Salmonella separation-detection integrated method based on nanofiltration and confined enzyme catalysis of integrated nanochannel electrode

Abstract

The application discloses a Salmonella separation-detection integrated method based on integrated nanochannel electrode nanofiltration and limited enzyme catalysis. A nanochannel membrane is used to prepare an integrated nanochannel-electrode chip INEC. Antibody and enzyme double-labeled magnetic beads DLMNPs are used to capture and enrich bacteria in a sample solution, and the enriched solution is added to the surface of the INEC, pressure is applied to filter the solution from the nanochannel. Free DLMNPs not combined with bacteria are removed, and DLMNPs-bacteria combinations are retained on the surface of the INEC. An enzyme substrate solution is added to the surface of the INEC, and the impedance change before and after enzyme catalysis is detected to realize quantitative detection of the bacteria. The application cooperates with the immune magnetic beads and the nanochannel nanofiltration to realize rapid separation of free magnetic beads of interfering substances and efficient enrichment of specific signal molecules. The micro electrolytic cell based on the INEC realizes limited catalysis signal amplification of the enzyme signal molecules, and the method has the advantages of rapid separation and high-sensitivity detection of the bacteria, is simple, and the electrode is easy to mass-produce, and has good universality.

Description

Technical Field

[0001] This invention relates to a method for bacterial isolation and detection in the field of nanochannel electrochemical analysis and sensing technology, specifically a method for the integrated isolation and detection of Salmonella based on nanofiltration and confined enzyme catalysis using integrated nanochannel electrodes. Background Technology

[0002] Salmonella is a type of Gram-negative bacterium belonging to the Enterobacteriaceae family. Poisoning can easily lead to abdominal pain, diarrhea, nausea, vomiting, bacteremia, and even death. In my country, there are approximately 3 million cases of Salmonella poisoning annually, ranking first among bacterial food poisoning incidents. my country stipulates that Salmonella must not be detected in food. Therefore, numerous detection methods have been developed to monitor Salmonella contamination in food. Traditional biochemical detection methods require pre-enrichment and isolation culture steps, making them cumbersome, time-consuming, and unable to provide timely feedback on the degree of bacterial contamination. Existing rapid detection methods, such as enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence PCR, while shortening the detection time, still require complex isolation and enrichment steps such as enrichment or nucleic acid extraction, and rely on expensive ELISA readers or PCR instruments, limiting their application. Therefore, there is an urgent need to develop simple, rapid, efficient, and portable microbial detection technologies and equipment.

[0003] In recent years, magnetic nanoparticles (MNPs) have been widely used for the purification and enrichment of target analytes due to their advantages such as ease of modification and superparamagnetic properties. To achieve specific signal output from the target analyte, a sandwich structure is typically formed by introducing a new label to eliminate interference from free magnetic beads. However, due to the limited number of target analyte recognition sites, this strategy struggles to achieve effective signal amplification and suffers from low sensitivity. Furthermore, this method is complex and time-consuming.

[0004] Solid-state nanochannels are artificial nanochannels developed based on biological nanochannels. Due to their advantages such as structural stability, tunable pore size, and ease of modification, they have attracted widespread research interest in fields such as DNA sequencing, biosensing, and energy conversion. Nanochannels can act as "nanosieves," enabling rapid sorting of molecules of different sizes. Simultaneously, nanochannels can also serve as reaction vessels, confining reactions within a nanoscale space, increasing mass transfer efficiency, improving reaction kinetics, and effectively amplifying reaction signals. Electrochemical sensing technology based on solid-state nanochannels offers high sensitivity and wide applications. However, in conventional detection systems, electrodes are often separated from the nanochannels, requiring the introduction of a large electrolytic cell. This results in poor portability, high background signal values, and signal molecules generated within the confined space of the nanochannel that can diffuse into the large-volume electrolytic cell, causing signal loss.

[0005] Therefore, developing a miniaturized nanochannel sensing platform and reducing the size of the electrolytic cell is expected to achieve both effective signal amplification and simple and efficient operation. Summary of the Invention

[0006] To address the challenges mentioned above, the present invention aims to provide an integrated method for the separation and detection of Salmonella based on nanofiltration and confined enzyme catalysis using integrated nanochannel electrodes. The method uses antibody- and enzyme-functionalized magnetic beads to separate and enrich target bacteria in a matrix, and integrates the high-efficiency separation function of nanochannels, the confined catalytic signal enhancement of a miniaturized electrolytic cell with nanochannel electrodes, and the high sensitivity of electrochemical sensing technology. It can achieve highly sensitive detection of Salmonella without the need for external signal markers.

[0007] like Figure 1 As shown, the specific technical solution adopted in this invention is as follows:

[0008] 1) An integrated nanochannel-electrode chip INEC was prepared using a nanochannel membrane and blocked with bovine serum albumin (BSA) solution;

[0009] 2) Prepare antibody- and enzyme-labeled magnetic beads (DLMNPs), and use the antibody- and enzyme-labeled magnetic beads (DLMNPs) to separate and enrich the target analytes in the sample solution to obtain the test solution;

[0010] 3) The test solution was filtered and detected using the integrated nanochannel-electrode chip INEC.

[0011] Step 1) specifically involves: sputtering conductive layers on both surfaces of the nanochannel membrane, connecting the conductive layers on both surfaces to an electrochemical workstation via their respective wires, and providing exposed conductive areas on both surfaces as electrodes. The nanochannel membrane after sputtering the conductive layers is then rinsed with deionized water and dried, subsequently immersed in bovine serum albumin (BSA) solution at room temperature for a period of time, and then rinsed with water to remove excess BSA solution, thereby producing the integrated nanochannel-electrode chip INEC.

