Graphene electrode for detecting food-borne pathogenic bacteria, sensor and application

Abstract

The invention provides a graphene electrode for detecting food-borne pathogenic bacteria, a sensor and application, and belongs to the technical field of analysis of electrochemical sensing. A defect-rich graphene conductive layer is created on the surface of a porous polymer membrane through laser irradiation, then carboxyl-rich magnetic nanoparticles are modified into recognition elements (immunomagnetic beads and IMBs) for capturing and enriching target bacteria in a sample, uncombined free IMBs are separated through nano-filtration of a membrane chip, and the target bacteria in the sample are detected. The larger IMBs-bacterial compound is retained, and the IMBs-bacterial compound retained on the LIG layer confinement structure generates impedance signal change in the micro-electrochemical cell, so that bacterial detection is realized. Meanwhile, the PES-LIG electrode provided by the invention can highly sensitively identify trace target bacteria in a complex matrix.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical electrochemical sensing technology, and in particular to a graphene electrode for detecting foodborne pathogenic bacteria, a sensor and application thereof. BACKGROUND

[0002] Foodborne pathogenic bacteria are considered to be the main cause of a large number of human and animal deaths worldwide. Rapid, specific and highly sensitive on-site detection of these microorganisms has become an urgent need in the fields of public health protection, medical diagnosis and food safety. In order to avoid bacterial hazards, sensitive and rapid-response sensors for detecting trace bacteria are essential. Although existing bacterial detection methods are widely recognized and reliable, they usually require pre-enrichment, separation and culture steps, making them time-consuming, labor-intensive, expensive and dependent on centralized laboratories, limiting their feasibility in rapid and on-site applications. Therefore, the focus of research has shifted to the development of rapid, economical, portable and user-friendly clinical on-site detection devices, eliminating the need for professional training.

[0003] Existing rapid detection methods, such as enzyme-linked immunosorbent assay (ELISA) and real-time fluorescent PCR, although shorten the detection time, still require complex separation and enrichment steps, such as bacterial enrichment or nucleic acid extraction, and rely on expensive equipment, such as enzyme labeling instruments or PCR machines, limiting their application. Therefore, there is an urgent need to develop simple, rapid, efficient and portable microbial detection technology and equipment.

[0004] Laser-assisted processing techniques have become widely used effective tools, including material manufacturing and surgical pathology. In this context, laser-induced graphene (LIG) as a graphene-containing material provides significant advantages, which can be made entirely from aromatic polymers (such as polysulfone and polyimide derivatives), and is commonly used to prepare graphene-based composite electrodes. Magnetic nanoparticles (MNPs) have excellent superparamagnetism, high specific surface area, easy functionalization and excellent biocompatibility, making them have great potential in the rapid separation, purification and enrichment of pathogenic bacteria.

[0005] To improve the detection selectivity of biosensors for target bacteria in complex samples, specific capture probes are applied as bacterial-specific capture units. Antibody-antigen interaction, aptamer binding, phage bio-recognition, antibiotic / antimicrobial peptide capture and other target recognition interactions are often applied to biosensors before bacterial detection. In most biosensors, the antibody-antigen sandwich method is widely used to capture intact bacterial cells by binding to external receptors of bacteria, without the need for cell lysis or release of enzyme labels for subsequent signal transduction and amplification. Functionalized magnetic nanoparticles are combined with target-specific bacterial antibodies to form immunomagnetic beads (IMBs), which are commonly used for the capture of bacterial cells.

[0006] Confinement refers to the containment of molecules in a specific geometric space, significantly improving their physicochemical properties, with significant advantages compared to bulk state. Nanostructures with active nanomaterials, located in nanoscale space, exhibit unique properties such as new crystal forms, enhanced stability, increased surface area, and improved catalytic activity. In traditional electrochemical detection systems, electrodes usually work in larger volume electrolytic cells, resulting in poor portability, high background signal, and signal molecules diffusing from the sensor surface to the larger electrolytic cell, which can easily cause signal loss.

[0007] Therefore, the development of a miniaturized LIG-based confined auxiliary sensing platform to reduce the volume of the electrolytic cell has great potential for effective signal amplification and simplified, efficient operation. SUMMARY

[0008] The purpose of the present application is to provide a graphene electrode, sensor and application for detecting foodborne pathogenic bacteria. The present application creates a defect-rich graphene conductive layer on the surface of a porous polymer membrane by laser irradiation, and then uses carboxyl-rich magnetic nanoparticles to modify the recognition element (immunomagnetic beads, IMBs) for capturing and enriching target bacteria in the sample. Unbound free IMBs (smaller in size) are separated by "nanofiltration" of the membrane chip, while larger IMBs-bacteria complexes are retained. The IMBs-bacteria complexes retained on the LIG layer confined structure produce impedance signal changes in the microelectrochemical cell, thereby achieving bacterial detection.

