and sonicated for 2 h. A 20 mg / mL dopamine hydrochloride solution was prepared using 50 mM Tris-HCl buffer as the solvent, and the pH was adjusted to 7.4. The Fe-HHTP dispersion and dopamine hydrochloride solution were mixed in equal volumes, stirred thoroughly, and then transferred to a sealed electrolytic cell. The reference electrode (Ag / AgCl, 3M KCl) and the counter electrode (platinum wire) were installed, and nitrogen gas was purged for 15-20 min. Then, a carbon microelectrode was installed, and cyclic voltammetry electrodeposition was performed. The electrodeposition parameters were as follows: potential window of 0-0.8 V, scan rate of 200 mV / s, and 20 scan cycles. After deposition, the electrode was dried in a 60℃ oven for 2 h and then stored for later use.

[0037] (4) Chemical modification of Fe-HHTP-SBMA antifouling layer A 50 mg / mL SBMA solution was prepared using 50 mM Tris-HCl buffer as the solvent. After adjusting the pH to 8.5, Fe-HHTP was added to bring the concentration to 0.2 mg / mL. The microelectrode modified with the Fe-HHTP-PDA composite substrate was fixed in a glass bottle containing the solution and placed on a constant temperature magnetic stirrer. The mixture was stirred at 55 °C for 12 h. After the reaction was completed, the electrode was removed, sonicated with ultrapure water for 2 min, and dried with nitrogen for later use.

[0038] (5) Sample preparation Preparation of mixed bacterial samples: Dip an inoculation loop into the mixed bacterial enrichment broth and streak it onto an LB nutrient agar plate; after incubation for 24 hours, individual colonies can be measured. Preparation of *Pseudomonas aeruginosa* biofilm for determining the spatial distribution of phenazine: A mixed cellulose filter membrane with a pore size of 0.22 μm was attached to an LB nutrient agar plate; purified colonies of the strain under study (approximately 1 mm in diameter) were picked from the agar plate and dispersed in 100 μL of PBS solution. The dispersion was then inoculated onto the filter membrane at a rate of 5 μL per spot; after absorption, the plate was placed in an incubator and incubated at 37 ℃ for 24-48 h, ready for spatial distribution detection; alternatively, a drug of interest (such as a certain concentration of antibiotics or disinfectant) could be added to the agar plate as needed, and after incubation for a specific time, it could be used to determine the spatial distribution of phenazine under specific treatment factors.

[0039] (6) Detection of phenazine in biofilms The detection process uses the carbon microelectrode prepared in step (2) as the working electrode, the silver wire coated with AgCl as the quasi-reference electrode, and the 0.5 mm platinum wire as the counter electrode. No solution system is required in the detection process. The agar plate inoculated with biofilm is fixed on the stage of SECM, the reference electrode and the counter electrode are inserted into the agar, and the working electrode is fixed on the stepper. The testing process is as follows: First, the potential is biased at -0.1 V. The probe is positioned to the central surface of the biofilm through a sudden jump in the approximation current under positive feedback mode. During this process, the working electrode approaches the biofilm surface in the air along the z-axis at a speed of 10 μm / s. The circuit is in an open-circuit state, and the probe stop current is set to 1 × 10⁻⁶. -9A. When the probe stops advancing, it just touches the biofilm surface. Then, the probe continues to advance 100 μm along the z-axis, ensuring the probe tip is in complete contact with the biofilm. Next, a differential pulse voltammetry (DPV) program is run to determine the levels of various phenazines (PYO, PCA, PCN, and 5-MCA) at the current site. The DPV parameters are as follows: initial potential 0.2 V, final potential -0.7 V, potential increment 0.008 V, amplitude 0.02 V, pulse width 0.02 s, sampling width 0.02 s, and pulse period 0.1 s. For mixed bacterial samples requiring strain identification, the probe is positioned at the center of the suspected colony for DPV measurement. The presence or absence of phenazine in the obtained DPV spectrum determines whether it is Pseudomonas aeruginosa. Combined with the phenazine spectrum, the strain type can be preliminarily determined. For biofilms requiring determination of the spatial distribution of phenazine, the probe is further positioned at each target measurement point on the diameter of the biofilm using the method described above, based on the required sampling density (i.e., the distance between measurement points). The DPV spectrum of each point is then measured, and the distribution of the three phenazines in the biofilm can be plotted using the measurement coordinates.

