Dual-mode food-borne pathogenic bacterium detection method and kit based on difunctional nano-enzyme

Abstract

According to the bifunctional nano-enzyme-based bimodal food-borne pathogenic bacterium detection method and kit disclosed by the invention, an identification probe is a bifunctional probe of an Au-coated PB nano-enzyme modified pathogenic bacterium specific antibody Ab2, and a colorimetric-Raman bimodal detection system is constructed by matching with an Ab1 modified magnetic capture probe and a Raman label and combining a magnetic separation technology; the dual-mode detection system has the advantages of high sensitivity (LDD = 1.9 CFU / mL), high specificity, simplicity and convenience in operation, rapidness and stability when being used for detection, the reliability and accuracy of detection are remarkably improved through mutual verification of two modes of Raman detection and colorimetric detection, and the dual-mode detection system is suitable for on-site rapid detection of food-borne pathogenic bacteria in food matrixes such as fruit juice and the like and has a wide application prospect. And an effective technical means is provided for food safety detection.

Description

Technical Field

[0001] This invention belongs to the field of foodborne pathogen detection technology, specifically to a dual-mode foodborne pathogen detection method and kit based on bifunctional nanozymes. Background Technology

[0002] Foodborne disease prevention and control is one of the core issues in the global public health field. Related testing technologies are mainly applied in areas such as food quality and safety supervision, food production and processing control, import and export food inspection and quarantine, and public health emergency response. Fruit juice, as a beverage with extremely high global consumption, has a production chain covering multiple stages including raw material planting, harvesting, processing, storage, transportation, and end-user sales, and its trade volume has consistently ranked among the top in the soft drink industry. However, the acidic and high-water-content characteristics of fruit juice make it highly susceptible to becoming a breeding ground for acid-resistant and heat-resistant microorganisms. Bacillus cyclophosphamide is a typical example—this type of bacteria can survive even after the high-temperature sterilization process during fruit juice processing and can subsequently multiply during product storage or sales, leading to spoilage phenomena such as off-flavors, turbidity, and bottle bulging. Beyond the fruit juice industry, contamination by such foodborne pathogens exists to varying degrees in multiple food sub-sectors, including processed fruit and vegetable products, dairy products, and bottled drinking water. This not only causes substantial economic losses for food companies but also directly threatens consumer health, causing acute gastrointestinal symptoms such as vomiting, diarrhea, and abdominal pain. In severe cases, it can lead to systemic infections, posing a threat to the lives of vulnerable groups (the elderly, children, and those with weakened immune systems). Therefore, developing technologies capable of rapidly and accurately detecting these pathogens on-site is of paramount practical importance for promptly preventing the circulation of contaminated food, reducing the risk of foodborne disease outbreaks, and safeguarding public health and the stable development of the food industry.

[0003] Currently, there are many pressing issues to be addressed in the practical application of foodborne pathogen detection. From the perspective of detection timeliness, streamlined quality control in food production lines, random inspections by market regulators, and emergency investigations of sudden contamination incidents all require results within a short timeframe to quickly decide whether to release products or initiate control measures. However, existing conventional testing methods struggle to meet this immediate need. From the perspective of adaptability to different testing scenarios, much of the testing work needs to be completed on-site, where professional laboratory equipment and clean environments are often lacking. This places high demands on the ease of operation and interference resistance of testing technologies; traditional methods relying on complex instruments cannot be flexibly adapted. From the perspective of detection accuracy, trace pathogens (i.e., extremely low concentrations of pathogens) are a significant source of foodborne disease outbreaks, especially in easily contaminated foods such as fruit juices. Even small amounts of pathogens can multiply rapidly during storage, requiring detection technologies capable of accurately capturing low concentrations of pathogens. However, many existing methods lack the sensitivity to achieve trace monitoring. In addition, the matrix of different food samples varies greatly (such as the pigments and proteins in fruit juice, and the fats in dairy products). These matrix components can easily interfere with the detection signal, leading to deviations in the detection results and affecting the accurate judgment of food contamination status. At the same time, a single detection mode can often only verify the detection results from one dimension. Once it is interfered with by external factors, misjudgments may occur, and the reliability of the detection cannot be guaranteed.