[0009] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a PES-LIG electrode for detecting foodborne pathogenic bacteria, comprising a PES-LIG membrane of a specified shape, which is divided into a sensing area, a hydrophobic closed area and a wire circuit connection area. The sensing area and the wire connection area are located at both ends of the PES-LIG membrane, respectively. The hydrophobic closed area is located at the connection between the sensing area and the wire connection area. The preparation method of the PES-LIG membrane is: preparing a graphene layer on the surface of a PES membrane by laser induction to obtain the PES-LIG membrane.

[0010] Preferably, the hydrophobic closed area is coated with shadowless glue, and the wire connection area is coated with conductive silver paste.

[0011] Preferably, the power of the laser induction is 8-12%, and the speed of the laser irradiation is 25-30 cm / s.

[0012] Preferably, the PES-LIG electrode is used in pairs.

[0013] Further preferably, when the two PES-LIG electrodes are used in pairs, one is used as a working electrode and the other is used as a reference electrode.

[0014] The application also provides a composite electrochemical biosensor for detecting foodborne pathogenic bacteria, comprising two PES-LIG electrodes, and using an insulating tape to encapsulate the two PES-LIG electrodes so that the distance between the two PES-LIG electrodes is constant and set, and at the same time, the sensing area and the wire connection area are kept exposed.

[0015] Preferably, the insulating tape is pre-provided with a through hole with a diameter of 1.5-2 mm; and the set distance is 2-2.5 mm.

[0016] The application also provides a method for detecting foodborne pathogenic bacteria, comprising the following steps: (1) adding magnetic particles to a solution to be tested containing foodborne pathogenic bacteria to perform immunomagnetic bead separation, and obtaining an enriched sample to be tested; (2) adding the enriched sample to be tested obtained in step (1) to the sensing area of the composite electrochemical biosensor, filtering the sample to be tested under pressure, and obtaining the concentration of foodborne pathogenic bacteria in the solution to be tested according to the electrochemical signal.

[0017] Preferably, the foodborne pathogenic bacteria in step (1) include Salmonella; and the time for immunomagnetic bead separation is 6-10 min.

[0018] Preferably, the pressure in step (2) is 0.4-0.6 bar.

[0019] Compared with the prior art, the application has the following beneficial effects: The application provides a graphene electrode, a sensor and an application for detecting foodborne pathogenic bacteria, the PES-LIG electrode combines the restriction effect between the LIG interlaced layers and the nanofiltration in the sensing platform, and can be used for efficient separation and sensitive detection of bacteria. The method selectively separates and enriches target bacteria from a complex matrix by using the combination of functionalized magnetic beads and recognition elements; by integrating the high separation capacity of IMBs, the signal enhancement effect of the basic electrochemical cell of the micro-LIG sensor, and the high sensitivity of electrochemical sensing, high-sensitivity detection of bacteria is realized without additional modification.

[0020] The present application creates a defect-rich graphene conductive layer on the surface of a porous polymer membrane by laser irradiation, thereby manufacturing an integrated laser-induced graphene (LIG) composite electrochemical biosensor, and adding an electrolyte to the electrode surface to develop a two-electrode system electrochemical platform based on a micro electrochemical cell. The present application uses carboxyl-rich magnetic nanoparticles, which are modified as recognition elements (immunomagnetic beads, IMBs) for capturing and enriching target bacteria in a sample. Unbound free IMBs (smaller in size) are separated by "nanofiltration" of the membrane chip, while larger IMBs-bacteria complexes are retained. The IMBs-bacteria complexes retained on the LIG layer confined structure generate impedance signal changes in the micro electrochemical cell, thereby achieving bacterial detection.

[0021] The PES-LIG electrode provided by the present application combines the following functions: ① selective separation and enrichment of target molecules using recognition element labeled magnetic microbeads; ② strong electron transfer kinetics achieved by laser-induced interwoven three-dimensional conductive graphene structure; ③ nanofiltration through a porous polymer membrane; ④ confined signal amplification strategy; and ⑤ two-electrode sensing system based on a micro electrochemical cell. The PES-LIG electrode realizes seamless integration of separation-enrichment, signal generation / amplification, and high-sensitivity signal acquisition, and has the advantages of high operation efficiency, strong portability, and wide applicability.

[0022] The present application combines a laser-induced three-dimensional graphene-based sensor with nanofiltration to manufacture a micro PES-LIG electrode, and develops a two-electrode system and a micro electrochemical cell. Functionalized magnetic beads with recognition molecules are used to separate and enrich target bacteria (such as Salmonella) from complex matrices. Free magnetic beads are rapidly separated through the porous electrode chip by "nanofiltration", while magnetic bead-bacteria complexes are retained. Subsequently, a micro electrochemical cell and a double LIG electrode sensing platform are integrated to achieve simple and high-sensitivity bacterial detection.

[0023] The present application combines the bacterial pretreatment and detection modules into a single platform, achieving rapid separation and high-sensitivity detection of bacteria. In addition, the simple electrode manufacturing process supports mass production. This innovation not only provides a new method for integrated separation-detection systems, but also promotes research progress in nanorestricted sensing technology and microelectrode design.