[0012] The wire is fixedly connected to the conductive layer and sealed with tape.

[0013] In practice, the conductive layers on both surfaces of the integrated nanochannel-electrode chip INEC are connected to an electrochemical workstation via their respective wires. The conductive region of the conductive layer on one surface serves as the working electrode, and the conductive region of the conductive layer on the other surface serves as the counter electrode, thereby establishing a dual-electrode electrochemical detection system for detection.

[0014] The detection using a dual-electrode electrochemical detection system involves dropping a trace amount of enzyme substrate solution onto the conductive layer electrodes on both surfaces of the INEC. The solutions on both surfaces of the integrated nanochannel-electrode chip INEC are connected through the conductive layer and nanochannels to form a micro-electrolysis cell. The concentration of Salmonella is obtained by detecting the impedance change caused by enzyme catalysis within the micro-electrolysis cell.

[0015] The nanochannel membrane has microscopically regular pores, specifically having pores arranged in parallel to each other along a direction perpendicular to the membrane surface as nanochannels.

[0016] The nanochannel membrane has a pore size in the range of 0.01-10 μm, including single nanopores and nanochannel arrays, and the types include, but are not limited to, porous anodic aluminum oxide membranes and polymer membrane channels (such as polyethylene terephthalate).

[0017] The conductive layer may be made of materials including, but not limited to, silver, gold, platinum, copper, and carbon.

[0018] The sample solution to be tested may contain Salmonella.

[0019] In step 1), the nanochannel membrane after the sputtered conductive layer is dried is treated with a 0.2% bovine serum albumin solution for 30 minutes to seal it.

[0020] In step 1), the Salmonella bacteria are larger than the pore size of the nanochannel membrane.

[0021] Step 2) specifically refers to:

[0022] 2.1) Antibacterial antibodies and enzyme signaling molecules were modified on the surface of carboxylated magnetic beads (MNPs-COOH), and then blocked with bovine serum albumin (BSA) solution to prepare antibody- and enzyme-labeled magnetic beads (DLMNPs);

[0023] 2.2) The target analytes in the sample solution were separated and enriched by a certain amount of antibody and enzyme dual-labeled magnetic beads (DLMNPs), followed by magnetic separation and multiple washing to obtain the test solution.

[0024] The target analyte is Salmonella to be tested. The enzyme signaling molecule can catalyze the production of a large number of ions from a poorly dissociated substrate or catalyze the consumption of ions in the system to produce poorly dissociated products, thereby causing a change in the ionic strength of the system.

[0025] The types of enzyme signaling molecules mentioned include, but are not limited to, urease, glucose oxidase, etc.

[0026] The preparation process in step 2.1) is as follows:

[0027] 2.1.1) Preparation of activation solution: Prepare a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide sodium sulfonate (NHSS) using 4-morpholine ethanesulfonic acid buffer solution (MEST) containing 0.01 v / v% Tween 20 to obtain the activation solution;

[0028] 2.1.2) The carboxylated magnetic beads MNPs-COOH were washed with 4-morpholine ethanesulfonic acid buffer solution MEST, and after magnetic separation, they were dispersed in the activation solution of step 2.1.1). After shaking and incubating for a period of time, the carboxylated activated magnetic beads were obtained.

[0029] 2.1.3) Wash the carboxyl-activated magnetic beads obtained in step 2.1.2) with borate buffer solution BST containing 0.01 v / v% Tween 20, disperse them in borate buffer solution BST containing a certain concentration of antibacterial antibody after magnetic separation, incubate briefly, and then add borate buffer solution BST containing a certain concentration of enzyme. After shaking and coupling for a period of time, a mixture of antibody and enzyme-labeled magnetic beads is obtained.

[0030] The enzyme solution is specifically diluted to a certain concentration with borate buffer solution BST, and the types of enzyme signaling molecules include, but are not limited to, urease, glucose oxidase, etc.

[0031] 2.1.4) Add an equal volume of bovine serum albumin (BSA) solution to the antibody and enzyme dual-labeled magnetic bead mixture obtained in 2.1.3). After reacting for a certain period of time, perform magnetic separation and wash and resuspend the precipitate with phosphate buffer solution (PBST) containing 0.01 v / v% Tween 20 to obtain BSA-blocked, antibody and enzyme dual-functionalized antibody and enzyme dual-labeled magnetic beads (DLMNPs). These can be stored at 4°C for later use.

[0032] Step 2.1) Adding Tween to the buffer solution increases the dispersibility of the magnetic beads and prevents them from adhering to the centrifuge tube wall. 2.1.2) The hydrated particle size of the carboxylated magnetic beads after labeling antibodies and enzymes is smaller than that of the nanochannel pores.

[0033] The separation and purification steps in step 2.2) are as follows: the antibody and enzyme dual-labeled magnetic beads DLMNPs are ultrasonically dispersed in the solution and then mixed with the sample solution to be tested and incubated for a period of time. Then, they are magnetically separated and washed and resuspended multiple times with phosphate buffer solution PBS to obtain the test solution purified and enriched by antibody and enzyme dual-labeled magnetic beads DLMNPs.

[0034] Step 3) specifically involves:

[0035] First, the integrated nanochannel-electrode chip (INEC) is fixed and filtered through a filtration device to remove the antibody- and enzyme-labeled magnetic beads (DLMNPs) that do not bind to Salmonella. The test solution obtained in step 2) is then dropped onto the upper surface electrode of the integrated nanochannel-electrode chip (INEC). The test solution is then filtered out from the lower surface by applying appropriate pressure through the nanochannels of the integrated nanochannel-electrode chip (INEC) to remove the antibody- and enzyme-labeled magnetic beads (DLMNPs) that do not bind to Salmonella, while retaining the DLMNPs-bacterial conjugates on the upper surface electrode of the integrated nanochannel-electrode chip (INEC).