[0040] Fe-HHTP is simultaneously doped into both the PDA substrate and the SBMA antifouling layer. The Fe-HHTP phase contact between the PDA substrate and the SBMA antifouling layer forms a conductive pathway, enabling antifouling measurement without sacrificing sensitivity and even improving it. PDA provides a good adhesion substrate for SBMA with antifouling properties, allowing it to adhere firmly to various interfaces and form a stable antifouling layer. However, during the modification of electrochemical interfaces, especially in the modification of microelectrodes that are highly sensitive to current changes, the insulating nature of the PDA-SBMA coating severely hinders electron transfer on the electrode, leading to a significant attenuation of the detected current signal.

[0041] This invention synthesizes a two-dimensional conductive MOF material, Fe-HHTP, which is then doped into a PDA-SBMA coating to improve the coating's conductivity. Fe-HHTP acts as a "conductive bridge" in the composite coating, ensuring electron transfer between the electrode and the analyte. Firstly, the unique two-dimensional ordered structure of Fe-HHTP itself endows it with high conductivity. Secondly, the iron metal nodes in Fe-HHTP are in a mixed valence state, which not only further enhances the material's conductivity through charge hopping but also has a specific catalytic effect on the electrochemical redox of phenazine compounds. Both factors synergistically amplify the current signal, thereby achieving anti-fouling modification without sacrificing electrode sensitivity, or even improving it.

[0042] This invention develops a cyclic voltammetric electrodeposition method to simultaneously deposit dopamine and Fe-HHTP onto the electrode surface. By controlling the solution concentration and electrodeposition parameters, stable modification of the electrode surface is achieved, allowing direct insertion into biomembranes for in-situ detection. Compared to drop-coating modification methods, this method eliminates electrode modification variations caused by human operation and batch variations, enabling stable batch production of modified microelectrodes with consistent quality. Furthermore, the electrodeposition process, through repeated application of oxidation potential, ensures sufficient polymerization of dopamine, avoiding the reduced oxidation efficiency after drop-coating when removed from the alkaline solution environment, which could lead to incomplete dopamine oxidation and polymerization, resulting in weak adhesion and easy detachment. Compared to solution-stirring modification methods, this method significantly shortens the modification time. The electrodeposition method developed in this invention can complete the modification of a microelectrode in just 3-4 minutes, while stirring modification based on the spontaneous oxidation polymerization of dopamine requires 8-10 hours to achieve the same modification effect.

[0043] The Fe-HHTP doped in this invention is designed for phenazine compounds. Its Fe metal nodes have a specific catalytic effect on the electrochemical redox of phenazine compounds. The specificity of the synthesized cMOF material for the target analyte varies depending on the metal node. When other redox-active metabolites need to be detected, cMOF materials with other metal nodes can be designed and doped accordingly.

[0044] When simultaneously measuring three phenazines—PYO, PCA, and PCN—using conventional carbon fiber electrodes, the similar reduction potentials of the three substances lead to close peak positions and continuous overlapping peaks, making the results difficult to interpret. Doping with Fe-HHTP increases the difference in reduction potentials among the three substances, thereby improving the peak separation in the DPV spectrum and facilitating more accurate quantification of the three phenazines.

[0045] Furthermore, this invention can be readily prepared by professionals in a scanning electrochemical microscope laboratory equipped with standard equipment, with the preparation cost of a single electrode being only about 20 yuan. Most importantly, due to the electrode's sensitivity and resistance to contamination, it can be directly inserted into the biofilm for in-situ detection. Continuous detection of multiple sites does not result in signal attenuation or signal deviation caused by diffusion; the collected information is truly in-situ. Compared to microelectrode arrays with fixed channel numbers and arrangements, this electrode, used as an SECM probe, can flexibly locate a series of sites of interest. The measurement sites can be adjusted as needed, thus allowing for more targeted and efficient acquisition of spatial distribution information of interest, avoiding the collection and processing of redundant site information.