[0024] The present application uses functionalized magnetic beads (IMBs) to rapidly separate and enrich target bacteria in complex matrices within 6-10 minutes through the recognition molecules on the magnetic beads. The process is simple, the magnetic beads have enhanced functionality, and high-sensitivity detection can be achieved without additional electrode surface modification. Moreover, the present application has high diversity: by changing the recognition elements (such as antibodies, aptamers, or DNA), various target analytes (larger than the membrane pore size) can be detected, and has wide application prospects.

[0025] This invention combines immunomagnetic beads with membrane-based nanofiltration, enabling rapid separation of interfering substances and efficient enrichment of target molecules. A LIG-based micro-electrochemical cell provides spatially confined signal amplification of the target molecules. By employing a micro-laser-induced graphene composite electrochemical biosensor, separation, signal amplification, and sensing modules are integrated into a single system. This design allows for seamless bacterial isolation and detection through a simple and efficient process. The device is portable, low-cost, and easy to manufacture, while avoiding time-consuming processing steps and eliminating the need for expensive instruments.

[0026] This invention utilizes the signal enhancement effect within the interwoven structure of laser-induced graphene, combined with a micro-electrolysis cell, to significantly improve the sensitivity of bacterial detection. This enables highly sensitive identification of trace target bacteria in complex matrices and can be integrated with a portable electrochemical workstation, demonstrating great potential for rapid on-site bacterial residue analysis and promising practical applications. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a flowchart illustrating the construction process of a PES-LIG electrode. Figure 2 A schematic diagram of a composite electrochemical biosensor constructed from PES-LIG electrodes for detecting Salmonella; Figure 3 The response results of the dual-electrode system before and after bacterial filtration; Figure 4 The results represent the morphological characterization, among which, Figure 4 In this context, A represents the laser processing interface. Figure 4 B in the image is a SEM image of the carbon layer of the PES-LIG electrode film. Figure 4 C in the figure represents the result of Raman spectroscopy analysis. Figure 4 In this context, D represents the XRD analysis result; Figure 5 The results are the electrochemical performance analysis results, among which, Figure 5 In the diagram, A represents the structural schematic of the PES-LIG electrode. Figure 5 In the figure, B represents the conductivity result between the two electrodes measured using an AVO digital multimeter. Figure 5 In this context, C represents the repeatability measurement result. Figure 5In the figure, D represents the stability test result of the PES-LIG electrode in K3Fe(CN)6 / K4Fe(CN)6 solution. Figure 5 E in the figure represents the CV characterization results of the PES-LIG electrode at different scan rates. Figure 5 F in the figure represents the linear calibration curves of the oxidation peak current and reduction peak current of the electrode as a function of the scan rate. Figure 6 The results are the determination results of electrochemical characterization, among which, Figure 6 In the diagram, A represents the dual-electrode detection mode of the PES-LIG electrode. Figure 6 In the figure, B represents the CV characterization results at different time points in the K3Fe(CN)6 / K4Fe(CN)6 solution. Figure 6 C in the figure represents the EIS curves at different time points in the K3Fe(CN)6 / K4Fe(CN)6 solution. Figure 6 In the image, D represents the SEM image of the PES-LIG electrode surface after filtering IMBs. Figure 6 E in the figure represents the concentration of 1×10⁻⁶. 2 LIG results after capturing Salmonella (IMBs-St) with CFU / mL immunomagnetic beads. Figure 6 In this context, F represents the EIS detection result of the three-electrode system. Figure 6 In the figure, G represents the CV voltammogram detected by EIS in a three-electrode system. Figure 6 H in the figure represents the filtered two-electrode system; Figure 7 The results are from electrochemical impedance spectroscopy (EIS) detection, where... Figure 7 In the figure, A represents the EIS detection results of the PES-LIG electrode after filtering 5 and 10 µg / mL AuNPs. Figure 7 In this context, B represents the corresponding resistance change obtained after filtering with AuNPs. Figure 7 In the figure, C represents the EIS response of the PES-LIG electrode after filtering 0.001, 0.01, and 0.1 mg / mL BSA. Figure 7 In this context, D represents the corresponding resistance change after filtration through different concentrations of BSA. Figure 7 In the figure, E represents the EIS detection result of the PES-LIG electrode after filtering Salmonella nucleic acid (St.(NA)). Figure 7 In this context, F represents the corresponding resistance change after filtering by IMBs-St; Figure 8 The results are the performance test results, among which, Figure 8 In the figure, A represents the EIS response curve of the PES-LIG electrode in a three-electrode system to different concentrations of IMBs and Salmonella. Figure 8 In the figure, B represents the calibration curve, and the inset shows the equivalent circuit. Figure 8In the figure, C represents the EIS response curve of the PES-LIG electrode to different bacteria. Figure 8 In this context, D represents the charge transfer resistance difference (ΔRct) obtained for different bacteria. Figure 8 In the figure, E represents a comparison of the sensor's response to 50nm and 500nm magnetic beads before and after bacterial capture. Figure 8 F in the figure represents the EIS response calibration curve of the PES-LIG electrode to Salmonella in a two-electrode system. Figure 9 The results show the EIS response of the PES-LIG electrode to different concentrations of Salmonella in a composite electrochemical biosensor. Figure 9 In the figure, A represents the EIS test result without the addition of Salmonella. Figure 9 In the figure, B represents Salmonella at a concentration of 1×10⁻⁶. 2 EIS test results at CFU / mL Figure 9 In the figure, C represents Salmonella at a concentration of 1×10⁻⁶. 3 EIS test results at CFU / mL Figure 9 In this context, D represents Salmonella at a concentration of 1×10⁻⁶. 4 EIS test results at CFU / mL Figure 9 In the figure, E represents Salmonella at a concentration of 1×10⁻⁶. 5 EIS test results at CFU / mL Figure 9 F in the figure represents Salmonella at a concentration of 1×10⁻⁶. 6 EIS test results at CFU / mL. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] The present invention provides a PES-LIG electrode for detecting foodborne pathogens, comprising a PES-LIG membrane of a predetermined shape, wherein the PES-LIG membrane is divided into a sensing region, a hydrophobic sealing region, and a wire circuit connection region. The sensing area and the wire connection area are located at both ends of the PES-LIG membrane, respectively. The hydrophobic sealing region is located at the junction of the sensing region and the wire connection region; The PES-LIG membrane is prepared by laser-induced preparation of a graphene layer on the surface of a PES membrane to obtain the PES-LIG membrane.