[0004] To address the aforementioned challenges in detecting foodborne pathogens, the industry has developed and implemented various detection technologies, primarily falling into three categories. The first category is microbial culture, currently considered the gold standard in foodborne pathogen detection. Its core principle involves inoculating bacteria from a sample onto a specific culture medium, culturing them under suitable temperature and environment to allow bacterial proliferation and the formation of visible colonies. The morphology and characteristics of these colonies are then used to determine the presence and quantity of the target pathogen. The second category is polymerase chain reaction (PCR) technology. This technology utilizes DNA polymerase to rapidly amplify unique DNA fragments specific to the target pathogen in vitro. The presence of the target pathogen is confirmed by detecting these amplified DNA fragments, essentially providing a magnified genetic identification of the pathogen. The third category is enzyme-linked immunosorbent assay (ELISA). This method utilizes the specific binding reaction between antigen and antibody. An enzyme is labeled onto the antibody, and the enzyme catalyzes a specific substrate to produce a color change signal, which indicates the presence and approximate concentration of the target pathogen in the sample. Furthermore, to address the limitations of single detection modes, some novel detection technologies have emerged, such as colorimetric methods based on nanozyme catalysis (using nanomaterials to simulate the catalytic function of enzymes, and determining the pathogen concentration by the intensity of the color of the solution after the reaction) and surface-enhanced Raman scattering (SERS), attempting to improve detection performance through different signal output modes.

[0005] While existing technologies have alleviated the demand for foodborne pathogen detection to some extent, many insurmountable shortcomings remain, and an ideal detection solution has yet to be formed. Specifically, while microbial culture methods provide reliable results, the detection cycle is too long, typically requiring 24 to 36 hours or even longer, far from meeting the needs of rapid screening and emergency testing. By the time the results are available, contaminated food may have already entered the market, causing adverse consequences. Although PCR technology offers faster detection than culture methods, it demands extremely high levels of expertise from both the operating environment and personnel, requiring sophisticated PCR amplification instruments, gel imaging systems, and other specialized equipment. Furthermore, the detection cost is high, and the sensitivity is limited by factors such as primer design and inhibitors in the sample, hindering its widespread adoption in grassroots and field testing scenarios. While ELISA technology is relatively simple to operate and has high specificity, its core reliance on natural enzymes as signal tags presents inherent problems such as poor stability, susceptibility to inactivation due to environmental factors like temperature and pH, high preparation costs, and significant performance variations between batches. This directly results in the detection sensitivity of ELISA technology typically only reaching 10. 4 CFU·mL -1 The current level of detection is insufficient to meet the requirements for precise monitoring of trace pathogens. Furthermore, the exploration of new detection technologies also has its shortcomings: colorimetric methods based on nanozyme catalysis are easily interfered with by the color of the sample matrix itself; for example, the color of dark fruit juice can mask the color change of the solution after the reaction, leading to inaccurate judgment of the detection results and limited quantitative precision. While SERS technology boasts extremely high sensitivity and resistance to water interference, the distribution of reporter molecules on the detection substrate is difficult to achieve completely uniformity, easily resulting in poor reproducibility of the detection signal. This means that the same batch of samples may produce different results under the same conditions, affecting the reliability of the detection.