[0036] Then, after adding deionized water or enzyme substrate solution to the upper surface electrode of the integrated nanochannel-electrode chip INEC and filtering it to thoroughly clean it, the integrated nanochannel-electrode chip INEC was removed from the filtration device. A small amount of enzyme substrate solution prepared with deionized water was added to the electrodes on both surfaces of the integrated nanochannel-electrode chip INEC, and the impedance change before and after enzyme catalysis was measured using a dual-electrode electrochemical system under constant humidity.

[0037] The specific detection steps in step 3) are as follows:

[0038] 3.1) Fix the integrated nanochannel-electrode chip (INEC) into the filtration device, take the test liquid and drop it onto the electrode on the upper surface of the filtered integrated nanochannel-electrode chip (INEC), apply negative pressure to the upper surface or positive pressure to the lower surface to filter out the test liquid through the nanochannel;

[0039] 3.2) Repeatedly add deionized water or enzyme substrate solution and filter to wash the integrated nanochannel-electrode chip INEC obtained in step 3.1), and obtain the integrated nanochannel-electrode chip INEC with antibody and enzyme dual-labeled magnetic beads DLMNPs-target complex interception and adsorption.

[0040] In step 3.2), washing the INEC with deionized water or enzyme substrate solution can effectively remove the antibody and enzyme dual-labeled magnetic beads DLMNPs that have not bound to bacteria, and also remove PBS salt ions from the INEC system, thus avoiding interference with subsequent impedance detection.

[0041] The enzyme substrate is the specific catalytic substrate of the enzyme used in step 2.1) to prepare antibody and enzyme dual-labeled magnetic beads.

[0042] 3.3) A small amount of enzyme substrate solution was dropped onto each of the two surfaces of the integrated nanochannel-electrode chip INEC obtained in step 3.2), and the chip was placed in a constant humidity sealed environment. The impedance change before and after enzyme catalysis was measured using a two-electrode electrochemical system.

[0043] 3.4) The impedance change measured in step 3.3) is compared with the standard curve model established by fitting the impedance change obtained in the pre-calibrated test with the Salmonella concentration to obtain the corresponding Salmonella concentration result.

[0044] In practice, the frequency corresponding to the maximum impedance change (decreasing or increasing) is used as the signal acquisition frequency. The impedance change response of different bacterial concentrations at this frequency is measured, and a standard curve model is established by fitting the impedance change response with the corresponding bacterial concentration. The same experiment is performed multiple times with different Salmonella concentrations to obtain the corresponding impedance changes, and the impedance changes are obtained by fitting the curve model with the corresponding Salmonella concentration.

[0045] In step 3.3), the conductive layers on both surfaces of the integrated nanochannel-electrode chip INEC are connected to the two electrodes of the electrochemical workstation, respectively. The conductive region of the conductive layer on one surface serves as the working electrode, and the conductive region of the conductive layer on the other surface serves as the counter electrode, thereby establishing a dual-electrode system. Then, a trace amount of enzyme substrate solution is dropped onto the working electrode and the counter electrode, so that only the nanochannel membrane is immersed on the two surfaces to form a liquid film. The enzyme substrate solution is connected through the conductive layer and the nanochannel to form a confined microelectrolysis cell. The impedance of the microelectrolysis cell is detected to obtain the corresponding concentration of Salmonella.

[0046] In this invention, the rapid separation of unbound bacterial free DLMNPs interfering substances and the efficient amplification and high-sensitivity acquisition of enzyme catalytic signals are both achieved based on the integrated nanochannel-electrode chip INEC, which is simple and efficient to operate.

[0047] This invention develops a microelectrolysis cell by sputtering a conductive layer onto a chip using a nanochannel membrane. It combines antibody and enzyme dual-labeled magnetic beads (DLMNPs) to enhance the confined catalytic signal within the microelectrolysis cell and applies it to the isolation and detection of Salmonella, achieving rapid, efficient, and accurate isolation and detection of Salmonella.

[0048] This invention sputters conductive layers on both sides of a nanochannel membrane to obtain an integrated nanochannel-electrode chip (INEC). Enzyme substrate solutions are added to both sides of the chip to develop a dual-electrode electrochemical sensing platform based on a microelectrolysis cell. Target bacteria in the sample are captured and enriched using bifunctionalized magnetic beads (DLMNPs) containing antibody recognition elements and enzyme signaling molecules. Small-sized unbound free DLMNPs are separated by nanofiltration on the membrane chip, while large-sized DLMNP-bacterial conjugates are retained. Bacterial detection is achieved by utilizing the confined catalytic output signal of enzyme signaling molecules on the surface of the retained DLMNPs within the confined microelectrolysis cell.

[0049] In this invention, the target bacteria in the sample solution are first captured and enriched by dual-functionalized magnetic beads (DLMNPs) containing both antibodies and enzymes. After magnetic separation and washing, a small amount of the dispersed magnetic bead resuspension (containing only unbound free DLMNPs (negative sample), or containing both DLMNPs-bacterial conjugates and unbound free DLMNPs (positive sample)) is added dropwise to the INEC surface, and pressure is applied to filter the solution through the nanochannels. After washing and filtration with deionized water or enzyme substrate solution, the unbound free DLMNPs are fully removed, while the DLMNPs-bacterial conjugates are retained on the electrode surface. A small volume of enzyme substrate solution is added dropwise to the electrode surface, and enzyme catalysis increases the ionic strength of the system. Finally, the quantitative detection of the target bacteria is achieved by detecting the decrease in impedance.