[0035] In this invention, the hydrophobic sealing region is coated with UV-curing adhesive, the thickness of which is preferably 0.2-0.5 mm, more preferably 0.3-0.4 mm, and the volume of which is preferably 8-10 µl, more preferably 9 µl. After coating with the UV-curing adhesive, it is preferred to use ultraviolet light for curing. The curing temperature is preferably 50-60°C, more preferably 54-58°C, and even more preferably 55°C. The curing time is preferably 8-12 s, more preferably 10 s. The UV light intensity during curing is preferably 25-35 mW / cm². 2 Further preferred is 28~32mW / cm 2 A further preferred value is 30mW / cm 2The wire connection area is coated with conductive silver paste; the thickness of the conductive silver paste coating is preferably 0.1~0.3mm, more preferably 0.2mm; the volume of the conductive silver paste coating is preferably 10~15µl, more preferably 12~14µl, and even more preferably 13µl; the laser-induced power is preferably 8~12%, more preferably 9~11%, and even more preferably 10%; the laser irradiation speed is preferably 25~30cm / s, more preferably 26~28cm / s, and even more preferably 27cm / s; the PES-LIG electrodes are used in pairs; when two PES-LIG electrodes are used in pairs, one is used as the positive electrode and the other as the negative electrode. The conductive silver paste ensures mechanical stability and reliable electrical connection with the measurement system.

[0036] The present invention also provides a composite electrochemical biosensor for detecting foodborne pathogens, comprising two PES-LIG electrodes, wherein the two PES-LIG electrodes are encapsulated with insulating tape, such that the distance between the two PES-LIG electrodes is constant at a set distance, while keeping the sensing area and the wire connection area exposed.

[0037] In this invention, the insulating tape preferably has a pre-formed through hole with a diameter of 1.5~2mm; the set distance is preferably 2~2.5mm, more preferably 2.2~2.4mm, and even more preferably 2.3mm.

[0038] The present invention also provides a method for detecting foodborne pathogens, comprising the following steps: (1) Magnetic microparticles are added to the test solution containing foodborne pathogens for immunomagnetic bead separation to obtain enriched test samples; (2) The enriched sample obtained in step (1) is added to the sensing area of ​​the composite electrochemical biosensor, the sample is filtered under pressure, and the concentration of foodborne pathogens in the solution is obtained according to the electrochemical signal.

[0039] In this invention, the foodborne pathogens mentioned in step (1) include Salmonella; the separation time of the immunomagnetic beads is preferably 6 to 10 minutes, more preferably 8 minutes; the pressure applied in step (2) is preferably 0.4 to 0.6 bar, more preferably 0.5 bar, and a small vacuum pump is preferably used to apply negative pressure when applying pressure.

[0040] Example 1

[0041] A polyethersulfone film was irradiated with a laser at 10% power and a speed of 27 cm / s to obtain a PES-LIG film containing a graphene conductive layer. The PES-LIG film containing the graphene conductive layer was then cut into uniform shapes using a blade. Conductive silver paste was applied to the wire connection area. UV adhesive (shadowless adhesive) was applied to the hydrophobic sealing area and cured using ultraviolet light to obtain a PES-LIG electrode (e.g., ...). Figure 1 (As shown). The enclosed sensing region and the wire connection region are always exposed; and the enclosed sensing region and the wire connection region are located at opposite ends of the PES-LIG electrode, with the hydrophobic enclosed region located at the junction of the enclosed sensing region and the wire connection region.

[0042] Two PES-LIG electrodes were encapsulated using insulating tape with a 1.5 mm diameter through-hole, and the spacing between the PES-LIG electrodes was set to 2 mm to obtain a composite electrochemical biosensor for detecting foodborne pathogens.

