An electrochemical sensor for salmonella detection, detection method and application

Abstract

The present application relates to the field of food biotechnology and analytical technology, in particular to an electrochemical sensor for detecting salmonella, a detection method and application thereof.The electrochemical sensor comprises: a conductive electrode substrate, a two-dimensional nanocomposite modification layer modified on the surface of the conductive electrode substrate, and a salmonella specific bacteriophage source recognition element fixed on the surface of the two-dimensional nanocomposite modification layer; the salmonella specific bacteriophage source recognition element is selected from at least one of a salmonella bacteriophage BPLT2 particle and a recombinant long tail fiber protein LTF-BPLT2.The electrochemical sensor has extremely high reproducibility and anti-matrix interference ability, and high detection accuracy.

Description

Technical Field

[0001] This invention relates to the fields of food biotechnology and analytical technology, and in particular to an electrochemical sensor, detection method and application for Salmonella detection. Background Technology

[0002] salmonella( Salmonella *S. spp.* is a widely distributed group of foodborne pathogens with complex serotypes. Livestock and poultry meat, eggs, and dairy products are highly susceptible to contamination by this bacterium, leading to serious public health problems such as acute gastroenteritis and septicemia in humans. Therefore, establishing highly sensitive and specific rapid on-site screening methods is crucial for ensuring food safety.

[0003] Currently, traditional bacterial culture methods remain the "gold standard" for pathogen detection, but the detection cycle is as long as 3-5 days, which is difficult to meet the real-time prevention and control needs of modern supply chains. Although rapid detection technologies based on molecular biology (such as PCR) and immunology (such as ELISA) have shortened the time, the former's nucleic acid amplification is easily interfered with by complex matrices and cannot distinguish between live and dead bacteria, while the latter's core component (antibody) has high production costs and poor environmental tolerance.

[0004] In recent years, biosensors, especially electrochemical biosensors, have attracted much attention in the field of rapid detection due to their rapid response and portability. Sensor performance is highly dependent on the transduction matching between the conductive transducer and the molecular recognition element. Bacteriophages and their derived receptor-binding proteins (RBPs) have become novel recognition probes due to their high host specificity and low preparation cost. However, in practical solid-phase electrode interface assembly, directly applying intact bacteriophages has two major drawbacks: first, the large size of intact viral particles (50~200 nm) causes steric hindrance, limiting the modification density; second, the inherent lytic activity of intact bacteriophages can lead to the rupture of captured host cells, and the release of intracellular ions can locally alter the interfacial environment, causing electrochemical signal drift. Furthermore, directly heterologously expressed bacteriophage long-tailed fibrils often readily form inactive inclusion body aggregates in prokaryotic expression systems, making them difficult to apply directly to the sensing interface.

[0005] Therefore, how to construct an efficient electron transport network on the surface of a solid electrode using novel two-dimensional nanomaterials, and how to collaboratively develop a solid-phase interface process that can capture across serotypes in a broad spectrum, avoid interference from biological lysis mechanisms, and achieve highly stable assembly, is a major technical bottleneck in the field of rapid electrochemical sensing detection of foodborne pathogens. Summary of the Invention

[0006] This invention addresses the shortcomings and limitations of existing technologies by providing an electrochemical sensor, detection method, and application for Salmonella detection. Instead of relying on the sequence evolution of a single recognition protein, this invention optimizes the design of two parallel nano-enhanced sensing solid-phase modification interfaces and curing assembly processes (covalent coupling system and directional coordination system) to efficiently and stably convert biocapture events into quantifiable electrochemical spatial barrier impedance signals.

[0007] To achieve the above objectives, the present invention provides the following solution: The present invention provides an electrochemical sensor for the detection of Salmonella, comprising: a conductive electrode substrate, a two-dimensional nanocomposite material modification layer on the surface of the conductive electrode substrate, and a Salmonella-specific phage source recognition element fixed on the surface of the two-dimensional nanocomposite material modification layer; wherein the Salmonella-specific phage source recognition element is selected from at least one of Salmonella phage BPLT2 particles and recombinant long-tailed fibrous protein LTF-BPLT2.

[0008] As a preferred embodiment, the recombinant long-tailed fibrous protein LTF-BPLT2 (molecular weight approximately 128.2 kDa) is an inclusion body protein obtained through prokaryotic heterologous expression. After denaturation and unfolding with high-concentration urea, its spatial conformation is reconstituted using a specific gradient-decreasing dialysis refolding process. Its final soluble effective concentration is 2.73 mg / mL, exhibiting non-lytic adhesion biological activity specifically binding to Salmonella.

[0009] Optionally, the pairing method between the two-dimensional nanocomposite material modification layer and the Salmonella-specific bacteriophage recognition element is selected from any one of the following two modes: The electrochemical sensor of mode one: the two-dimensional nanocomposite material modification layer is a carboxylated reduced graphene oxide film, the Salmonella-specific phage source recognition element is Salmonella phage BPLT2 particles, and the Salmonella phage BPLT2 particles are solidified on the surface of the carboxylated reduced graphene oxide film through covalent crosslinking amide bonds. The electrochemical sensor in Mode 2: The two-dimensional nanocomposite modification layer is a two-dimensional transition metal carbide composite film modified with gold nanoparticles (Au-MXene composite film); the Salmonella-specific phage recognition element is a recombinant long-tailed fibrous protein LTF-BPLT2 with a histidine tag; the recombinant long-tailed fibrous protein LTF-BPLT2 is transmitted via Ni… 2+ -His affinity coordination is directionally cured on the surface of Au-MXene composite film.