[0050] The innovation of this invention lies in the preparation of an integrated nanochannel-electrode chip to develop a micro-electrode system for a micro-electrolysis cell. It integrates the three functions of nanochannel size sieving, nanochannel reaction vessel and electrode sensing detection into one, achieving a high degree of integration of the three modules of separation and enrichment, signal generation and amplification and high-sensitivity signal acquisition. It is highly efficient, portable and versatile.

[0051] The positive effects of this invention are:

[0052] This invention integrates nanochannels with electrodes to fabricate miniaturized electrode chips, thereby developing dual-electrode systems and miniaturized electrolytic cells. Simultaneously, it modifies magnetic beads with antibody recognition molecules and enzyme signaling molecules to separate and enrich target bacteria in complex matrices. Free magnetic beads are rapidly separated by nanofiltration through the nanochannels of the electrode chip, while retaining the magnetic bead-bacterial complex. Subsequently, based on the enzyme catalytic response in the miniaturized electrolytic cell and the dual-electrode system platform, a simple and highly sensitive detection of Salmonella is achieved.

[0053] This invention achieves rapid separation of interfering substances and efficient enrichment of signal molecules through the synergistic use of immunomagnetic beads and nanochannel "nanofiltration". At the same time, the microelectrolysis cell based on INEC realizes the confined catalytic signal amplification of enzyme signal molecules.

[0054] The integrated nanochannel-electrode chip of this invention highly integrates bacterial pretreatment and detection modules, achieving both rapid bacterial isolation and highly sensitive detection. In addition, the electrode is simple to fabricate and easy to mass-produce. This not only provides a new approach to the field of integrated separation-detection, but also brings new ideas to the research of nanochannels and miniaturized electrodes.

[0055] The positive effects of the method of this invention compared with existing methods are:

[0056] (1) The method of the present invention utilizes magnetic beads with dual functions of antibody recognition molecules and enzyme signaling molecules to rapidly separate and enrich target bacteria in complex matrices. It only takes 30 minutes, is simpler to operate, has richer magnetic bead functions, and can obtain specific signals without the need for additional signal markers.

[0057] (2) The method of the present invention is based on a miniaturized nanochannel electrode chip that highly integrates separation, signal amplification and sensing detection modules, which can realize the separation and detection of bacteria in one step. It is simple and efficient to operate, the equipment is portable and inexpensive and easy to prepare, avoiding cumbersome and time-consuming processing steps and the use of a variety of expensive instruments.

[0058] (3) The method of the present invention combines the high efficiency of enzyme catalysis with the enhancement of enzyme catalytic signal in the confined space of the INEC micro electrolytic cell, which can significantly improve the sensitivity of bacterial detection and realize the highly sensitive detection of trace target bacteria in complex matrices.

[0059] (4) The method of the present invention has good universality. By changing the antibody, different target substances (larger than the pore size of nanochannels) can be detected. Changing the enzyme can change the signal output mode, including electrochemical signals (impedance or steady-state current) and optical signals, and has a wide range of applications.

[0060] (5) The method of the present invention is expected to be combined with a portable electrochemical workstation, which has the potential to rapidly analyze bacterial residues on site and shows good application prospects. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the present invention.

[0062] Figure 2 These are scanning electron microscope images of the porous anodic aluminum oxide film before and after gold sputtering on both sides in this invention, wherein (a) is a top view before gold sputtering, (b) is a top view after gold sputtering, (c) is a cross-sectional view before gold sputtering, and (d) is a cross-sectional view after gold sputtering.

[0063] Figure 3 This is a schematic diagram illustrating the fabrication of the integrated nanochannel electrode chip in this invention.

[0064] Figure 4 The image shows the electrochemical activity characterization of INEC in this invention, where (a) is the result of cyclic voltammetry and (b) is the result of electrochemical impedance spectroscopy, compared with a commercial gold rod electrode.

[0065] Figure 5 The diagram shows the electrochemical detection performance evaluation of the dual-electrode system of the microelectrolysis cell in this invention, where (a) is the evaluation result of cyclic voltammetry and (b) is the evaluation result of electrochemical impedance spectroscopy, compared with commercial gold rod electrodes.

[0066] Figure 6 Figure 1 shows the characterization results of DLMNPs preparation in this invention. Figure 2 shows the labeling effect of the antibody by enzyme-linked immunosorbent assay (ELISA), Figure 3 shows the labeling effect of the enzyme by ultraviolet-visible light scanning, and Figure 4 shows the particle size distribution before and after magnetic bead modification.

[0067] Figure 7 These are scanning electron microscope (SEM) images of the membrane surface of the INEC filtered sample in this invention, where Figure (a) is the blank control solution and Figure (b) is the solution containing 10... 4 CFU mL -1 St's sample solution.

[0068] Figure 8 The results of St detection in the buffer solution of this invention are shown in Figure (a), where 0-10 are the values. 5 CFU mL -1 Figure (b) shows the impedance change curve corresponding to St. Figure (c) shows the standard curve model of this method for the detection of St in buffer solution.