[0043] In this composite electrochemical biosensor, one PES-LIG electrode serves as the working electrode, exposed to the test solution for filtration and sensing, while another PES-LIG electrode simultaneously serves as both a reference and auxiliary electrode. This structure creates a miniature electrochemical cell capable of achieving highly sensitive and accurate detection of foodborne pathogens using extremely small amounts of detection solution (approximately 10 µl). Figure 6 A).

[0044] Example 2

[0045] A method for detecting foodborne pathogens, comprising the following steps: (1) Immobilize Salmonella-specific antibodies with amino functional groups onto carboxyl-rich magnetic beads to prepare immunomagnetic beads (IMBs). Then, add the IMBs to a sample solution containing Salmonella, incubate to achieve specific binding (form IMBs-St), and then separate the complex by an external magnetic field to obtain the enriched test sample; (2) The enriched sample obtained in step (1) is dropped into the closed sensing area of ​​the composite electrochemical biosensor prepared in Example 1, pressure is applied, and the sample is washed with PBST to remove unbound IMBs (magnetic particles). The concentration of foodborne pathogens in the test solution is obtained based on the electrochemical signal.

[0046] Experimental Example 1: Optimization of Laser Parameters The key parameters affecting the conductivity of the LIG layer are laser power, laser scanning speed, and pulses per inch (PPI), with optimized values ​​of 10%, 27 cm / s, and 1000, respectively.

[0047] This invention optimizes surface laser parameters to form a uniform graphene layer on the surface of a PES membrane. Through cutting and the introduction of hydrophobic sealing regions and wire connection regions, an integrated PES-LIG electrode is constructed. Two separate PES-LIG electrodes are encapsulated to form a dual-electrode system, which, combined with the membrane filtration and screening function of IMBs, achieves a sensitive and rapid response to Salmonella. Benefiting from the controllability of the laser processing and the large specific surface area of ​​graphene, the prepared composite electrochemical biosensor exhibits excellent stability, repeatability, and good electrochemical activity during performance characterization and detection, which is beneficial for improving the specific and sensitive response capability to Salmonella.

[0048] Laser power and probe movement speed significantly affect graphene formation. Excessive power or inappropriate speed can lead to film damage or graphene inhomogeneity. Ultimately, 10% power, 27 cm / s speed, and 1000 pulses per inch (PPI) were selected to fabricate the PES-LIG electrode.

[0049] Experimental Example 2: Morphological Characterization The morphology of the PES-LIG film was characterized using scanning electron microscopy (SEM). Three-dimensional graphene networks were observed at different magnifications in the acquired images. The formation of graphene was further confirmed by Raman spectroscopy and X-ray diffraction (XRD) analysis. Results are as follows... Figure 4 As shown.

[0050] The results showed that the LIG-treated film surface exhibited clear boundary features, indicating that laser processing has precise positional control capabilities. At high magnification, a three-dimensional porous graphene structure was observed on the PES-LIG film surface, significantly increasing the specific surface area of ​​the electrode.

[0051] The Raman spectrum of the PES-LIG film showed three characteristic peaks: the D peak was located at approximately 1331 cm⁻¹. –1 This reflects the defects and tortuosity of the membrane during LIG preparation; the G peak is located at approximately 1597 cm⁻¹. –1 This confirmed the presence of a graphitized structure on the membrane surface; the 2D peak is located at approximately 2664 cm⁻¹. –1 This indicates the formation of multilayer graphene. Meanwhile, the PES-LIG film exhibits a lower ID / IG value and a weaker 2D peak, which is because the graphene on the PES-LIG film is induced by a lower-power laser.

[0052] XRD diffraction patterns of the PES-LIG film show a broad peak at approximately 18.5°, generated by the PES substrate; a strong peak at approximately 31.6°, corresponding to the interlayer spacing between (002) planes; and a smaller peak at approximately 42.3°, related to (100) reflection in the high-graphite material. However, the peak positions of the PES-LIG film are slightly shifted compared to those in the literature, which may be due to the influence of the film substrate and the doping of oxygen and nitrogen atoms.

[0053] Experimental Example 3: Electrochemical Performance Analysis To prevent damage to the graphene layer on the PES-LIG membrane surface during electrochemical detection, conductive silver paste was used as a protective coating at the electrode connection sites. Simultaneously, the filter membrane was hydrophobically treated to limit solution diffusion and create an independent detection area. Finally, the two treated single electrodes were encapsulated and combined to construct a planar dual-electrode system without direct electrical connections, ensuring accurate subsequent detection. Actual detection conditions were simulated by repeatedly filtering PBST buffer, and the effect of scan rate on electrode performance was investigated. Cyclic voltammetry (CV) was used, and CV curves were plotted after each filtration step using PBST (containing 0.1% Tween 20 phosphate-buffered saline) buffer. After three repeated cycles, all curves completely overlapped. The results are as follows: Figure 5 As shown.