[0046] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0047] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0048] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0049] In this invention, SBMA, abbreviated as sulfobetaine methacrylate, has the chemical name 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, and is an amphoteric monomer with the chemical formula C. 11 H 21 NO5S, CAS number is 3637-26-1.

[0050] HHTP is 2,3,6,7,10,11-hexahydroxytriphenylene, with the molecular formula C2. 18 H 12 O6, CAS number is 4877-80-9.

[0051] The Fe-HHTP of this invention is a two-dimensional conductive metal-organic framework material with an ordered two-dimensional network structure. The intersections of the two-dimensional network contain mixed-valence Fe metal nodes.

[0052] Example: An anti-fouling microelectrode for identifying *Pseudomonas aeruginosa* strains and detecting the spatial distribution of phenazine ions comprises a carbon fiber electrode substrate, an Fe-HHTP-PDA composite substrate layer, and an Fe-HHTP-SBMA anti-fouling layer. The Fe-HHTP-PDA composite substrate layer is modified onto the surface of the carbon fiber electrode substrate by electrodeposition, and the Fe-HHTP-SBMA anti-fouling layer is modified onto the surface of the Fe-HHTP-PDA composite substrate layer by chemical grafting. The Fe-HHTP-SBMA anti-fouling layer includes an SBMA anti-fouling layer and Fe-HHTP doped on the SBMA anti-fouling layer. The Fe-HHTP-PDA composite substrate layer includes a polydopamine substrate and Fe-HHTP doped on the polydopamine substrate. The Fe-HHTP on the Fe-HHTP-PDA composite substrate layer and the Fe-HHTP on the Fe-HHTP-SBMA anti-fouling layer are in contact to form a conductive path. The diameter of the carbon fiber electrode substrate is 7–30 μm, and its length exposed outside the insulating glass layer is 50–150 μm. Fe-HHTP is obtained by reacting HHTP with a ferrous salt, which is selected from at least one of ferrous acetate, ferrous chloride, ferrous sulfate, and ferrous nitrate. Fe-HHTP is a two-dimensional conductive metal-organic framework material containing Fe metal nodes in mixed valence states.

[0053] The method for preparing the anti-fouling microelectrode in this embodiment is as follows: I. Reagents, materials and instruments used in this embodiment Ferrous chloride tetrahydrate, 2,3,6,7,10,11-hexahydroxytriphenyl, N,N-dimethylformamide (Adams, China), Pseudomonas aeruginosa, phenazine-1-carboxylic acid, phenazine-1-carboxamide (MCE, China), potassium ferricyanide, hexaammineruthenium chloride (Sigma-Aldrich, USA), Tris-HCl buffer (Sangon Biotech, China), LB nutrient agar (Qingdao Haibo, China). Carbon fiber (WPI, USA), borosilicate glass capillary (Sutter Instruments, USA), polydimethylsiloxane (Dow Corning, USA). Scanning electrochemical microscope (CHI 920D, Shanghai Chenhua), vertical needle puller (Narishige, PC-100, Japan).

[0054] II. Preparation of Sensitive and Anti-fouling Microelectrodes 1. Synthesis of Fe-HHTP Using N,N-dimethylformamide (DMF) as solvent, prepare 25 mM HHTP solution and 50 mM FeCl2 solution. Take 20 mL of each and pour them into a glass bottle, mix thoroughly, and sonicate for 5 min. Place the glass bottle containing the mixture on a thermostatic magnetic stirrer and stir at 85 ℃ for 24 h. Figure 1A). After synthesis, the solution was cooled to room temperature for 2 hours, centrifuged to discard the supernatant, and then washed three times each with DMF, anhydrous ethanol, and water, respectively. It was then dried in a vacuum drying oven at 60°C for 12 hours. The dried material was ground in an agate mortar and pestle and passed through a 200-mesh sieve for later use.