[0006] In summary, existing detection technologies generally fail to simultaneously achieve high sensitivity, high specificity, ease of operation, rapid stability, and strong anti-interference capabilities. In particular, they lack integrated sensing platforms that can organically combine different signal output modes and improve detection reliability through mutual verification. This makes it difficult to meet the urgent needs for efficient and accurate detection of foodborne pathogens in scenarios such as food quality and safety supervision and emergency prevention and control. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a dual-mode detection method and kit for foodborne pathogens based on bifunctional nanozymes. The recognition probe is a bifunctional probe of Au@PB nanozyme modified with pathogen-specific antibody Ab2, which is combined with a magnetic capture probe modified with Ab1 and a Raman tag. Combined with magnetic separation technology, a colorimetric-Raman dual-mode detection system is constructed to achieve portable and rapid detection of foodborne pathogens.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a bifunctional recognition probe for detecting foodborne pathogens, comprising an Au@PB nanozyme with peroxidase catalysis and Raman-enhanced substrate function, and an antibody Ab2 specifically targeting the foodborne pathogen modified on the surface of the Au@PB nanozyme and containing streptavidin, wherein: The Au@PB nanozyme comprises a gold nanoparticle core and a Prussian blue shell.

[0009] Furthermore, the preparation of the Au@PB nanozyme is as follows: Gold nanoparticles with a particle size of 50 nm were obtained by redox reaction of tetrachloroauric acid and sodium citrate. The core of gold nanoparticles was etched with potassium ferricyanide to obtain an Au@CN nanoparticle solution; Equal volumes of potassium ferrocyanide aqueous solution and ferric chloride hexahydrate aqueous solution were added to the Au@CN nanoparticle solution, and the mixture was stirred to react. Prussian blue was generated in situ on the surface of the Au@CN nanoparticles, yielding Au@PB nanozyme.

[0010] Furthermore, the preparation of the gold nanoparticle core is specifically as follows: By mass-volume percentage, 300 μL of 1% tetrachloroauric acid solution and 30 mL of ultrapure water were taken, stirred and mixed, and heated to boiling. Immediately after boiling, 180 μL of 1% sodium citrate solution was added. After the reaction was completed, the solution was cooled to room temperature to obtain the gold nanoparticle core solution. The preparation of Au@PB nanozymes is as follows: 5 mL of AuNP seed solution and 200 μL of 0.5 mM K4[Fe(CN)6] aqueous solution were mixed and treated for 5 min to form Au@CN nanoparticles; Subsequently, 1 mL of 0.1 mM K₄[Fe(CN)₆] aqueous solution and 1 mL of 0.1 mM FeCl₃ were added to the Au@CN nanoparticle solution. The product was collected by stirring vigorously at 25°C for 3 hours in an aqueous solution of 6H2O, followed by centrifugation at 10000 rpm / min for 5 minutes, and the product was washed to obtain Au@PB nanozyme.

[0011] This invention also provides a dual-mode detection kit for foodborne pathogens, comprising a capture probe, a recognition probe, a Raman tag, colorimetric detection reagents, and Raman detection reagents, wherein: The identification probe is the aforementioned identification probe; The capture probe is a magnetic microsphere modified with a specific antibody Ab1 against the foodborne pathogen to be detected; The Raman tag is a polystyrene microsphere loaded with a Raman reporter, and the surface of the polystyrene microsphere is modified with single-stranded DNA; the single-stranded DNA is a single-stranded DNA modified with both amino and biotin. The colorimetric detection reagents include: buffer system reagents and colorimetric reaction reagents; The Raman detection-related reagents include: buffer system reagents and microsphere dissociation reagents.

[0012] Furthermore, in the colorimetric detection reagents: the buffer system reagent is PBST solution, and the colorimetric reaction reagent is BR buffer solution, TMB solution, and H2O2 solution at pH=4.35; In the Raman detection reagents: the buffer system reagent is PBS solution, and the microsphere dissociation reagent is THF reagent.

[0013] Furthermore, the linear range of this kit is 1×10⁻⁶. 1 -1×10 7 The minimum detection concentration for Raman spectroscopy is 1.9 CFU / mL, and the minimum detection concentration for colorimetric spectroscopy is 13.5 CFU / mL.