[0069] Figure 9 The results of St detection in the chicken extract of this invention are shown in Figure (a), where 0-10 are the values. 6 CFU mL -1 The impedance change curve corresponding to St is shown in Figure (b), which is the standard curve model for the detection of St in chicken extract using this method. Detailed Implementation

[0070] To enable those skilled in the art to better understand the technical solution of the present invention, the method provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0071] The embodiments of the present invention are as follows:

[0072] (1) Fabrication of integrated nanochannel electrode chip INEC:

[0073] First, gold layers were sputtered onto both sides of a porous anodic aluminum oxide (AAO) film with a pore size of approximately 300 nm and a thickness of 60 μm to obtain an AAO / Au film. The structure and morphology of the AAO / Au film were characterized using field emission scanning electron microscopy (SEM). Figure 2 The results showed that the AAO / Au membrane maintained its cylindrical channel structure, with a bottom inlet diameter of approximately 250 nm and a top inlet diameter of approximately 350 nm. The deposition process did not alter the morphology and pore size of the AAO, ensuring the smooth operation of nanofiltration.

[0074] Then, as Figure 3As shown, the gold conductive layers on both sides of the AAO / Au membrane were connected by wires and sealed with tape. The tape was pre-drilled (Φ = 2 mm) to expose portions of the gold conductive layers on both sides of the AAO / Au membrane to act as electrodes, thus obtaining the integrated nanochannel-electrode chip INEC. After rinsing and drying the INEC with deionized water, it was immersed in a 0.2% (m / v) bovine serum albumin (BSA) solution for 30 minutes. Excess BSA was then rinsed with water and dried for later use.

[0075] A conventional three-electrode system (using the exposed conductive layer of INEC as the working electrode, a saturated calomel electrode as the reference electrode, and a carbon rod as the counter electrode) was employed to characterize the electrochemical performance of INEC in 1 mM K3Fe(CN)6 / K4Fe(CN)6 enzyme substrate solution through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements. The results were compared with those obtained using a commercially available gold rod electrode (Φ = 2 mm). CV measurement parameters were: scan rate 100 mV / s. -1 The scanning potential range is -0.1 to 0.5 V. ZIS test parameters are: frequency range 1 to 1 × 10⁻⁶. 4 Hz, open-circuit voltage 0V, amplitude 10mV. For example... Figure 4 Compared to gold rod electrodes, INEC exhibits a higher redox peak current. Figure 4 a) The electrode internal resistance is also lower ( Figure 4 b) indicates that INEC exhibits slightly superior electrochemical activity compared to commercial gold rod electrodes. Although the diameter of the exposed conductive layer electrode in INEC is the same as that of commercial gold rod electrodes, the former significantly increases the specific area of ​​its surface gold electrode due to the presence of numerous cylindrical nanochannels. Figure 2 b) can promote electron transfer on the electrode surface, thereby endowing INEC with excellent electrochemical activity.

[0076] (2) Construct a dual-electrode system and develop a micro-electrolysis cell:

[0077] A dual-electrode electrochemical detection system was developed by connecting the gold conductive layers on both surfaces of an INEC electrode to an electrochemical workstation via their respective wires. One conductive layer served as the working electrode, and the other as the reference and counter electrode. Simultaneously, 2.5 μL of enzyme substrate solution was dropped onto both electrode surfaces, and the enzyme substrate solution was connected to the two electrode surfaces via nanochannels, thus constructing a microelectrolysis cell. Using this microelectrolysis cell dual-electrode system, the CV and ZIS responses of INEC to a 1 mM K3Fe(CN)6 / K4Fe(CN)6 enzyme substrate solution were tested and compared with those of a commercially available gold rod electrode to evaluate the electrochemical detection performance of the dual-electrode system. The CV test parameters were: scan rate 100 mV / s. -1 The scanning potential range is -0.4V to 0.4V. ZIS test parameters are: frequency range 1-1×10⁻⁶.5 Hz, open-circuit voltage 0V, amplitude 10mV. A slight decrease (around 2μA) in the redox peak current of the electrolyte was observed due to the two-electrode system. Figure 5 a) The internal resistance of the electrode increases slightly (by about 1Ω). Figure 5 (b) However, the comparison results between INEC and gold rod electrodes are consistent with those obtained from the three-electrode system, demonstrating that the micro-electrolysis cell sensing platform coupled with the two-electrode system exhibits superior electrochemical sensing performance. This may be due to the fact that the confined micro-electrolysis cell environment facilitates the diffusion of electrochemical probes, while the extremely short distance between the electrodes on the two surfaces of INEC (only 60 μm thick for the nanochannel film) further increases the collision probability between the electrochemical probe and the electrode, promoting electron transfer.

[0078] (3) Preparation of DLMNPs:

[0079] The specific steps are as follows: After magnetic separation of carboxylated magnetic beads (20 nm), they were washed multiple times with 4-morpholinoethanesulfonic acid buffer solution (25 mM, pH 6.0, MEST) containing 0.01% (v / v) Tween 20. Then, they were dispersed in MEST solution containing 10 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 15 mM N-hydroxysuccinimide sodium sulfonate (NHSS), and incubated at room temperature with shaking for 30 min to activate the carboxyl groups. After activation, the supernatant was removed by magnetic separation, and the beads were washed multiple times with borate buffer solution (10 mM, pH 7.4, BST) containing 0.01% (v / v) Tween 20. The precipitate was finally dispersed in a solution containing 50 μg / mL... -1 In a BST solution containing anti-St lipopolysaccharide monoclonal antibody, the mixture was incubated with shaking at room temperature for 1 hour, and then 0.5 mg / mL was added. -1 Urease (derived from sword bean, EC3.5.1.5, 2,000 U mg) -1 (Purity >95%), coupling reaction was carried out overnight at 4℃. The next day, 5% (m / v) BSA solution prepared by BST was added, and the mixture was shaken at room temperature for 1 h to block residual carboxyl groups on the surface of the magnetic beads. After blocking, magnetic separation was performed and the mixture was washed with phosphate buffer solution (10 mM, pH 7.4, PBST) containing 0.01% (v / v) Tween 20 until the supernatant did not turn the phenol red and urea mixture pink within 30 min. The precipitate was finally dispersed in 200 μL of PBST solution to obtain antibody and urease bifunctional magnetic beads DLMNPs, which were stored at 4℃.