[0010] Optionally, the preparation method of the electrochemical sensor of Mode 1 includes the following steps: 1) A composite dispersion of rGO-COOH and Nafion was drop-coated onto the polished and cleaned gold disk electrode surface, and dried to form a film, thus obtaining an rGO-COOH / GDE modified electrode; wherein the optimal modification concentration of rGO-COOH was 1.0 mg / mL, and the final concentration of Nafion in the composite dispersion was 0.1 wt%; 2) The rGO-COOH / GDE modified electrode was immersed in a MES buffer containing EDC and NHS to activate the carboxyl functional groups. After washing, it was placed in a container with a titer of 3.0 × 10⁻⁶. 10 Cross-linked electrodes were obtained by incubating the PFU / mL phage BPLT2 solution for 2 h. 3) Remove the cross-linked electrode, clean it, and then block the non-specific sites with 1% bovine serum albumin solution to obtain the electrochemical sensor of mode one. The electrochemical sensor (mode one) is a complete phage sensor.

[0011] Optionally, the preparation method of the electrochemical sensor of mode two includes the following steps: 1) Au-MXene was coated onto the surface of a gold disk electrode to obtain an Au-MXene / GDE modified electrode; wherein the optimal modification concentration of Au-MXene was 0.25 mg / mL; 2) The Au-MXene / GDE modified electrode was immersed in SH-PEG-NTA solution overnight to construct an NTA-functionalized electrode; 3) After washing the NTA-functionalized electrode, it was immersed in a 10 mM nickel ion solution for coordination chelation for 30 min, and then incubated in a 2.73 mg / mL recombinant long-tail fibroin LTF-BPLT2 solution for 60 min. The electrode was then subjected to Ni... 2+ -His affinity enables the targeted immobilization of specifically recognized proteins, thus yielding the Mode 2 electrochemical sensor. The Mode 2 electrochemical sensor is a recombinant long-tail fibrous protein electrochemical sensor.

[0012] An electron transport network was constructed by drop-coating 0.25 mg / mL Au-MXene highly conductive nanocomposite material onto the surface of a gold disk electrode. An NTA-PEG-SH monolayer was then self-assembled via Au-S bonds and chelated with Ni. 2+ Utilizing Ni 2+ Directional immobilization is achieved through coordination with the N-terminal His-tag of the recombinant protein. This mode ensures that the receptor-binding region is fully oriented towards the solution phase. Since the protein lacks viral lysis activity, it does not induce host lysis after bacterial capture, thus completely eliminating the background current signal rise caused by lysis.

[0013] This invention provides the application of electrochemical sensors in the detection of Salmonella for non-diagnostic purposes and / or in the preparation of products for detecting Salmonella.

[0014] This invention provides a rapid quantitative detection method for non-diagnostic Salmonella using an electrochemical sensor, comprising the following steps: 1) The electrochemical sensor is used as the working electrode, and together with the platinum wire auxiliary electrode and the silver / silver chloride reference electrode, a three-electrode system is formed; 2) The working electrode is co-incubated with a suspension containing Salmonella to be tested, so that the recognition element on the electrode surface specifically captures the target bacteria, and a non-conductive steric barrier layer is constructed on the electrode surface. 3) After capturing the target bacteria, the working electrode is transferred to a device containing potassium ferricyanide [Fe(CN)6]. 3- / 4- In the electrolyte substrate of the redox probe ions, differential pulse voltammetry (DPV) was triggered by an electrochemical workstation to scan and record the oxidation peak current response signal at the electrode interface; 4) Based on the attenuation amplitude of the oxidation peak current response signal, compare it with the standard curve to obtain the concentration of Salmonella in the sample to be tested.

[0015] Optionally, the Salmonella to be tested includes Salmonella typhimurium, wherein the Salmonella typhimurium is a type of Salmonella typhimurium that can be lysed by bacteriophage BPLT2.

[0016] Optionally, when using the electrochemical sensor of Mode 1 for detection, the optimal capture incubation time for the target bacteria is strictly limited to 15 min to avoid electrochemical baseline signal drift caused by the natural lysis mechanism of intact bacteriophages.

[0017] Optionally, when the electrochemical sensor of Mode 2 is used for detection, its electrochemical response signal is related to the Salmonella concentration at 9.2 × 10⁻⁶. 1 -9.2×10 7 It exhibits a good linear relationship within the CFU / mL range, and the standard curve regression equation is: y =-0.74653 x +7.19476, of which y This represents the peak current value of the DPV circuit, in microamperes (µA). x The value is the logarithm of the Salmonella concentration, lg C, with a detection limit as low as 3 CFU / mL, and the relative standard deviation of the response current for five consecutive scans at a concentration of 500 CFU / mL is no greater than 1.3%.

[0018] Optionally, when using the electrochemical sensor of Mode 1 for detection, the linear detection range for Salmonella is 1.2 × 10⁻⁶. 1 -1.2×10 7 CFU / mL.

[0019] The present invention discloses the following technical effects: This invention provides an electrochemical sensor for Salmonella based on a two-dimensional nanocomposite interface and phage-derived dual-mode recognition, which has the following advantages: 1. Synergistic Enhancement through Multiple Materials and Mechanisms: This invention utilizes the large specific surface area of ​​rGO-COOH and the ultra-high conductivity layered network of Au-MXene to effectively overcome the problem of high charge transfer resistance in traditional sensing electrodes. By transforming biocapture into a steric barrier, it effectively blocks [Fe(CN)6]. 3- / 4- Mass transfer of probe ions enables ultra-high sensitivity.