[0014] The present invention also provides a dual-mode detection method for foodborne pathogens, which uses the above-mentioned dual-mode detection kit for foodborne pathogens.

[0015] Furthermore, when performing colorimetric testing: The capture probe was added to the buffer system and vortexed for 30 seconds. The sample solution and recognition probe were added and vortexed for 30 seconds. After incubation at 37°C for 45 minutes, the sample was adsorbed by a magnet, washed, and resuspended in the buffer system to obtain the immune complex solution. The immune complex solution and colorimetric reagent were mixed and incubated for 15 min. The supernatant was then taken and its absorbance was measured at 652 nm. The ratio of the identification probe to the capture probe is 4:1, and the concentration of the capture probe is 5 mg / mL. The volume of the sample solution used is 50 μL; The ratio of the immune complex solution to the colorimetric reagent is 1:10.

[0016] Furthermore, Raman spectroscopy is performed after colorimetric detection: The immune complex solution was mixed with the Raman tag, incubated at 37°C with shaking for 25 min, and then magnetically separated and washed before being resuspended in a buffer system to obtain mixture 1. Add the microsphere dissociation reagent to mixture 1, and react to obtain mixture 2; Two drops of the mixture were placed on a glass slide wrapped in aluminum foil, and Raman spectroscopy was performed. The ratio of the immune complex solution, Raman tag, and microsphere dissociation reagent is 50:3:30. The amount of the mixture 2 is 2 μL, and the laser power for Raman detection is 10 mW.

[0017] Furthermore, the foodborne pathogen is *Bacillus cyclophosphamide*. A.acidoterrestris The laser power for Raman detection was 10mW.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a bifunctional recognition probe for detecting foodborne pathogens, comprising an Au@PB nanozyme integrating peroxidase-like catalytic activity and surface-enhanced Raman scattering (SERS) enhancement capability. Its bifunctional characteristics stem from a unique core-shell structure and component synergistic effects, exhibiting significant advantages. The bifunctional mechanism is well-defined and highly efficient. (1) Au@PB nanozymes exhibit peroxidase-like activity, the core of which originates from the Fe in the Prussian blue (PB) shell of its core-shell structure. 3+ / Fe 2+ Synergistic effect of redox cycle and electron conduction in gold nucleus (Au): Fe in PB shell 3+ It can react with hydrogen peroxide (H2O2) and be reduced to Fe. 2+ At the same time, it generates hydroxyl radicals with strong oxidizing power. OH), while Fe 2+ It can also be re-oxidized to Fe by H2O2. 3+ This forms a continuous catalytic cycle, thereby efficiently catalyzing the oxidation and color development of 3,3,5,5-tetramethylbenzidine (TMB); the excellent electronic conductivity of the gold core can accelerate the Fe... 3+ with Fe 2+ Electron transfer between the core and shell further enhances catalytic efficiency, and the stability of the core-shell structure ensures that the catalytic activity remains stable over a wide pH range (4.0-9.0) and temperature range (4℃-40℃). Compared with natural horseradish peroxidase (HRP), it is less susceptible to inactivation due to environmental factors, providing an efficient and stable catalytic basis for colorimetric detection.

[0019] (2) Au@PB nanozymes can serve as Raman enhancement substrates, primarily due to the synergistic effect of their core-shell structure and components, forming a multi-enhancement mechanism: the gold core (Au) generates localized surface plasmon resonance (LSPR) under laser irradiation, creating a strong localized electromagnetic field hotspot, which lays the foundation for Raman signal enhancement; the Prussian blue (PB) shell, through Fe... 3+ / Fe 2+The redox cycle regulates the electron density on the gold core surface, further enhancing the LSPR effect. At the same time, its cyano (-CN) group can form a weak interaction with Raman reporter (such as 4-NTP), increasing the molecular adsorption capacity. In addition, the rough and porous morphology of the Au@PB nanozyme surface increases the number of hot spots and expands the effective adsorption area, allowing more reporter to be in the strong electric field region. Combined with the high concentration of 4-NTP released after Raman tag depolymerization and the precise anchoring mediated by biotin-streptavidin, the Raman signal is ultimately efficiently amplified, generating a strong and stable SERS signal, providing a guarantee for trace detection.