[0080] The antibody modification effect was characterized using enzyme-linked immunosorbent assay (ELISA). Figure 6 As shown in a, DLMNPs, even after being diluted 10-fold and 30-fold, still exhibited high binding affinity to St immobilized on the ELISA plate, even at St concentrations as low as 10-fold. 3 CFU mL -1At that time, the titer value was still as high as 1.337. This was because the antibody dosage was 50 μg / mL. -1 With a dilution factor of 1, assuming that antibody labeling on MNPs does not affect their affinity, the labeling rate of DLMNPs antibodies can be estimated to be approximately 33% by comparing it with the original antibody titer value. The labeling effect of urease was characterized by the color change of the pH indicator phenol red caused by DLMNPs catalyzing urea. Figure 6 As shown in b, the phenol red solution alone and the phenol red + urea mixture only showed an absorption peak at 430 nm. Upon addition of urease, the enzyme catalyzed the conversion of urea to ammonia, causing the solution pH to rise and the solution to change from yellow to pink, with a strong absorption peak appearing at 560 nm. The addition of DLMNPs also resulted in an absorption peak at 560 nm, while the addition of MNPs only showed an absorption peak at 430 nm, indicating that MNPs cannot catalyze the increase in solution pH caused by urea, while DLMNPs can. Furthermore, the addition of DLMNPs resulted in a characteristic broad peak of MNPs in the 300-400 nm range. These results confirm that urease was successfully labeled onto MNPs.

[0081] The particle size distribution of MNPs and DLMNPs was further characterized by dynamic optical scintillation (DLS). Figure 6 c shows that after antibody, urease, and BSA labeling, the average hydrodynamic diameter of MNPs increased from 90 nm to 122 nm, further confirming the successful preparation of DLMNPs. Since DLS reflects the hydrodynamic diameter of particles rather than their actual size, the measured size is larger than the true particle size. However, the size of DLMNPs is still about twice the size of the AAO pore size (250-350 nm), ensuring sufficient filtration.

[0082] (4) Magnetic bead separation and enrichment of bacteria:

[0083] First, bacteria were cultured to obtain an inactivated bacterial solution of a certain concentration. The specific steps are as follows: *Salmonella typhimurium* (ATCC14028) was thawed from the ultra-low temperature freezer. 0.5 mL of the bacterial solution was added to 9.5 mL of brain heart extract medium and incubated overnight at 37°C and 170 rpm on a shaker. The cultured bacterial solution was washed three times by centrifugation (6,000 rpm, 5 min) with 0.01 M phosphate buffered saline (PBS, 10 mM, pH 7.4). Subsequently, the solution was diluted with PBS to obtain a series of 10-fold serial dilutions. 100 μL of the appropriate concentration of bacterial solution was plated onto XLT4 agar medium and incubated at 37°C for 24 h. Colonies were counted to determine the final bacterial concentration. The bacterial solution was then boiled for 10 min to inactivate the bacteria.

[0084] Then, bacteria were isolated and enriched using DLMNPs. The specific steps are as follows: The inactivated bacterial solution was diluted to a certain concentration with 0.01M PBS or sample extraction buffer. 1 mL of sample solution was mixed with 100 μL of DLMNPs and incubated for 30 min. Bacteria were captured by lipopolysaccharide antibody to obtain the DLMNPs-St complex. After magnetic separation, the supernatant was discarded, and the precipitate was washed with PBS and resuspended to obtain the test solution.

[0085] (5) Electrochemical detection of Salmonella:

[0086] 5.1) Detection of Salmonella in buffer solutions:

[0087] INEC was fixed in a self-made filtration device. A 10 μL drop of the test solution was placed on the surface of the INEC electrode. A vacuum pump was used to pressurize the solution, forcing it through the INEC nanochannels for filtration. The solution was then thoroughly rinsed with deionized water. SEM characterization results showed that, because the size of DLMNPs (hydrodynamic diameter 122 nm) is smaller than the nanochannel pore size, unbound free DLMNPs could be filtered out through the BSA-sealed nanochannels. Figure 7 a). For positive samples, because rod-shaped Salmonella (approximately 0.8 μm wide and 1-2 μm long) are captured by DLMNPs and induced to aggregate, a large number of DLMNP-bacterial conjugates are trapped on the electrode surface. Figure 7 b).

[0088] The INEC was then removed from the filter, and 2.5 μL of a low-ionic-strength urea aqueous solution was added dropwise to both sides of the electrode. An enzyme-catalyzed reaction was then carried out under constant humidity, causing a change in the ionic strength of the solution. The impedance value was measured after 15 min using a dual-electrode system. The detection conditions were: one side of the INEC containing the DLMNPs-St complex was used as the working electrode, and the other side as the counter electrode; the frequency range was 10⁻¹ × 10⁻¹⁰. 4 Hz, open-circuit voltage 0V, amplitude 10mV. The relative impedance drop at 400Hz (1-Z0 / Z) 15 , %) as a quantitative signal.