[0020] 2. Precise and controllable kinetic time control: This invention elucidates for the first time the dual electrochemical properties of intact bacteriophages capturing the host at the electrochemical interface. The electrochemical sensor (Mode 1), namely the covalently coupled bacteriophage sensor, ensures maximum signal attenuation (attenuation up to 48.55%) by strictly limiting the capture incubation time to the critical saturation period of 15 min, while successfully avoiding ion background fluctuations triggered by the lysis cycle.

[0021] 3. Excellent stability and ultra-low detection limit: The electrochemical sensor (mode 2), namely the directed assembly recombinant protein sensor, further reduces the detection limit to 3 CFU / mL. The RSD is only 1.3% after 5 consecutive cycles of electrochemical scanning, showing extremely high reproducibility and resistance to matrix interference. The recovery rate is as high as 97.2%~102% in actual samples spiked with milk and eggs.

[0022] Biological Preservation Certificate bacteriophage ( Salmonella sp . phage BPLT2 was deposited on June 24, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20251473. Attached Figure Description

[0023] 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.

[0024] Figure 1 The figures show the CV and DPV characterization curves for the layer-by-layer assembly of the electrochemical sensor. A represents the electrochemical behavior characterization of the layer-by-layer modification process of the sensor interface using cyclic voltammetry (CV), and B represents the electrochemical behavior characterization of the layer-by-layer modification process of the sensor interface using differential pulse voltammetry (DPV). Figure 2The graph shows the DPV response and standard fitting curves of the electrochemical sensor (mode 1) for detecting Salmonella. A is the differential pulse voltammetry (DPV) detection results of the electrochemical sensor capturing different concentrations of Salmonella, and B is the linear relationship between the attenuation of the DPV response current of the electrochemical sensor and the concentration of Salmonella. Figure 3 Time-kinetic optimization curves for the electrochemical sensor (Mode 1); Figure 4 The graph shows the DPV response of the electrochemical sensor (mode 2) for detecting Salmonella and the standard fitting curve. A is the DPV detection result of the electrochemical sensor capturing different concentrations of Salmonella, and B is the linear relationship between the attenuation of the DPV response current of the electrochemical sensor and the concentration of Salmonella. Figure 5 This is a graph showing the sensor's broad-spectrum recognition of multiple serotypes of Salmonella and its specificity evaluation of non-target bacteria. Figure 6 Schematic diagram of co-expression plasmid construction. Detailed Implementation

[0025] 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.

[0026] 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. Any stated value or intermediate value within a stated range, as well as each smaller range between 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Example 1: Fabrication and Reactivation of Recognition Element 1. Purification of intact bacteriophage BPLT2: Bacteriophage BPLT2 was amplified using Salmonella Typhimurium LT2 as the host. The supernatant was collected and precipitated with polyethylene glycol (PEG 8000). Further purification was performed by density gradient ultracentrifugation with cesium chloride (CsCl) (30000 r / min, 3 h). The white enrichment band was then transferred to PBS to obtain the bacteriophage BPLT2 solution. The final titer of bacteriophage BPLT2 in the solution was determined to be 3.0 × 10⁻⁶. 10 PFU / mL.

[0031] 2.1) Construction of co-expression plasmid: gp37 and gp38 are components of the BPLT2 long-tailed fibrin, and gp92 is a chaperone protein that assists in the folding of the long-tailed fibrin and the formation of its correct spatial conformation. The co-expression plasmid was constructed using pETDuet-1, which has dual cloning sites, as a vector. The gp37-gp38 fragment was inserted into cloning site 1 (MCS1). BamHI and NotI restriction endonucleases were used for directional insertion. A 6×His tag was added to the N-terminus of MCS1. The gp92 fragment was inserted into cloning site 2 (MCS2) using NdeI and XhoI restriction endonucleases for directional insertion, resulting in the recombinant long-tailed fibrin plasmid pETDuet-1-LTF-BPLT2. A schematic diagram of the co-expression plasmid construction is shown below. Figure 6 .

[0032] 2) Transformation parameters: The recombinant long-tailed fiber plasmid pETDuet-1-LTF-BPLT2 was added to 100 μL of Escherichia coli BL21(DE3)pLysS competent cells. After gently tapping the tube wall to mix, the cells were incubated on ice for 10 min, then heat-shocked in a 42℃ water bath for 90 s, and quickly transferred to ice and incubated for 3 min. 1 mL of antibiotic-free LB broth was added to the system, and the cells were incubated at 37℃ and 180 r / min with shaking for 45 min to regenerate the cells. 60 μL of the regenerated bacterial culture was spread onto LB agar plates containing 50 μg / mL ampicillin. The plates were incubated upside down overnight in a 37℃ incubator. Positive single clones were finally picked for subsequent protein expression induction.