[0020] This invention provides a dual-mode detection kit for foodborne pathogens, employing a sandwich-like immune recognition mode of capture probe-target bacteria-recognition probe. Relying on the specific binding of antigen and antibody, it achieves precise capture of the target pathogen. Specifically: (1) High recognition specificity: The antibodies modified by the capture probe (MB-Ab1) and the recognition probe (Au@PB-Ab2) target different antigenic epitopes on the surface of Bacillus cyclophosphamide, respectively. This dual specificity effectively avoids cross-reaction with other foodborne pathogens (such as Escherichia coli and Salmonella). Specificity experiments show that the signal response values ​​of non-target bacteria are no different from those of blank samples, and the detection specificity is over 99%.

[0021] (2) High binding affinity: The binding constant of the antibody to the antigen is much higher than that of the nucleic acid probe to the target sequence. It can quickly form a stable sandwich complex in complex food matrices, shortening the incubation time (specific binding is completed within 45 minutes).

[0022] (3) Strong resistance to matrix interference: Combined with magnetic separation technology, the sandwich complex can be quickly separated from interfering components such as proteins, pigments, and bacteria in the sample matrix, reducing the influence of matrix background on the signal. In fruit juice samples, the detection recovery rate remained at 99.28%-101.30%, and the relative standard deviation (RSD) was ≤9.88%.

[0023] Furthermore, the Raman tag employs a design where polystyrene (PS) microspheres encapsulate the Raman reporter (4-NTP). Through signal enrichment and controlled release mechanisms, it significantly enhances the sensitivity of SERS detection, as detailed below: (1) High and stable reporter loading: 200nm PS microspheres have a high specific surface area, and a single microsphere can load approximately 1.2 × 10⁻⁶. 6 Each 4-NTP molecule, compared to direct adsorption Raman tags, increases the reporter loading by more than 50 times. The encapsulation effect of PS microspheres prevents the loss of 4-NTPs during storage and detection, ensuring signal stability. After 25 days of storage at 4°C, the reporter retention rate reaches over 90%.

[0024] (2) Controlled signal release and enrichment: By depolymerizing PS microspheres with THF, a high concentration of 4-NTPs can be rapidly released from the surface of Au@PB nanozymes, creating a localized high-concentration reporter environment. Combined with the SERS enhancement effect of the nanozymes, secondary signal amplification is achieved. This mechanism reduces the SERS detection limit to as low as 1.9 CFU / mL, compared to traditional ELISA methods (where the detection limit is typically 10 CFU / mL). 4 The sensitivity of CFU / mL is improved by three orders of magnitude.

[0025] (3) Anti-nonspecific adsorption: The single-stranded DNA (ssDNA) modified on the surface of PS microspheres binds specifically to the recognition probe only through biotin-streptavidin, avoiding nonspecific adsorption of Raman tags to sample matrix or magnetic beads and reducing background signal.

[0026] This invention provides a dual-mode detection method for foodborne pathogens, constructing a dual-mode detection model of rapid colorimetric screening + precise SERS quantification. This effectively avoids the shortcomings of single-mode detection and adapts to various detection needs: the colorimetric method is simple to operate, requiring no professional instruments; rapid on-site screening can be achieved by visually observing the color change of the solution (colorless → blue), with qualitative judgment completed within 15 minutes. It is suitable for on-site scenarios such as food production lines and market supervision, lowering the detection threshold. The SERS method provides precise quantification. Utilizing the signal amplification effect of Raman tags and the enhancement effect of Au@PB, it can achieve precise quantification over a wide concentration range, meeting the needs of laboratory trace analysis and emergency testing. The two modes mutually validate each other: the colorimetric method quickly identifies positive samples, while the SERS method accurately determines the concentration, effectively reducing the risk of false positives and false negatives.