[0054] Tests show that the filtration process has no impact on the sensor response. The PES-LIG electrode maintains good stability after multiple filtrations, with an Rct coefficient of variation of only 1.7%, verifying its reliability in practical applications. Meanwhile, the peak current increases significantly with increasing scan rate, and the absolute values ​​of both the oxidation and reduction peak currents show a good linear relationship with the square root of the scan rate (Rct). 2 The characteristic of >0.99 indicates that the electrochemical reactions occurring on the PES-LIG electrode surface are typical diffusion-controlled processes. Calculations show that the electrochemically active surface area of ​​the PES-LIG electrode is 1.4 times its actual surface area, indicating high roughness and specific surface area, which in turn improves the electron transfer rate, confirming its excellent electrochemical performance.

[0055] Experimental Example 4: Electrochemical Characterization 4.1 To verify the electrochemical performance of the PES-LIG electrode, cyclic voltammetry (CV) was first used to characterize the electrochemical activity and fabrication stability of a three-electrode system comprising an Ag / AgCl reference electrode, a platinum auxiliary electrode, and a PES-LIG working electrode. The results are as follows: Figure 6 B and Figure 6 As shown in C.

[0056] The results showed that Fe(CN)6 3- / Fe(CN)6 4- The ratio of the anodic oxidation peak current (approximately 9.82 ± 0.21 μA) to the cathodic reduction peak current (approximately 9.17 ± 0.15 μA) is close to 1, indicating that it promotes the reversible redox reaction of the electroactive probe and meets the basic requirements for electrochemical detection.

[0057] 4.2 Under the same test conditions as in 4.1, CV and EIS scans were performed on a single electrode.

[0058] The results show that the peak current variation coefficient is 0.56%. This result demonstrates the good analytical stability of the PES-LIG electrode, providing a reliable guarantee for its electrochemical sensing applications.

[0059] 4.3 To verify the specific response of the PES-LIG electrode to Salmonella (St.), SEM was used to characterize the electrode surface after treatment with negative samples (containing only free IMBs) and positive samples. The results are as follows: Figure 6 D to Figure 6 As shown in G.

[0060] After filtering the negative sample, only a small amount of IMBs remained on the PES-LIG electrode surface. This is likely due to the adsorption and interception effect of the irregular graphene layer on the PES-LIG electrode surface on IMBs. The PES-LIG electrode surface not only successfully retained the IMBs-St complex but also observed obvious IMBs aggregates. This may be because the presence of bacteria reduced the dispersibility of IMBs, making them more prone to aggregation. These results indicate that a simple membrane filtration process can, to some extent, distinguish between negative and positive samples.

[0061] 4.4 The electrochemical response of the PES-LIG electrode in two-electrode and three-electrode detection systems was evaluated using EIS and CV techniques. The results are as follows: Figure 6 As shown in H in the diagram.

[0062] The results showed that the IMBs-St complex significantly increased the Rct value. The response was most stable at 10,010 Hz, making it suitable as the detection frequency. Furthermore, due to the high specific surface area and adsorption capacity of the graphene layer, its specific response mechanism requires further investigation.

[0063] Experimental Example 5: Electrochemical Impedance Spectroscopy (EIS) Detection 5.1 Since both IMBs and the IMBs-St complex are insulating materials, and more of the complex covers the electrode surface after filtering IMBs-St, the response caused by the insulating particles covering the electrode surface is considered first. To verify this hypothesis, filtration experiments were conducted using conductive AuNPs to compare the effect of different amounts of conductive particles on the change in electrode impedance. A new aqueous solution of gold nanoparticles was prepared. Specifically, gold nanoparticles (purchased from Aladdin, 10 nm in diameter) were dispersed in 0.1 mM phosphate-buffered saline (PBS) to obtain aqueous solutions with concentrations of 5 µg and 10 µg, respectively. The Rct values ​​were recorded using electrochemical impedance spectroscopy. The results are as follows: Figure 7 A and Figure 7 As shown in B in the diagram.

[0064] The results showed that the electrode impedance decreased significantly with increasing amount of filtered AuNPs, because AuNPs promoted charge transfer in the electrode. Therefore, the electrochemical response of the PES-LIG electrode is partly due to the influence of electrode surface particles on the electron transfer process. Different amounts of insulating particles (such as different amounts of retention after filtering different concentrations of IMBs-St) will hinder charge transfer to varying degrees, leading to an increase in impedance.

[0065] 5.2 To investigate the effect of biomolecules on the electrode response, the surface of the PES-LIG electrode was modified with BSA. Specifically, BSA protein solutions with concentrations of 0.001, 0.01, and 0.1 mg / mL were prepared. After filtering each sample, the corresponding charge transfer resistance (Rct) value was measured by electrochemical impedance spectroscopy. The results are as follows: Figure 7 C and Figure 7 As shown in D in the diagram.

[0066] The results showed that the Rct value increased significantly with the increase of BSA, indicating that the PES-LIG electrode has a significant response to biomolecules.