[0055] The obtained materials are as follows Figure 2 As shown in A, the particles are two-dimensional lamellar structures with stacked layers. The average particle size is approximately 150 nm, and the three elements C, O, and Fe are uniformly distributed. Figure 2 (BD). The valence state of iron is as follows: Figure 3 As shown, it contains Fe 2+ and Fe 3+ The atomic content of iron in both valence states can be calculated by peak integration, and the atomic content of each valence state is 38.66% (Fe). 2+ ) and 61.34% (Fe 3 + This is an important reason why the material has good conductivity, and it is also the active site for catalyzing the electrochemical redox reaction of phenazine on the electrode surface.

[0056] 2. Fabrication of carbon microelectrodes Take a carbon fiber approximately 1.5 cm long (30 μm or 7 μm in diameter) and place it inside a glass capillary tube (outer diameter 1.2 mm, inner diameter 0.69 mm). Use a copper wire to move the carbon wire to the middle of the tube. Insert a 0.5 mm diameter copper wire into the tube to block one end of the glass tube opening, securing the carbon wire in the middle of the tube to prevent displacement or detachment. Mount the glass tube on a vertical needle drawing apparatus (brand: Narisuge, model: PC-100), with the copper wire end facing down. Perform a two-step drawing process using four counterweights. The heating temperatures for the two steps are 75% and 80% respectively, with the upper and lower slide positions at 4 mm and 5 mm respectively. After drawing, trim the exposed carbon fiber tip to approximately 100 μm under a microscope (adjustable to any length as needed). Then, use a needle grinding instrument to further fine-tune the exposed carbon fiber length and tip flatness. Finally, carbon powder is filled into the tube to one-third to one-half of its volume, and then a copper wire with a diameter of 400 μm is inserted. The open end of the glass tube is sealed with epoxy resin to fix the position of the copper wire.

[0057] 3. Electrodeposition modification of Fe-HHTP-PDA composite substrate ( Figure 1 B) (1) Prepare a 20 mg / mL Fe-HHTP dispersion with ultrapure water, sonicate for 2 h and then use it.

[0058] (2) Prepare a 20 mg / mL dopamine hydrochloride solution using 50 mM Tris-HCl buffer as solvent and adjust the pH to 7.4.

[0059] (3) Mix the solutions prepared in steps (1) and (2) in equal volumes, mix them thoroughly by blowing, and then transfer them to a sealed electrolytic cell. After installing the reference electrode (Ag / AgCl, 3M KCl) and the counter electrode (platinum wire), blow nitrogen gas for 15-20 min, and then install the carbon microelectrode for cyclic voltammetry electrodeposition.

[0060] (4) The electrodeposition parameters are as follows: potential window is 0~0.8V, scanning speed is 200 mV / s, and number of scans is 20.

[0061] (5) After deposition, rinse with ultrapure water, then place the electrode in an oven and dry at 60°C for 2 hours for later use.

[0062] 4. Chemical modification of SBMA antifouling layer ( Figure 1 B) Prepare a 50 mg / mL SBMA solution using 50 mM Tris buffer, adjust the pH to 8.5, and then add Fe-HHTP to achieve a concentration of 0.2 mg / mL. Fix the microelectrode in a glass bottle containing this solution and place it on a thermostatic magnetic stirrer at 55°C for 12 h. After the reaction is complete, remove the electrode, sonicate it with ultrapure water for 2 min, and then dry it under nitrogen for later use.

[0063] A schematic diagram of electrode antifouling detection of phenazine compounds in biofilms is shown below. Figure 1 C.

[0064] 5. Characterization of electrodes SEM images of the electrodes before and after modification, as shown below Figure 4 As shown in AC, compared to the smooth carbon fiber surface, the modified layer exhibits a rough texture and is composed of Fe-HHTP nanoparticles encapsulated in PDA-SBMA. C, O, N, S, and Fe (… Figure 4 DH is uniformly distributed on the electrode surface, with N mainly coming from PDA, S mainly from SBMA, and Fe mainly from Fe-HHTP.

[0065] To understand the antifouling properties of the prepared electrodes, the bare electrodes, FeHHTP / PDA, and FeHHTP / PDA / SBMA were immersed in 1% BSA solution for 1 hour for biofouling treatment. The reduction current of the electrodes in 10 μM PYO before and after the treatment was observed. Figure 5 It is evident that after BSA contamination treatment, the current signals measured by both the bare electrode and the FeHHTP / PDA-modified electrode decreased, while the current signal measured by the FeHHTP / PDA / SBMA-modified electrode did not change significantly, confirming its anti-contamination effect.