[0033] Extraction and gradient refolding of recombinant long-tailed fibrous protein LTF-BPLT2: Positive monoclonal antibodies were cultured to obtain overnight pETDuet-1-LTF-BPLT2 bacterial culture. This overnight culture was transferred to LB broth containing ampicillin at a volume ratio of 1%, and cultured until the OD600 reached approximately 0.6. IPTG was then added to a final concentration of 0.4 mM, and expression was induced at 16℃ and 120 r / min for 20 h. The bacterial cells were collected by centrifugation and sonicated, then centrifuged at 4℃ and 15000 r / min for 30 min. The supernatant was discarded, and the inclusion body precipitate was collected. The inclusion body precipitate was dissolved in 8 M urea denaturation and dissolution buffer, then transferred to a dialysis bag and subjected to a gradient dialysis with decreasing urea concentrations (6 M, 4 M, 2 M, and 0 M urea buffers) at 4℃ for 8-12 h per step, yielding a refolded crude LTF protein solution. The obtained renatured LTF solution was slowly loaded onto a 5 mM Ni-NTA affinity chromatography column pre-equilibrated with low-temperature binding buffer for purification. The concentration of the purified target protein was determined to be 2.73 mg / mL by BCA assay, with a molecular weight of approximately 128 kDa. Enrichment with Anti-His magnetic beads and PCR amplification of the invA gene confirmed that the renatured protein LTF possessed Salmonella-specific capture activity.

[0034] GP37 amino acid sequence (SEQ ID NO.1): MAVGEIQISALPQAALPIDLSDIFHLKQGIEDKRCTLEQLLAPHSSLRNNPHGVTKTQIGLDNVINALQLVAANNLSDITNVDEARANLQIMSSEEVNNLVQQHINDKSNPHNTTKAQVGLSNVQNWTTSNLYNEDADKYATARAVNNLYKAVQASYPVGTIHLSMNPANPSTYLICGGTWELVSKGRALVGYD SDSRPVGSNFGSSSVSLSSNLPSHSIYLTGGGHTHGAAITIDGFDYGNKSTNSFDYGTKATDTTGAHTHSVSGSTNNTGAHTHTVGGHYGGDSIGGKLRVQVYGTEQVSSVAGDHSHTISGSTNTTGNHQHTVAIGAHTHTVAIGAHTHKGTVTLQSSEHTHSGTTGTTGAGQAFSVEQPSFVVYVWQRTA.

[0035] GP38 amino acid sequence (SEQ ID NO.2): MADYKLSELNSIGTIRSDDLLHVRVKKRPEMLGDEDRRMTYQDFLASFKLERFVQIAGSTMTGDLGIVKLLYGGKTVFDPTGSSEINIGDVLKTFKINANGLKLTIADASRSATVYHTLNKPSPNELGMRTNEENDARYARLAVTNTFSGTQNIQGDANLLRLRNQNANNAQYIEGVNLDGSARWWVGIGSNGSDEVKLYNNKYNSVLTVASNISVNKSLAITGQVQPSDFSNLDARYFTQTVANQRFAQLAANNTFSGLNTFSTSVSISSNAAAIMLKNKAVDESLFIQAKDVENNNLWYIGKGGANDNITFYDYKGDTSIVLNSSGAAFNKTVKITGQVQPSDWDNIDSRYIPAATLSTIARTNAQNTFKSGQTIVADGQALLLRSATDSPSLYVRGQNQSGTNKWYVGFSTSNVLGLSNEASGTTLTLDSAGINSNKNLNITGQVKPSDFSNLDARYFTQSASDSRYLRIRSTNFNNGNDDKWAKIATVVMPPSASTAVIEVFGGSGYNYGFPYQASKTEIVLRAGGDSPKGLNIVAWKNSENLIITDIGYVNTSGDTYDIYCRVGPFQNDTTSRVQSSSNASVQLFEAPQTFDDAPQGIVKGTIARYYTSMQKPTPSDIGAYTKAETDQKIAQAVSDSTDLNKIYPVGIVTWFNSNVNPNTALPGLTWTYLNNGVGRTIRIAAANGSDVATTGGSDSVTLAVGNLPSHTHSFSATTSSFDYGTKTTNTTGAHTHQFGGYINSFYGDSSHTSFQPGGGAWTQAAGDHSHSVGIGAHSHTVSGNTGSTGSGSAFSVTNQFYKLMAWVRTA.

[0036] Chaperone protein GP92 amino acid sequence (SEQ ID NO.3): MKTESRVINGKKVNIILLGARDGIKMSMKLGKIVVPTFAQMLSSLTDKDKKEVPMVPFKELVEACFDRIEEINLEEMATMLFQGATVDDFPLNIDTYFQANYGEFI DYLAFALEANFGSFFEASIFKSLTSQMGNTLQTPLTDAAVEATYEEADEMKFVLAIYGMEGCKETLDQLFAMTFSDLLSLRQFLEIQRSYKEEIAYNELRRAGKM.

[0037] The amino acid sequence of the 6×His tag (SEQ ID NO.4): HHHHHH.

[0038] The nucleotide sequence encoding the 6×His tag is 5'-CATCATCACCATCACCAT-3' (SEQ ID NO.5); The nucleotide sequence encoding the gp37 protein is shown in SEQ ID NO.6:

[0039] The nucleotide sequence encoding the gp38 protein is shown in SEQ ID NO.7:

[0040] The nucleotide sequence encoding the gp92 protein is shown in SEQ ID NO.8: .

[0041] Example 2: Model 1 Electrochemical Sensor: Construction and Quantitative Analysis of a Covalently Coupled Phage Sensor 1) The surface of the gold disk electrode (GDE, 3 mm in diameter) was polished with 0.05 μm alumina powder, ultrasonically cleaned sequentially with anhydrous ethanol and ultrapure water, and dried for later use. Nafion membrane solution was added to a 1 mg / mL carboxylated reduced graphene oxide aqueous dispersion (rGO-COOH) to achieve a final concentration of 0.1 wt%. A mixed dispersion of Nafion and rGO-COOH was obtained, and 5 μL of this mixed dispersion was drop-coated onto the electrode surface and dried to form a film. Subsequently, the electrode was immersed in a mixed solution containing 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.1 M N-hydroxysuccinimide (NHS) to activate the carboxyl groups for 30 min, followed by cleaning after activation.