[0089] like Figure 8 As shown in Figure a, the impedance change at 400 Hz (Z0-Z) increases with increasing bacterial concentration. 15 The higher the bacterial concentration, the greater the number of DLMNPs-St conjugates, and the more DLMNPs are retained on the electrode surface. Furthermore, the enzyme signaling molecules on the surface of DLMNPs can catalyze the production of HCO3- from poorly dissociated urea (pKb 13.82) within the confined microenvironment. - OH - and NH4 +This significantly increases the ionic strength of the system and substantially reduces the impedance. A curve was constructed with the logarithm of the *Salmonella typhimurium* concentration on the x-axis and the impedance drop at 400 Hz on the y-axis. The linear range for the detection of *St* in buffer solutions using this method is 10⁻¹⁰. 5 CFU mL -1 The linear regression equation is: y = 10.9log x - 3.37 (R²) 2 =0.978), the detection limit is 9.3 CFU / mL. -1 (3S / N) (e.g.) Figure 8 (b) The method of this invention has high sensitivity for the detection of St in buffer solutions, which is mainly due to two factors: (1) Because the mass transfer path of small substrate molecules in the confined microreactor (about 5 μL) is shortened, the probability of collision with the enzyme is increased, making the enzyme kinetics faster than that of the traditional open electrolytic cell. Therefore, even low concentrations of enzyme (bacteria) can effectively catalyze the substrate, leading to a decrease in impedance. (2) The signal molecules (ions) generated by enzyme catalysis are fully retained in the micro-liter electrolytic cell. A small amount of ions can cause a significant increase in local ionic strength, thereby causing a decrease in impedance.

[0090] 5.2) Detection of Salmonella in chicken breast extract:

[0091] Chicken breast samples were verified as Salmonella negative using the standard Salmonella culture method. 25g of chopped chicken breast was weighed into a homogenizer, 225mL of 0.01M phosphate-buffered saline (PBS) was added, and the mixture was homogenized for 1 min using a beater homogenizer. The resulting crude chicken extract was centrifuged at 6,000 rpm for 15 min at room temperature. The supernatant was collected and diluted twice with 0.01M PBS to obtain the final chicken extract. Different concentrations of Salmonella were added to the chicken extract, and the Salmonella in the chicken extract was detected using the invented INEC dual-electrode system microelectrolysis cell sensing platform according to step 5.1).

[0092] like Figure 9 As shown in Figure a, the impedance change at 400 Hz increases with increasing bacterial concentration. A curve was constructed with the logarithm of Salmonella concentration on the x-axis and the impedance decrease at 400 Hz on the y-axis, revealing a linear range of 10 for the detection of St in chicken breast extract using this method. 2 -10 5 CFU mL -1 The linear regression equation is: y = 16.29log x - 12.95 (R²) 2 =0.994), detection limit is 42 CFU / mL -1 (3S / N) (e.g.) Figure 9 b) indicates that the method of the present invention also has high sensitivity for the detection of Salmonella in chicken matrix and good resistance to interference from chicken matrix.

[0093] 5.3) Specificity analysis:

[0094] By detecting Salmonella typhimurium (10) 2 CFU mL -1 And other pathogenic bacteria, including Escherichia coli O157:H7 (ATCC43888), Listeria monocytogenes (EGDe), Vibrio parahaemolyticus (KP9), and Staphylococcus aureus (ATCC25923) (all at a concentration of 10). 4 CFU mL -1 ()( Figure 8 c) It can be concluded that the method of the present invention has high specificity against Salmonella Typhimurium. This is mainly due to the specificity of the anti-Salmonella Typhimurium lipopolysaccharide monoclonal antibody. Therefore, by modifying the magnetic beads with specific antibodies against other target bacteria, the method of the present invention is also expected to achieve highly sensitive and specific detection of other pathogenic bacteria.

[0095] As demonstrated by the above implementation examples, the integrated method for Salmonella isolation and detection based on nanofiltration and confined enzyme catalysis using integrated nanochannel electrodes proposed in this invention avoids the use of additional signal markers by employing bifunctional magnetic beads; it utilizes nanochannel "nanofiltration" to rapidly separate interfering magnetic beads and enrich signal molecules; it develops a micro-electrolysis cell for enzyme-confined catalysis to generate and efficiently amplify signals; and it combines this with an electrode chip to output readable electrochemical signals, achieving highly sensitive detection of Salmonella typhimurium in buffer solutions and chicken matrices. The entire process takes only about 1 hour. This method is simple, rapid, highly specific, sensitive, and versatile, and the functional magnetic beads and integrated electrodes are easy to prepare and mass-produce. Therefore, this method is expected to become a rapid on-site detection method with promising development prospects.