[0066] The microelectrode prepared by this invention not only has anti-fouling effects, but also, compared with bare electrodes, increases the peak separation between phenazines when multiple phenazines are measured simultaneously, reducing the difficulty and uncertainty of signal analysis. For example... Figure 6 As shown, in the DPV spectrum (black) obtained by the bare electrode in a mixture containing three phenazine compounds (PYO, PCA, and PCN), the reduction peaks of the three substances are too close, causing peak overlap and broadening. However, in the DPV curve obtained using the electrode prepared according to this invention, the elution position of PCN shifts significantly to the right to -0.45V, appearing as a single peak. Although the peak positions of PYO and PCA are still relatively close, the number of overlapping peaks is reduced to two, greatly reducing the uncertainty of Gaussian peak fitting and enabling more accurate analytical results. Figure 7 The calibration curves of the three phenazine solutions measured by the developed electrode show that the reduction current signals of the three phenazines are all proportional to their concentrations.

[0067] 6. Sample Preparation (1) Preparation of mixed bacterial samples for strain identification Use an inoculation loop to collect the mixed bacterial enrichment broth and streak it onto an LB nutrient agar plate. After 24 hours of incubation, individual colonies can be analyzed.

[0068] (2) Preparation of Pseudomonas aeruginosa biofilm for determining the spatial distribution of phenazine A mixed cellulose filter membrane with a pore size of 0.22 μm was attached to an LB nutrient agar plate. Purified *Pseudomonas aeruginosa* (bred for 24 h) was picked from the agar plate. P. aeruginosa Colonies (approximately 1 mm in diameter) were dispersed in 100 μL of PBS solution. The dispersion was then inoculated onto a filter membrane at a rate of 5 μL per spot. After absorption, the plate was inverted in an incubator and incubated at 37 °C for 24–48 h. This incubation was then used to determine the spatial distribution of phenazine. Alternatively, depending on the research requirements, the drug of interest (such as a certain concentration of antibiotics or disinfectant) could be added to the agar plate. After incubation for a specific time, the spatial distribution of phenazine under specific treatment factors could be determined.

[0069] 7. Identification of bacterial strains and detection of spatial distribution of phenazine in biofilms This detection process uses a Fe-HHTP / PDA / SBMA-modified carbon microelectrode as the working electrode, an AgCl-coated silver wire as the reference electrode, and a 0.5 mm platinum wire as the counter electrode. No solution system is required during the detection process. An agar plate inoculated with bacterial colonies or biofilms is fixed onto the SECM stage. The reference and counter electrodes are inserted into the agar, and the working electrode is fixed to the SECM stepper.

[0070] First, the potential is biased at -0.1 V, and the probe is positioned to the center point of the biofilm surface through a positive feedback approximation current jump. During this process, the working electrode approaches the biofilm surface at a speed of 10 μm / s along the z-axis. The circuit is in an open circuit state before contacting the biofilm, and the background current is 1×10⁻⁶. -11 Level A, set the probe stop current value to 1×10 -9 A. When the probe stops advancing, it just touches the biofilm surface. Then, the probe continues to advance 100 μm along the z-axis to ensure the probe tip is in complete contact with the biofilm. Next, the differential pulse voltammetry (DPV) program is run to measure the reduction current signals of the three phenazines (PYO, PCA, PCN) at the current site. The DPV parameters are as follows: initial potential 0.2 V, final potential -0.7 V, potential increment 0.008 V, amplitude 0.02 V, pulse width 0.02 s, sampling width 0.02 s, and pulse period 0.1 s.