[0042] 2) After cleaning in step 1), place the electrode in a solution with a concentration of 3.0 × 10⁻⁶. 10 The electrode was incubated in a PFU / mL phage BPLT2 solution for 2 h. The carboxyl groups on the electrode surface were cross-linked with the amino groups of the phage capsid protein by a covalent coupling reaction, resulting in a covalent amidation reaction. Finally, non-specific binding sites were blocked with 1% bovine serum albumin (BSA) for 30 min to obtain a covalently coupled phage sensor (i.e., the modified gold disk electrode BPLT2 / rGO-COOH / GDE).

[0043] Application testing: rGO-COOH / GDE: The surface of the gold disk electrode (GDE, 3 mm in diameter) was polished with 0.05 μm alumina powder, ultrasonically cleaned sequentially with anhydrous ethanol and ultrapure water, and dried for later use. A 1 mg / mL aqueous dispersion of carboxylated reduced graphene oxide (rGO-COOH) was drop-coated onto the electrode surface and dried to form a film. The electrode was then immersed in a mixed solution containing 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.1 M N-hydroxysuccinimide (NHS) to activate the carboxyl groups for 30 min. After activation, the electrode was cleaned to obtain the prepared rGO-COOH / GDE gold disk electrode.

[0044] LT2 / BPLT2 / rGO-COOH / GDE: The prepared rGO-COOH / GDE gold disk electrode was placed in a solution with a concentration of 3.0 × 10⁻⁶ g / L. 10 Incubate in BPLT2 solution with PFU / mL for 2 h, and finally block non-specific binding sites with 1% bovine serum albumin (BSA) for 30 min to obtain BPLT2 / rGO-COOH / GDE gold disk electrode. Further fix LT2 to form LT2 / BPLT2 / rGO-COOH / GDE gold disk electrode.

[0045] 1) Electrochemical characterization and detection were performed using a standard three-electrode system, with each modified gold disk electrode as the working electrode, platinum wire as the auxiliary electrode, and silver / silver chloride (Ag / AgCl) as the reference electrode. CV characterization used a 5mM K3[Fe(CN)]6 solution containing 0.1 M KCl as the base solution, with a scan rate of 100 mV / s. DPV testing was performed in 10 mM PBS containing 5 mM K3[Fe(CN)]6, with a potential scan range of -0.1 V to +0.6 V, a pulse amplitude of 50 mV, and a pulse period of 0.1 s.

[0046] The electrochemical behavior of the layer-by-layer modification process of the sensor interface was characterized using cyclic voltammetry (CV). Figure 1 (A). In the case of [Fe(CN)]6 3- / 4- In the electrolyte, the modified rGO electrode exhibited the largest and most significant redox peak current, indicating that rGO possesses good electron transport rate and excellent conductivity. When BPLT2 was immobilized on the rGO surface, the redox peak current decreased significantly, and the peak potential difference increased. This is mainly because the biomolecule BPLT2 has a relatively high impedance, hindering electron transfer at the electrode surface. Further immobilization of LT2 to form an LT2 / BPLT2 / rGO-COOH / GDE modified electrode resulted in a further decrease in the response current, demonstrating that the sensor successfully captured Salmonella Typhimurium LT2.

[0047] To further verify the sensor interface construction process, differential pulse voltammetry (DPV) tests were performed on the electrodes at each modification stage. Figure 1 (B) The results showed a distinct oxidation peak at approximately 0.24 V. With progressive modification of the electrode surface, the DPV peak current exhibited a stepwise decreasing trend: the rGO electrode showed the highest response current, which decreased after modification with BPLT2, and further decreased after capturing Salmonella Typhimurium LT2. This trend is highly consistent with the CV test results, fully confirming the gradual and successful construction of the sensor electrode interface.

[0048] 2) The prepared covalently coupled phage sensor was immersed in a serially diluted suspension of Salmonella typhimurium to carry out the capture reaction.

[0049] Serial dilution of bacterial suspension: The host strain Salmonella phage BPLT2, cultured overnight, was resuspended in 0.01 M PBS (pH 7.4), and its initial concentration was determined by plate counting. 8 Subsequently, a 10-fold serial dilution was performed using PBS to obtain concentrations of 10... 1 10 2 10 3 104 10 5 10 6 10 7 10 8 Prepare a bacterial suspension of CFU / mL for later use.

[0050] Sensor incubation response: Take the gold disk electrode BPLT2 / rGO-COOH / GDE prepared in Example 2, add 10 μL of the bacterial suspension of different concentrations mentioned above, and incubate at 37°C for 30 min. After incubation, gently rinse the electrode surface with PBS buffer to remove non-specifically bound bacteria.

[0051] Electrochemical testing: Electrodes incubated with bacterial solutions of different concentrations were used as working electrodes, with silver / silver chloride as the reference electrode and a platinum wire electrode as the counter electrode, forming a three-electrode system. DPV testing was performed in 10 mM PBS containing 5 mM K3[Fe(CN)]6. The potential scan range was set to -0.1 to +0.6 V, the pulse amplitude was 50 mV, and the pulse period was 0.1 s. Peak current signals were recorded. The blank electrode signal was used as the reference electrode. The electrode signal after binding with bacteria is Calculate the difference in current response .