[0027] In summary, the detection system constructed using this invention can achieve simultaneous, rapid, and portable detection of foodborne pathogens, with excellent detection performance and strong practicality. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the experimental principle of a dual-mode foodborne pathogen detection method based on bifunctional nanozymes according to the present invention.

[0029] Figure 2 This is a TEM image of the Au@PB nanozyme.

[0030] Figure 3 (A) The ultraviolet spectrum of Au@PB nanozyme; (B) The Raman spectrum of Au@PB nanozyme.

[0031] Figure 4 (A) Catalytic activity of Au@PB nanozymes; (B) Raman activity of Au@PB nanozymes.

[0032] Figure 5Zeta potential analysis for capture probe (A) and recognition probe (B).

[0033] Figure 6 SEM images: (A) PS; (B) PS@4-NTP; (C) PS@4-NTP@ssDNA; (D) Raman spectra before and after Raman tag depolymerization.

[0034] Figure 7 The sensitivity and detection limit (LOD) of colorimetric detection (A)(B) and Raman detection (C)(D) are evaluated.

[0035] Figure 8 For specific detection: (A) colorimetric method; (B) Raman method.

[0036] Figure 9 For testing real samples: (A) colorimetric method; (B) Raman method.

[0037] Figure 10 For sensor stability analysis: (A) Storage stability of identification probe; (B) Storage stability of Raman tag. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0039] This invention provides a dual-mode detection method for foodborne pathogens based on bifunctional nanozymes, mainly comprising: 1) preparing a bifunctional Au@PB nanozyme with peroxidase catalytic function and a substrate for enhancing Raman signal; 2) preparing a recognition probe (Au@PB-Ab2) for the target analyte using the Au@PB nanozyme; 3) preparing a capture probe (MB-Ab1) for the target analyte using magnetic microspheres MB; 4) preparing a specific Raman tag (PS@4-NTP@ssDNA); 5) establishing a dual-mode detection method using colorimetry and Raman spectroscopy based on the recognition probe and the capture probe. By optimizing experimental parameters and combining magnetic separation technology, this method is more time-saving and faster than traditional enzyme-linked immunosorbent assay (ELISA) methods, enabling rapid quantitative detection of foodborne pathogens. The specific implementation steps of the above method are as follows: 1) Preparation of Au@PB nanozymes with peroxidase catalysis and Raman-enhanced substrate functions Gold nanoparticles (AuNPs) with a particle size of 50 nm were synthesized using a redox reaction of tetrachloroauroic acid and sodium citrate. Using these AuNPs as the core, gold nanoparticles were then synthesized using potassium ferricyanide (K3[Fe(CN)6]) as the CN core. AuNPs were etched, and then potassium ferrocyanide (K4[Fe(CN)6]) aqueous solution (0.1 mM, 1 mL) and an equal volume of ferric chloride hexahydrate (FeCl3·6H2O) aqueous solution were added as ligands and stirred to prepare Au@PB nanozyme. After centrifugation and washing, it was stored at 4℃ for later use. 2) Preparation of recognition probe Au@PB-Ab2 Au@PB nanozyme was mixed with phosphate-buffered saline (PBS) and Ab2, a specific antibody containing streptavidin, and incubated for 1 h. Then, bovine serum albumin (BSA) aqueous solution was added and incubated for another 1 h. The final product was collected by centrifugation (4 °C, 8000 rpm, 5 min), washed twice with PBST, and then dispersed in PBST for later use at 4 °C.