[0071] For mixed bacterial samples requiring strain identification, the probe is positioned at the center of the suspected colony, and DPV is measured. The presence or absence of phenazine in the obtained DPV spectrum can determine whether it is Pseudomonas aeruginosa. Combined with the phenazine spectrum, the strain type of Pseudomonas aeruginosa can be preliminarily determined. Figure 8 The DPV spectra of six bacterial colonies show that *Pseudomonas aeruginosa* typically exhibits at least 2-3 characteristic peaks of phenazines simultaneously: a PCN peak at -0.45V, a PCA peak at -0.32V, a PYO peak at -0.28V, and 5-MCA peaks at -0.1V and 0V. The peak positions of the first three substances are determined by calibration using the corresponding standards. However, 5-MCA currently lacks commercially available standards, and based on literature reports, it is believed to have the aforementioned two peak positions. Specifically, *Pseudomonas aeruginosa* strains PAO1 and PA14 have similar phenazine characteristic peak spectra, both showing peaks of four phenazines. Because the PCA peak partially overlaps with the PYO peak, PCA does not appear as a separate peak but rather raises the curve to the left of the PYO peak. Compared to PAO1, PA14 has a higher PCN peak than the PYO peak, a more pronounced PCA peak elevation, and a higher 5-MCA content. PA is *Pseudomonas aeruginosa* ATCC15442, a non-PYO-secreting strain of *P. aeruginosa* isolated from the natural environment, exhibiting characteristic peaks of three phenazine compounds: PCN, PCA, and 5-MCA. ΔPYO is an *P. aeruginosa* strain with the PhzS gene, which controls PYO secretion, artificially knocked out. This bacterium exhibits PCN and PCA reduction peaks and a weak 5-MCA reduction peak. SA is *Staphylococcus aureus*, and EC is *Escherichia coli*. The former shows a weak reduction peak at -0.26V, and the latter shows a reduction peak at -0.47V; neither of these are characteristic phenazine peaks, and based on the spectra, they can be identified as non-*P. aeruginosa* strains.

[0072] For purified Pseudomonas aeruginosa biofilms that require spatial distribution determination of phenazines, the probe is positioned at each target point on the diameter using the center of the circle as the x-axis point 0 and the radius of the biofilm as the total distance traveled on the x-axis, with 200 μm as the sampling interval. The DPV spectrum of each point is then measured, and the spatial distribution of various phenazines in the biofilm is plotted in combination with the coordinates of the measurement points. Figure 9 This is a spatial distribution map of the levels of four phenazine compounds in the *Pseudomonas aeruginosa* biofilm after 24 hours of culture. The map shows that PYO, PCN, and 5-MCA all exhibited lower secretion levels at the center of the biofilm. As the location moved away from the center, PYO showed a trend of initially increasing rapidly and then gradually decreasing, eventually reaching the same level as the center at the outermost layer of the biofilm. PCA secretion levels showed a trend of high levels at the center and low levels at the outer layers, maintaining a similar level in the 0–600 μm range before gradually decreasing, maintaining a similar low level in the 1200–2600 μm range. PCN initially increased rapidly, maintaining a similar level in the 400–1000 μm range from the center, then gradually decreasing to a level below the center. 5-MCA increased rapidly in the approximately 200–600 μm range from the center, maintaining a similar level from 600 μm to the outermost layer of the biofilm. Since phenazine metabolites are closely related to the metabolic heterogeneity, antibiotic resistance mechanisms, redox balance, and morphological development regulation of *Pseudomonas aeruginosa* biofilms, different spatial distribution patterns exist under different survival pressures (such as the application of antibiotics and disinfectants). This information is of great significance for revealing the biofilm's coping mechanisms under corresponding treatment factors and analyzing its drug resistance evolution. The microelectrode developed in this invention can provide rich spatial measurement information for related research.

[0073] In summary, this invention synthesizes 2D-cMOFFe-HHTP, which specifically catalyzes the electrochemical redox reaction of phenazine compounds. This 2D-cMOFFe-HHTP is used as a conductive bridge in a PDA-SBMA antifouling modification layer, recovering or even enhancing the current signal drop caused by the insulation properties of the PDA-SBMA antifouling layer. This results in the development of an antifouling microelectrode that does not sacrifice sensitivity, suitable for in-situ determination of bacterial biofilms prone to biofouling. The co-electrodeposition method using PDA and Fe-HHTP achieves controllable modification of the microelectrode, overcoming the challenge of stable modification. This microelectrode is used as a working probe for SECM to obtain spatial distribution information of phenazine compounds in bacterial biofilms; the spatial resolution can be adjusted as needed, offering flexibility and convenience.