[0052] Fitting the standard curve: using the logarithm of the target bacterial concentration ( ,in The unit is () is the x-axis, and the current change value is the y-axis. ( Plot a curve with ) as the ordinate. Linear fitting results show that at 10 1 -10 7 Within the range, and They exhibit a good linear relationship, and the linear equation is: Correlation coefficient .according to ( The standard deviation of the blank control measurements is given. The sensor's lowest detection limit (LOD) is calculated using the slope of the standard curve. 95 )for The DPV response of the electrochemical sensor (mode 1) for detecting Salmonella is shown in the standard fitting curve. Figure 2 B, R 2 =0.997, the linear regression equation is y =-1.21668 x +23.85674.

[0053] Optimal bacterial capture time optimization: Take the gold disk electrode BPLT2 / rGO-COOH / GDE prepared in Example 2, and add 10 μL of a 1.2×10⁻⁶ concentration. 6 Salmonella suspensions were incubated at 37°C for 5, 10, 15, 20, 25, and 30 min, respectively. After incubation, the electrode surface was gently rinsed with PBS buffer to remove non-specifically bound bacteria.

[0054] The DPV detection method is the same as above. The DPV oxidation peak current decreases stepwise with the extension of target incubation and capture time. When the incubation time is 15 min, the current decay reaches its maximum (drops to 15.75 μA), at which point the effective recognition and binding sites on the electrode surface tend to saturate. When the incubation time exceeds 15 min (20-25 min), due to the lysis of host bacteria mediated by intact bacteriophages, the release of intracellular ions causes a nonspecific rebound in the detection current (rebounds to 21.91 μA). Therefore, the target capture time of this system is strictly determined to be 15 min in this invention.

[0055] Standard curve and detection limit test: Serial dilution of bacterial suspension: The preparation method is the same as above, preparing concentrations of 1.2 × 10⁻⁶ in sequence. 1 1.2×10 2 1.2×10 3 1.2×10 4 1.2×10 5 1.2×10 6 1.2×10 7 1.2×10 8 Prepare the bacterial suspension for later use.

[0056] Sensor incubation response: Take the gold disk electrode BPLT2 / rGO-COOH / GDE prepared in Example 2, add 10 μL of the bacterial suspension of different concentrations mentioned above, and incubate at 37°C for 15 min according to the above-mentioned optimal capture time (see Figure 3 After incubation, gently rinse the electrode surface with PBS buffer to remove any non-specifically bound bacteria.

[0057] The DPV detection method is the same as above, and the DPV detection results are as follows: Figure 2 As shown in Figure A. Under optimal kinetic conditions for 15 min, the linear range for Salmonella detection by the sensor is 1.2 × 10⁻⁶. 1 -1.2×10 7 CFU / mL, Limit of Detection (LOD) 95 It is 5 CFU / mL.

[0058] Example 3: Mode 2 Electrochemical Sensor: Construction and High-Stability Detection of a Directed Chelation Recombinant Protein Sensor Preparation of gold nanoparticle-modified two-dimensional transition metal carbide (Au-MXene) composite films: Two-dimensional transition metal carbide (Ti3C2T) films were etched and exfoliated. x MX ene A monolayer suspension was ultrasonically dispersed uniformly. Chloroauric acid (HAuCl4) solution was added dropwise to the above suspension in a ratio of 10 mL of 1 mg / mL MXene to 1 mL of 10 mM HAuCl4. In-situ reduction was carried out using the abundant reducing functional groups on the surface of MXene. The reaction was carried out at room temperature (25°C) for 30 min, so that gold nanoparticles grew in situ and were uniformly modified on the surface of MXene nanosheets. After the reaction was completed, the Au-MXene composite material dispersion, i.e., Au-MXene composite solution, was obtained by centrifugation, washing, and resuspending.

[0059] SH-PEG-NTA: A single-molecule self-assembly precursor containing thiol and hyponitrotriacetic acid.

[0060] 1) Take 5 μL of Au-MXene composite solution with a concentration of 0.25 mg / mL and drop it onto the surface of gold disk electrode (GDE, diameter 3 mm). After the solvent evaporates and dries, immerse the electrode in SH-PEG-NTA aqueous solution with a concentration of 1 mM overnight. Construct NTA functionalized monolayer on the surface of gold nanoparticles through Au-S bonds to complete monolayer self-assembly.

[0061] 2) After washing the electrode, immerse it in a Ni solution with a concentration of 10 mM. 2+ The chelation reaction was carried out in the solution for 30 minutes; after rinsing, unbound Ni was removed. 2+ The electrode was then immersed in a 2.73 mg / mL recombinant LTF-BPLT2 protein solution and incubated for 60 min. The His tag then interacted with Ni... 2+ The coordination interactions complete the directional immobilization and assembly of the protein, resulting in a directionally chelated recombinant protein sensor.

[0062] 3) The prepared directional chelation recombinant protein sensor was applied to the quantitative detection of Salmonella Typhimurium. The detection results are as follows: Figure 4 As shown in Figure A, the results indicate that at 9.2 × 10 1 -9.2×10 7Within the CFU / mL concentration range. Since long-tailed fibrin does not possess viral lysis activity, even when the incubation time is extended to 60 min, the interface signal can still maintain excellent insulation barrier layer stability; after five consecutive DPV scans at a Salmonella concentration of 500 CFU / mL, the relative standard deviation (RSD) of the obtained oxidation peak current was only 1.3%, completely solving the signal baseline drift problem caused by intact bacteriophage-mediated lysis.