[0040] The concentrations of PBS and PBST were 0.01 M, with a pH of 7.4; the concentration of BSA was 1% (w / v); and the concentration of Ab2, a specific antibody containing streptavidin, was 8 μg / mL. 1 The specific antibody Ab2 is Bacillus cyclophosphamide (Cyclocarya) A.acidoterrestris Antibody detection.

[0041] 3) Preparation of the capture probe MB-Ab1 The magnetic microsphere mother liquor was sonicated for 10 minutes to fully disperse it. Transfer 400 μL of magnetic microspheres (5 mg / mL) to a centrifuge tube, perform magnetic separation, wash twice with MES (0.015 M, pH=5.5), and bring the volume to 5 mg / mL (0.4 mL). Vortex and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (20 µL each, 10 mg / mL), vortex until mixed, and incubate at 37 °C for 30 min. Magnetic separation removes the supernatant, wash once with MES, and resuspend to 0.4 mL. Vortex and add 40 µg of capture antibody Ab1, and incubate at 37 °C for 4 h. Wash twice with PBST, perform magnetic separation, add 0.2 mL of 1% BSA (prepared with PBST), and incubate at 37 °C for 1 h for blocking. Magnetic separation removes the supernatant, bring the volume to 0.4 mL with PBST, aliquot, and store at -20 °C.

[0042] Among them, the capture antibody Ab1 is Bacillus cyclophosphamide (Cyclocarya) A.acidoterrestris The capture antibody Ab1 was directly coupled to magnetic microspheres, and the concentrations of EDC and NHS were both 10 mg / mL.

[0043] 4) Preparation of Raman tags Polystyrene nanospheres (PS) were mixed with deionized water and vortexed for 1 min. Then, 4-nitrobenzenethiophenol (4-NTP) was added, and the mixture was vortexed at room temperature for 5 min. After centrifugation (10,000 rpm, 30 min) and washing, PS@4-NTP was obtained and suspended in deionized water.

[0044] PS@4-NTP was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (crosslinking agent, EDC), N-hydroxysuccinimide (crosslinking agent, NHS), and 2-morpholinoethanesulfonic acid (buffer, MES) to activate the carboxyl groups on the surface of polystyrene microspheres. After centrifugation, washing, and resuspending, amino- and biotin-modified single-stranded DNA (ssDNA) was added. The single-stranded DNA was coupled to the activated carboxyl groups through amino modification to construct the Raman tag. After incubation at 37°C, washing yielded PS@4-NTP@ssDNA, which was suspended in PBS and stored at 4°C. Streptavidin and the biotin in ssDNA achieved Raman tag coupling with the recognition probe through a specific affinity reaction. The ssDNA sequence is as follows: 5`(Biotin)-GTCCTCGCTCTTTCCGCATTTTCCCGTATGCGCTTTGTATTATTT-3`(NH2); The concentration of 200nm PS microspheres was 10% (w / v), and the concentration of 4-NTP was 2×10⁻⁶. -3 M, EDC concentration is 40mM, NHS concentration is 100mM, and ssDNA concentration is 8nM; 2. A dual-mode rapid detection method for foodborne pathogens, comprising the following steps: 1) Colorimetric detection Add 15 μL of the prepared capture probe and 45 μL of PBST to a 2 mL centrifuge tube and vortex for 30 s. Then, add 5 μL of sample solution and 50 μL of recognition probe, vortex for 30 s, and incubate at 37 °C for 45 min. The antibodies on the recognition and capture probes can bind to the surface antigen epitopes of the pathogenic bacteria. In this way, the pathogenic bacteria are sandwiched between the capture and recognition probes, initially forming a sandwich structure. After the reaction, the unbound elements are removed by magnetic adsorption and washing, and the mixture is resuspended in 50 μL of PBST to obtain a sandwich-type immune complex solution.

[0045] The sandwich-type immune complex solution, 380 μL of pH 4.35 BR buffer, 100 μL of LMB (3,3',5,5'-tetramethylbenzidine) solution, and 20 μL of H2O2 solution were incubated for 15 min. The supernatant was then used to measure the absorbance at 652 nm.