Claims

1. An anti-fouling microelectrode for Pseudomonas aeruginosa strain identification and detection of its phenazine spatial distribution, characterized in that, The anti-pollution microelectrode comprises a carbon fiber electrode base, a Fe-HHTP-PDA composite substrate layer and a Fe-HHTP-SBMA anti-pollution layer; the Fe-HHTP-PDA composite substrate layer is modified on the surface of the carbon fiber electrode base by an electrodeposition method; the Fe-HHTP-SBMA anti-pollution layer is modified on the surface of the Fe-HHTP-PDA composite substrate layer by a chemical grafting method; the Fe-HHTP-SBMA anti-pollution layer comprises a SBMA anti-pollution layer and Fe-HHTP doped on the SBMA anti-pollution layer; the Fe-HHTP-PDA composite substrate layer comprises a polydopamine substrate and Fe-HHTP doped on the polydopamine substrate; the Fe-HHTP on the Fe-HHTP-PDA composite substrate layer and the Fe-HHTP-SBMA anti-pollution layer forms a conductive path in contact; and the Fe-HHTP is a two-dimensional conductive metal organic framework material containing mixed-valence Fe metal nodes.

2. The anti-fouling microelectrode of claim 1, wherein, The diameter of the carbon fiber electrode base is 7-30 μm, and the length exposed outside the insulating glass layer is 50-150 μm.

3. The anti-fouling microelectrode according to claim 1 or 2, wherein, The Fe-HHTP is obtained by mixing reaction of HHTP and ferrous salt, the ferrous salt is at least one selected from ferrous acetate, ferrous chloride, ferrous sulfate and ferrous nitrate; the atomic ratio of Fe 2+ and Fe 3+ in the mixed valence Fe metal node is 38-40:60-62.

4. A method for preparing the anti-fouling microelectrode according to any one of claims 1 to 3, characterized in that, The anti-pollution microelectrode comprises the following steps: (1) dissolving HHTP and ferrous salt in N,N-dimethylformamide respectively, mixing and ultrasonicating, stirring and reacting, cleaning, drying, grinding, sieving to obtain Fe-HHTP powder; (2) fixing the carbon fiber in a glass capillary, drawing by a two-step vertical needle instrument method, loading carbon powder and inserting a copper wire, and sealing and fixing with epoxy resin to obtain a carbon fiber electrode base; (3) preparing Fe-HHTP dispersion liquid, mixing with an equal amount of dopamine hydrochloride solution, taking the carbon fiber electrode base as a working electrode, and forming a Fe-HHTP-PDA composite substrate layer on the surface of the carbon fiber electrode base by cyclic voltammetry electrodeposition under nitrogen protection; (4) grafting the carbon fiber electrode modified with the Fe-HHTP-PDA composite substrate layer in a SBMA solution containing Fe-HHTP, ultrasonic cleaning, nitrogen drying to form a Fe-HHTP-SBMA anti-pollution layer on the surface of the Fe-HHTP-PDA composite substrate layer, thereby obtaining the anti-pollution microelectrode.

5. The preparation method according to claim 4, characterized in that, In step (1), the stirring and reaction temperature is 80-90 ℃, and the time is 12-24 h; the cleaning is performed with N,N-dimethylformamide, anhydrous ethanol and water in sequence, and the drying is performed at 50-80 ℃ under vacuum for 12-24 h; and the sieving is performed through a 200-1000 mesh sieve; In step (2), the diameter of the carbon fiber is 7-30 μm, and the two-step heating temperature levels of the two-step vertical needle instrument method are 70-80% and 75-85% in sequence; and the upper and lower slide rod positions are 3-5 mm and 4-6 mm, respectively.