[0096] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the method of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An integrated method for the isolation and detection of Salmonella based on integrated nanochannel electrode nanofiltration synergistic confined enzyme catalysis, characterized in that: The method includes the following steps: 1) An integrated nanochannel-electrode chip INEC was prepared using a nanochannel membrane and blocked with bovine serum albumin (BSA) solution; 2) Prepare antibody- and enzyme-labeled magnetic beads (DLMNPs), and use the antibody- and enzyme-labeled magnetic beads (DLMNPs) to separate and enrich the target analytes in the sample solution to obtain the test solution; 3) The test solution was filtered and detected using the integrated nanochannel-electrode chip INEC; Step 1) specifically involves: sputtering conductive layers on both surfaces of the nanochannel membrane, connecting the conductive layers on both surfaces to an electrochemical workstation via their respective wires, and providing exposed conductive areas on both surfaces as electrodes. The nanochannel membrane after sputtering the conductive layers is then rinsed with deionized water and dried, subsequently immersed in bovine serum albumin (BSA) solution for room temperature sealing, and then rinsed with water to remove excess BSA solution, thereby producing the integrated nanochannel-electrode chip INEC. The pore size of the nanochannels in the nanochannel membrane is in the range of 0.01-10 μm; the materials of the conductive layer include, but are not limited to, silver, gold, platinum, copper, and carbon. In step 1), the nanochannel membrane after the sputtered conductive layer was dried was treated with a bovine serum albumin solution with a mass fraction of 0.2% for 30 min. Step 2) specifically refers to: 2.1) Antibacterial antibodies and enzyme signaling molecules were modified on the surface of carboxylated magnetic beads (MNPs-COOH), and then blocked with bovine serum albumin (BSA) solution to prepare antibody- and enzyme-labeled magnetic beads (DLMNPs); 2.2) The target analytes in the sample solution were separated and enriched using antibody- and enzyme-labeled magnetic beads (DLMNPs), followed by magnetic separation and multiple washing to obtain the test solution. The preparation process in step 2.1) is as follows: 2.1.1) Preparation of activation solution: Prepare a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide sodium sulfonate (NHSS) using 4-morpholine ethanesulfonic acid buffer solution (MEST) containing 0.01 v / v% Tween 20 to obtain the activation solution; 2.1.2) The carboxylated magnetic beads MNPs-COOH were washed with 4-morpholine ethanesulfonic acid buffer solution MEST, and after magnetic separation, they were dispersed in the activation solution of step 2.1.1). After shaking and incubation, the carboxylated activated magnetic beads were obtained. 2.1.3) Wash the carboxyl-activated magnetic beads obtained in step 2.1.2) with borate buffer solution BST containing 0.01 v / v % Tween 20, disperse them in borate buffer solution BST containing antibacterial antibody after magnetic separation, incubate them, add more borate buffer solution BST, and oscillate to obtain a mixture of antibody and enzyme dual-labeled magnetic beads. 2.1.4) Add an equal volume of bovine serum albumin (BSA) solution to the antibody- and enzyme-labeled magnetic bead mixture obtained in 2.1.3). After the reaction, perform magnetic separation and wash and resuspend the precipitate with phosphate buffer solution (PBST) containing 0.01 v / v % Tween 20 to obtain antibody- and enzyme-labeled magnetic beads (DLMNPs).

2. The integrated method for Salmonella isolation and detection based on integrated nanochannel electrode nanofiltration synergistic confined enzyme catalysis as described in claim 1, characterized in that: The separation and purification steps in step 2.2) are as follows: the antibody and enzyme dual-labeled magnetic beads DLMNPs are ultrasonically dispersed in the solution and then mixed and incubated with the sample solution to be tested. Then, they are magnetically separated and washed and resuspended multiple times with phosphate buffered PBS to obtain the test solution purified and enriched by antibody and enzyme dual-labeled magnetic beads DLMNPs.

3. The integrated method for Salmonella isolation and detection based on integrated nanochannel electrode nanofiltration synergistic confined enzyme catalysis as described in claim 1, characterized in that: Step 3) specifically involves: First, the integrated nanochannel-electrode chip (INEC) is fixed and filtered through a filtration device. The test liquid obtained in step 2) is dropped onto the upper surface electrode of the integrated nanochannel-electrode chip (INEC). The test liquid is filtered out by pressure through the nanochannels of the integrated nanochannel-electrode chip (INEC) from the lower surface, while the DLMNPs-bacterial conjugate is retained on the upper surface electrode of the integrated nanochannel-electrode chip (INEC). Then, after adding deionized water or enzyme substrate solution to the upper surface electrode of the integrated nanochannel-electrode chip INEC and filtering it to thoroughly clean it, the integrated nanochannel-electrode chip INEC was removed from the filtration device. The enzyme substrate solution prepared with deionized water was added to the electrodes on both surfaces of the integrated nanochannel-electrode chip INEC, and the impedance change before and after enzyme catalysis was measured using a dual-electrode electrochemical system under constant humidity.

4. The integrated method for Salmonella isolation and detection based on integrated nanochannel electrode nanofiltration synergistic confined enzyme catalysis as described in claim 3, characterized in that: The specific detection steps in step 3) are as follows: 3.1) Fix the integrated nanochannel-electrode chip (INEC) into the filtration device, take the test liquid and drop it onto the electrode on the upper surface of the filtered integrated nanochannel-electrode chip (INEC), apply negative pressure to the upper surface or positive pressure to the lower surface to filter out the test liquid through the nanochannel; 3.2) Repeatedly add deionized water or enzyme substrate solution and filter to clean the integrated nanochannel-electrode chip (INEC) obtained in step 3.1); 3.3) The enzyme substrate solution was dropped onto the two surfaces of the integrated nanochannel-electrode chip INEC obtained in step 3.2), and the chip was placed in a constant humidity sealed environment. The impedance change before and after enzyme catalysis was measured using a two-electrode electrochemical system. 3.4) The impedance change measured in step 3.3) is compared with the standard curve model established by fitting the impedance change obtained in the pre-calibrated test with the Salmonella concentration to obtain the corresponding Salmonella concentration result.

5. The integrated method for Salmonella isolation and detection based on integrated nanochannel electrode nanofiltration synergistic confined enzyme catalysis as described in claim 4, characterized in that: In step 3.3), the conductive layers on both surfaces of the integrated nanochannel-electrode chip INEC are connected to the two electrodes of the electrochemical workstation, respectively. The conductive region of the conductive layer on one surface serves as the working electrode, and the conductive region of the conductive layer on the other surface serves as the counter electrode, thereby establishing a dual-electrode system. Then, the enzyme substrate solution is dropped onto the working electrode and the counter electrode. The enzyme substrate solution is connected through the conductive layer and the nanochannel to form a confined microelectrolysis cell. The impedance of the microelectrolysis cell is detected to obtain the corresponding concentration of Salmonella.

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