[0067] 5.3 The sensor responses of the bare electrode, Salmonella (St.) filter electrode, IMBs-St complex, and sensors with equal concentrations of nucleic acid were compared and analyzed using EIS. The results are as follows: Figure 7 E and Figure 7 As shown in F in the diagram.

[0068] When the PES-LIG electrode was used for the detection of unenriched Salmonella and its nucleic acids, an electrochemical response was generated, but the signal was significantly lower than that of the IMBs-St complex enriched by immunomagnetic separation. This difference is mainly due to the larger volume and molecular weight of the enriched complex, resulting in greater electrode retention and enhanced response, further validating the effectiveness of immunomagnetic separation in improving detection sensitivity.

[0069] Experimental Example 6: Performance Testing of Composite Electrochemical Biosensors 6.1 A three-electrode system consisting of a PES-LIG working electrode, an Ag / AgCl reference electrode, a platinum auxiliary electrode, and a 5 mL electrochemical cell was prepared. Electrochemical impedance spectroscopy (EIS) analysis was performed using the three-electrode system, and the results were recorded for the blank electrode and after passing through different concentrations of bacteria (range 1 × 10⁻⁶). 2 Up to 1×10 6 CFUmL -1 The electrochemical response of the filtered electrode was studied, and the linear concentration range for Salmonella detection was determined based on the obtained calibration curve. Results are as follows: Figure 8 A and Figure 8 As shown in B in the diagram.

[0070] The results showed that the increasing trend of Rct indicates an increase in the accumulation of the IMBs-St complex on the electrode surface. According to the calibration curves in the figure, the linear detection range (LDR) of the composite electrochemical biosensor for Salmonella is 1 × 10⁻⁶. 2 Up to 1×10 6 CFU / mL (R) 2 =0.995), and the limit of detection (LOD) was calculated to be 12 CFU / mL (S / N=3).

[0071] 6.2 To evaluate the specificity of the PES-LIG electrode-based composite electrochemical biosensor, three common foodborne bacteria—Escherichia coli (E. coli), Listeria monocytogenes (L. monocytogenes, Lm.), and Vibrio parahaemolyticus (Vp.)—were tested under the same conditions at a concentration of 1×10⁻⁶. 3 CFU / mL. The specific method was as follows: 5 µL of Salmonella-specific antibody-conjugated magnetic beads (IMBs-St) was added to each 500 µL sample solution containing bacteria, accounting for 1% of the total sample solution volume. Each sample solution was enriched using immunomagnetic separation and filtered through the porous membrane of a PES-LIG electrode. The electrode response to the bacterial samples was evaluated using electrochemical impedance spectroscopy (EIS). The results are as follows: Figure 8 C and Figure 8 As shown in D in the diagram.

[0072] The results showed that the impedance response of these three bacteria was similar to that of the negative control group (IMBs), and significantly lower than that of Salmonella at 1×10⁻⁶. 3The increase in Rct caused by CFU / mL concentration. The results indicate that the PES-LIG electrode detection system has good specificity for Salmonella, mainly attributed to the selective recognition and capture of Salmonella by the antibodies on the IMBs, while other pathogens are effectively removed during magnetic separation and washing. Furthermore, the electrode itself exhibits a very low response to IMBs.

[0073] 6.3 To optimize detection performance, the responses of IMBs prepared using 50 nm and 500 nm MNPs-COOH were compared in detection. Salmonella was captured using two different sizes of magnetic beads (50 nm and 500 nm) via immunomagnetic separation. The resulting bacterial solutions were then filtered through electrodes, and the electrochemical response of each electrode was evaluated using electrochemical impedance spectroscopy (EIS). The results are as follows: Figure 8 As shown in E in the figure.

[0074] The results showed that the increase in Rct caused by 50 nm IMBs was approximately 1.31 times that caused by 500 nm IMBs. When using larger magnetic beads, the distance between the IMBs and the target molecules increases, thereby weakening their interaction and leading to a decrease in capture efficiency. In contrast, smaller magnetic beads (50 nm) can cover more electrode surface, thus enhancing signal transmission.

[0075] 6.4 The PES composite electrode was evaluated in a two-electrode system, in which one electrode was exposed to the test solution via filtration (as the working electrode), while the bare electrode was used as the reference electrode. Electrochemical impedance spectroscopy (EIS) was used to measure the electrochemical response. Results are as follows: Figure 8 As shown in F in the diagram.

[0076] The results showed that its linear detection range (LDR) was 1×10⁻⁶. 2 Up to 1×10 6 CFU / mL, correlation coefficient R 2 =0.962. The limit of detection (LOD) is 32 CFU / mL (S / N=3).

[0077] Experimental Example 7: The Effect of Different Concentrations of Salmonella on Detection Results Using the dual-electrode system in Example 1, at 1×10 2 Up to 1×10 6 The detection performance of the PES-LIG electrode for Salmonella was evaluated within the concentration range of CFU / mL. To quantify the detection performance, a linear fitting curve was plotted with the logarithm of the St. concentration on the x-axis and the percentage decrease in impedance after sample filtration on the y-axis. The results are as follows: Figure 9 As shown.