[0063] 4) Serial dilution of bacterial suspension: The host strain Salmonella phage BPLT2, cultured overnight, was resuspended in 0.01 M PBS (pH 7.4), and its initial concentration was determined by plate counting method. 8 Subsequently, a 10-fold serial dilution was performed using PBS to obtain concentrations of 10... 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Prepare the bacterial suspension for later use.

[0064] Sensor incubation response: Take the phage-modified electrode (i.e., the directional chelation recombinant protein sensor) prepared in Example 3, and add dropwise... The bacterial suspensions of different concentrations were incubated at 37°C for 30 min. After incubation, the electrode surface was gently rinsed with PBS buffer to remove non-specifically bound bacteria.

[0065] Electrochemical testing: Electrodes incubated with bacterial solutions of different concentrations were used as working electrodes, with silver / silver chloride as the reference electrode and a platinum wire electrode as the counter electrode, forming a three-electrode system. DPV testing was performed in 10 mM PBS containing 5 mM K3[Fe(CN)]6. The potential scan range was set to -0.1 to +0.6 V, the pulse amplitude was 50 mV, and the pulse period was 0.1 s. Peak current signals were recorded. The blank electrode signal was used as the baseline. The electrode signal after binding with bacteria is Calculate the difference in current response .

[0066] Fitting the standard curve: using the logarithm of the target bacterial concentration ( ,in The unit is () is the x-axis, and the current change value is the y-axis. ( Plot a curve with ) as the ordinate. Linear fitting results show that at 10 1 -107 Within the range of CFU / mL It exhibits a good linear relationship with lgC, and the linear equation is: Correlation coefficient .according to ( The standard deviation of the blank control measurements is given. The sensor's lowest detection limit (LOD) is calculated using the slope of the standard curve. 95 )for The results are shown below. Figure 4 The linear relationship between the differential pulse voltammetry response current and the Salmonella concentration is shown in the graph (RB). 2 =0.995), the linear regression equation is y =-0.74653 x +7.19476, Limit of Detection (LOD) 95 The concentration dropped to 3 CFU / mL.

[0067] Example 4: Broad-spectrum and specific detection of multiple serotypes of Salmonella using a bacteriophage electrochemical sensor. 1. Preparation and concentration adjustment of test strains Thirty-one Salmonella strains covering the core pathogenic serogroups of Salmonella B, C, D, E, and F were selected. The strain numbers were XC-5-IZ, PG-1-20JX, tho-1, FS-1-7Y, LT2, DX-4-JX, HD-3-JX, 1009, L98, Z90, XC-5-JC, CY-1-6Z, Stanly, SY-1-22N, CP-1-3JC, SM22KDE, SM61KDE, SJS-2-JX, Z35, 11, FS-1-10Z, P159-1, p34-1, Z91, L35, Sen-1, 19SC14RX29, FT-6-5JT, XT-24, P00-1-4-2, and SJS-1-JC. Each bacterial strain was inoculated into LB liquid medium and cultured at 37°C with constant shaking at 180 r / min until the logarithmic growth phase. The bacterial cells were collected by centrifugation, washed three times with 0.01 M PBS (pH 7.4), resuspended, and the concentration of each strain was uniformly adjusted to 1.0 × 10⁻⁶ using the plate count method. 8 CFU / mL, for later use.

[0068] 2. Sensor incubation and specific capture The gold disk electrode BPLT2 / rGO-COOH / GDE prepared in Example 2 was immersed in the above 31 1.0×10 8The electrodes were incubated at 37°C for 15 min in a CFU / mL Salmonella suspension. A blank control group of pure PBS solution without Salmonella was also prepared. After incubation, the electrode surface was gently rinsed with sterile deionized water to remove non-specifically attached impurities and free bacteria.

[0069] 3. Electrochemical detection and signal acquisition The sensor, after incubation, was used as the working electrode, with a platinum wire electrode as the auxiliary electrode and silver / silver chloride (Ag / AgCl) as the reference electrode. It was placed in a 10mM PBS electrolyte containing 5mM K3[Fe(CN)6]. Electrochemical scanning was performed using DPV with the following parameters: potential scan range -0.1~+0.6V, pulse amplitude 50mV, pulse period 0.1s, and three parallel tests per group. n =3).

[0070] 4. Test Results As attached Figure 5 As shown, when the test bacteria were Salmonella lysed by BPLT2 (a total of 31 different serotypes), the DPV oxidation peak current decreased significantly due to the sensor's specific capture of the bacteria and the formation of a non-conductive barrier on the electrode surface. The current values ​​of all test strain groups decreased significantly to approximately 14-16 μA, showing a highly significant decrease compared to the blank control group (approximately 25.3 μA) (***). p <0.001). This result confirms that the phage electrochemical sensor described in this invention has excellent cross-serotype capture and broad-spectrum specific recognition capabilities for Salmonella.

[0071] Application example: reproducibility and resistance to matrix interference testing To verify the practical application value of the electrochemical biosensor, the response of the gold disk electrode BPLT2 / rGO-COOH / GDE prepared in Example 2 to milk and egg samples contaminated with different concentrations of Salmonella was investigated. Three samples were set for each concentration, and each sample was measured three times. The experiment was independently repeated three times. The results were measured by DPV and are shown in Table 1. It can be seen that the electrochemical sensor exhibits extremely high reproducibility and resistance to matrix interference, with a recovery rate of 97.2%~102% in actual samples of milk and eggs spiked with the electrode.