6. The production method according to claim 4, characterized by, In step (3), the concentration of the Fe-HHTP dispersion solution is 2-20 mg / mL, and the ultrasonic dispersion is performed for 1-2 h; the dopamine hydrochloride solution has a concentration of 2-20 mg / mL and is dissolved in 50 mM Tris-HCl buffer, and the pH value is adjusted to 7.2-7.6; the reference electrode is an Ag / AgCl electrode containing 3 M KCl, and the counter electrode is a platinum wire; the potential window of the cyclic voltammetry electrodeposition is 0-0.8 V, the scanning speed is 50-200 mV / s, and the scanning number is 15-30; after the deposition is completed, the carbon fiber electrode is placed in a 50-60℃ oven for drying for 1-2 h, thereby obtaining a carbon fiber electrode modified with a Fe-HHTP-PDA composite base layer.

7. The production method according to any one of claims 4 to 6, characterized by, In step (4), the SBMA solution containing Fe-HHTP has a concentration of 30-150 mg / mL of SBMA and 0.05-0.5 mg / mL of Fe-HHTP, and the concentration ratio of Fe-HHTP to SBMA is 1:100-1000; the grafting reaction is performed at 35-60℃ for 12-24 h under stirring.

8. Use of the anti-fouling microelectrode according to any one of claims 1 to 3 or of the anti-fouling microelectrode obtained by the method according to any one of claims 4 to 7 for detecting phenazines and their spatial distribution in bacterial biofilms, characterized in that, The method for the application comprises the following steps: taking the anti-fouling microelectrode according to any one of claims 1-3 or obtained by the preparation method according to any one of claims 4-7 as a working electrode, taking a silver wire coated with AgCl as a quasi-reference electrode, and taking a platinum wire as a counter electrode; positioning the working electrode to multiple to-be-detected sites on the surface of a bacterial biofilm to be detected by using a scanning electrochemical microscope; detecting each to-be-detected site by using a differential pulse voltammetry method to obtain a voltammogram containing the redox current signals of multiple phenazine substances; and quantifying the contents of the multiple phenazine substances according to the voltammogram and combining the spatial coordinates of each site to obtain spatial distribution information of the multiple phenazine substances in the bacterial biofilm.

9. Use according to claim 8, characterized in that, The parameters of the differential pulse voltammetry method are as follows: an initial potential of 0-0.2 V, a final potential of -0.6 to -0.8 V, a potential increment of 0.005-0.02 V, an amplitude of 0.01-0.05 V, a pulse width of 0.01-0.04 s, a sampling width of 0.01-0.1 s, and a pulse period of 0.05-0.2 s. The parameters of the scanning electrochemical microscope positioning are: potential bias -0.1~0.1V, z-axis approaching speed 5~20μm / s, stop current value 1×10 -10 ~1×10 -9 A, continue to advance 50~150μm after approaching to the surface of the biofilm. The bacteria are Pseudomonas aeruginosa (ATCC® 10145™) P. aeruginosa and the phenoxazine includes at least one of pyocyanine, phenoxazine-1-carboxylic acid, phenoxazine-1-carboxyamide, and 5-methylphenoxazine-1-carboxylic acid.

10. An anti-fouling microelectrode according to any one of claims 1 to 3, or an anti-fouling microelectrode obtained by any one of claims 4 to 7, for the identification of Pseudomonas aeruginosa (… P. aeruginosa The application of ) or / and its strain type, characterized in that, The method for the application comprises the following steps: taking the anti-fouling microelectrode according to any one of claims 1-3 or obtained by the preparation method according to any one of claims 4-7 as a working electrode, taking a silver wire coated with AgCl as a quasi-reference electrode, and taking a platinum wire as a counter electrode; positioning the working electrode to multiple to-be-detected sites on the surface of a bacterial biofilm to be detected by using a scanning electrochemical microscope; detecting each to-be-detected site by using a differential pulse voltammetry method to obtain a voltammogram containing the redox current signals of multiple phenazine substances; and quantifying the contents of the multiple phenazine substances according to the voltammogram and combining the spatial coordinates of each site to obtain spatial distribution information of the multiple phenazine substances in the bacterial biofilm. The parameters of the differential pulse voltammetry method are as follows: an initial potential of 0-0.2 V, a final potential of -0.6 to -0.8 V, a potential increment of 0.005-0.02 V, an amplitude of 0.01-0.05 V, a pulse width of 0.01-0.04 s, a sampling width of 0.01-0.1 s, and a pulse period of 0.05-0.2 s.

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