[0078] The increased concentration of Salmonella in the sample led to an increased rate of change of electrochemical impedance at 10,010 Hz. This is consistent with existing literature (e.g., Li, H., Huang, S., Ling, T. and et al. Rapid detection of Salmonella in milk by a label-free electrochemical immunosensor based on CoFe-MOFs@MWCNTs modified electrode. International Dairy Journal 166, 2025, 106242. DOI: 10.1016 / j.idairyj.2025.106242: * limit of detection (LOD) for Salmonella: 2.89 × 10⁻⁶ 3 CFU / mL; Xu, M., Wang, R., Li, Y. Rapid detection ofEscherichia coli O157:H7 and Salmonella Typhimurium in foods using anelectrochemical immunosensor based on screen-printed interdigitatedmicroelectrode and immunomagnetic separation. Talanta 148, 2016, 200-208.DOI: 10.1016 / j.talanta.2015.10.082:* limit of detection (LOD) for Salmonella:1.04×10 3 CFU / mL; Brandão, D., Liébana, S., Campoy, S. and et al. Immunomagnetic separation of Salmonella with tailored magnetic micro andnanocarriers. A comparative study. Talanta, 143, 2015, 198-204. DOI: 10.1016 / j.talanta.2015.05.035:* limit of detection (LOD) for Salmonella: 5.38×10 2CFU / mL; Fei, J., Dou, W., Zhao, G. A sandwich electrochemical immunosensorfor Salmonella pullorum and Salmonella gallinarum based on a screen-printedcarbon electrode modified with an ionic liquid and electrodeposited goldnanoparticles. Microchim Acta 182, 2015, 2267–2275. DOI: 10.1007 / s00604-015-1573-x:*limit of detection (LOD) for Salmonella: 3×10 3 Compared to most methods described in the literature (CFU / mL), this system exhibits higher sensitivity and enables rapid detection without labeling. The detection limits of the aforementioned methods are approximately 10. 3 The detection limit of the detection method provided by this invention is 32 CFU, which is significantly lower than the detection limit of previous studies.

[0079] As described in the above embodiments, this invention provides a PES-LIG (graphene) electrode, a composite electrochemical biosensor, and its applications for detecting foodborne pathogens. This invention first captures and enriches target bacteria in a sample solution using IMBs. After magnetic separation and washing, the resuspended magnetic bead solution—containing either unbound IMBs (negative samples) or IMBs-bacterial complexes and unbound IMBs (positive samples)—is applied to the LIG surface. Pressure is applied to filter the solution through a porous membrane; after washing with PBST, unbound IMBs are completely removed, while the IMBs-bacterial complexes remain on the LIG electrode surface. Quantitative detection of the target bacteria is achieved by measuring the decrease in impedance.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PES-LIG electrode for detecting foodborne pathogens, characterized in that, It includes a PES-LIG membrane with a defined shape, which is divided into a sensing area, a hydrophobic sealing area, and a wire circuit connection area. The sensing area and the wire connection area are located at both ends of the PES-LIG membrane, respectively. The hydrophobic sealing region is located at the junction of the sensing region and the wire connection region; The PES-LIG membrane is prepared by laser-induced preparation of a graphene layer on the surface of a PES membrane to obtain the PES-LIG membrane.

2. The PES-LIG electrode according to claim 1, characterized in that, The hydrophobic sealing area is coated with UV-curing adhesive, and the wire connection area is coated with conductive silver paste.

3. The PES-LIG electrode according to claim 1, characterized in that, The power induced by the laser is 8-12%; the speed of the laser irradiation is 25-30 cm / s.

4. The PES-LIG electrode according to claim 1, characterized in that, The PES-LIG electrodes are used in pairs.

5. The PES-LIG electrode according to claim 4, characterized in that, When two PES-LIG electrodes are used in pairs, one serves as the working electrode and the other as the reference electrode.

6. A composite electrochemical biosensor for detecting foodborne pathogens, characterized in that, The device includes two PES-LIG electrodes as described in claim 1, which are encapsulated with insulating tape to keep the distance between the two PES-LIG electrodes constant at a set distance, while keeping the sensing area and the wire connection area exposed.

7. The composite electrochemical biosensor according to claim 6, characterized in that, The insulating tape has pre-drilled through holes with a diameter of 1.5~2mm; the set distance is 2~2.5mm.

8. A method for detecting foodborne pathogens, characterized in that, Includes the following steps: (1) Magnetic microparticles are added to the test solution containing foodborne pathogens for immunomagnetic bead separation to obtain enriched test samples; (2) The enriched sample obtained in step (1) is added to the sensing area of ​​the composite electrochemical biosensor of claim 6, the sample is filtered under pressure, and the concentration of foodborne pathogens in the solution is obtained according to the electrochemical signal.

9. The method according to claim 8, characterized in that, The foodborne pathogens mentioned in step (1) include Salmonella; the immunomagnetic bead separation time is 6-10 min.

10. The method according to claim 8, characterized in that, The pressure applied in step (2) is 0.4~0.6 bar.

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