[0072] Table 1 Recovery rates of Salmonella in different food samples using electrochemical biosensors The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An electrochemical sensor for Salmonella detection, characterized in that, include: The conductive electrode substrate, the two-dimensional nanocomposite material modification layer on the surface of the conductive electrode substrate, and the Salmonella-specific phage source recognition element fixed on the surface of the two-dimensional nanocomposite material modification layer; the Salmonella-specific phage source recognition element is selected from at least one of Salmonella phage BPLT2 particles and recombinant long-tailed fibrous protein LTF-BPLT2.

2. The electrochemical sensor according to claim 1, characterized in that, The conductive electrode substrate includes a gold disk electrode.

3. The electrochemical sensor according to claim 1, characterized in that, The pairing method between the two-dimensional nanocomposite material modification layer and the Salmonella-specific bacteriophage recognition element is selected from any one of the following two modes: The electrochemical sensor of mode one: the two-dimensional nanocomposite material modification layer is a carboxylated reduced graphene oxide film, the Salmonella-specific phage source recognition element is Salmonella phage BPLT2 particles, and the Salmonella phage BPLT2 particles are solidified on the surface of the carboxylated reduced graphene oxide film through covalent crosslinking amide bonds. The electrochemical sensor in mode two: the two-dimensional nanocomposite modification layer is an Au-MXene composite film, and the Salmonella-specific phage recognition element is a recombinant long-tailed fibrin LTF-BPLT2 with a histidine tag. The recombinant long-tailed fibrin LTF-BPLT2 is transmitted via Ni... 2+ -His affinity coordination is directionally cured on the surface of Au-MXene composite film.

4. The electrochemical sensor according to claim 3, characterized in that, The preparation method of the electrochemical sensor of Mode 1 includes the following steps: 1) A composite dispersion of rGO-COOH and Nafion was drop-coated onto the polished and cleaned gold disk electrode surface, and dried to form a film, thus obtaining an rGO-COOH / GDE modified electrode; wherein the modification concentration of rGO-COOH was 1.0 mg / mL, and the final concentration of Nafion in the composite dispersion was 0.1 wt%. 2) The rGO-COOH / GDE modified electrode was immersed in a MES buffer containing EDC and NHS to activate the carboxyl functional groups. After washing, it was placed in a container with a titer of 3.0 × 10⁻⁶. 10 Cross-linked electrodes were obtained by incubating the PFU / mL phage BPLT2 solution for 2 h. 3) Remove the cross-linked electrode, clean it, and then block the non-specific sites with 1% bovine serum albumin solution to obtain the electrochemical sensor of mode one.

5. The electrochemical sensor according to claim 3, characterized in that, The preparation method of the electrochemical sensor of mode two includes the following steps: 1) Au-MXene was coated onto the surface of a gold disk electrode to obtain an Au-MXene / GDE modified electrode; wherein the modification concentration of Au-MXene was 0.25 mg / mL; 2) The Au-MXene / GDE modified electrode was immersed in SH-PEG-NTA solution overnight to construct an NTA-functionalized electrode; 3) After washing, the NTA-functionalized electrode was immersed in a 10 mM nickel ion solution for coordination chelation for 30 min, and then incubated in a 2.73 mg / mL recombinant long-tail fibroin LTF-BPLT2 solution for 60 min. The electrode was then subjected to Ni... 2+ -His affinity enables the targeted immobilization of specific recognition proteins, thus obtaining the electrochemical sensor of mode two.

6. The use of the electrochemical sensor according to any one of claims 1-5 in the detection of Salmonella for non-diagnostic purposes and / or in the preparation of products for detecting Salmonella.

7. A rapid quantitative detection method for non-diagnostic Salmonella based on the electrochemical sensor described in any one of claims 1-5, characterized in that, Includes the following steps: 1) The electrochemical sensor was co-incubated with a suspension containing the target Salmonella to obtain the working electrode after capturing the target bacteria; 2) The working electrode after capturing the target bacteria is combined with a platinum wire auxiliary electrode and a silver / silver chloride reference electrode to form a three-electrode system; 3) Transfer the working electrode after capturing the target bacteria to a container containing potassium ferricyanide [Fe(CN)6]. 3- / 4- In the electrolyte substrate of the redox probe ions, differential pulse voltammetry was used to scan and record the oxidation peak current response signal at the electrode interface using an electrochemical workstation. 4) Based on the attenuation amplitude of the oxidation peak current response signal, compare it with the standard curve to obtain the concentration of Salmonella in the sample to be tested.

8. The rapid quantitative detection method according to claim 7, characterized in that, The Salmonella to be tested includes Salmonella Typhimurium, which is a Salmonella Typhimurium that can be lysed by bacteriophage BPLT2.

9. The rapid quantitative detection method according to claim 7, characterized in that, The electrochemical sensor includes an electrochemical sensor of mode one and / or an electrochemical sensor of mode two; when the electrochemical sensor of mode one is used for detection, the capture incubation time for the target bacteria is 15 min. When the electrochemical sensor of Mode 2 is used for detection, its electrochemical response signal corresponds to the Salmonella concentration at 9.2 × 10⁻⁶. 1 -9.2×10 7 It exhibits a good linear relationship within the CFU / mL range, and the standard curve regression equation is: y =-0.74653 x +7.19476, of which y This represents the peak current value of the DPV circuit, in microamps. x lg C represents the logarithm of the Salmonella concentration.

10. The rapid quantitative detection method according to claim 9, characterized in that, When the electrochemical sensor of Mode 1 is used for detection, the linear range for Salmonella detection is 1.2 × 10⁻⁶. 1 -1.2×10 7 CFU / mL.

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