[0046] 2) Raman detection Add 3 μL of Raman tag PS@4-NTP@ssDNA to the sandwich immune complex solution from step 1). Anchor the Raman tag to the sandwich complex via streptavidin-biotin affinity reaction. Incubate at 37°C with shaking for 25 min. After magnetic separation and washing, resuspend in 5 μL of PBS. Add 30 μL of THF (tetrahydrofuran) to depolymerize the PS microspheres and release the Raman reporter 4-nitrobenzenethiophenol (4-NTP). A strong SERS signal is generated on the surface of Au@PB nanozyme. Take 2 μL of the mixed solution and drop it onto a glass slide wrapped in aluminum foil for SERS measurement.

[0047] The linear range of detection in this invention is 1×10⁻⁶. 1 -1×10 7 The minimum detection concentration for Raman spectroscopy is 1.9 CFU / mL, and the minimum detection concentration for colorimetric spectroscopy is 13.5 CFU / mL.

[0048] This invention uses 50nm gold nanoparticles synthesized by sodium citrate reduction as the core, which are then etched with K3[Fe(CN)6] and then K4[Fe(CN)6] and FeCl3 are added. Using an aqueous solution of 6H₂O as a ligand, the reaction was stirred, followed by centrifugation and washing to obtain Au@PB nanozymes possessing both peroxidase-like activity and SERS-enhancing capabilities. Based on this, the Au@PB nanozymes were embedded in a biosensor for the rapid detection of foodborne pathogens. The main principle (e.g.) Figure 1): Antibodies Ab1 and Ab2 targeting different antigenic epitopes were coupled onto the surfaces of magnetic beads and Au@PB nanozymes, respectively, to obtain capture probe (MB-Ab1) and recognition probe (Au@PB-Ab2). The capture probe (MB-Ab1) specifically binds to the surface antigen of Bacillus cyclophosphamide through antibody, and the recognition probe (Au@PB-Ab2) binds to the other end of the pathogenic bacteria with the same antigen-antibody specificity, forming an MB-Ab1-pathogenic bacteria-Au@PB-Ab2 sandwich complex. Utilizing the magnetic response characteristics of the magnetite microspheres in the capture probe, the sandwich complex is rapidly separated by an external magnetic field to remove interfering components in the sample matrix, thereby achieving enrichment and purification of the target bacteria. (1) Colorimetric signal: Au@PB bifunctional nanozymes have peroxidase-like activity, which can catalyze the substrate TMB to react with H2O2 to produce a blue product. Qualitative and quantitative detection can be achieved by visual observation or measurement of absorbance at 652nm. The higher the concentration of the target bacteria, the stronger the color intensity. (2) SERS signal: The Ab2 in the recognition probe is modified with streptavidin, which specifically binds to biotin on the Raman tag (PS@4-NTP@ssDNA), anchoring the Raman tag to the sandwich complex. Adding THF depolymerizes the PS microspheres, releasing the Raman reporter 4-NTP. Under the SERS enhancement effect of Au@PB nanozyme, a strong characteristic signal is generated. Highly sensitive quantitative detection is achieved by reading the intensity of the characteristic peak; the concentration of the target bacteria is positively correlated with the intensity of the characteristic peak.

[0049] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0050] Example 1 Au@PB nanozyme synthesis identification and catalytic function determination Preparation of Au: First, the AuNP seed solution was prepared using the sodium citrate reduction method. 300 μL of 1% (w / v) HAuCl4 and 30 mL of ultrapure water were stirred. The mixture was rapidly heated to boiling while stirring at maximum speed, ensuring no splashing. Immediately after boiling, 180 μL of 1% (w / v) sodium citrate was added. After 6 minutes, stirring was stopped, and the Erlenmeyer flask was removed from the heating device. The color change of the AuNPs at this point indicated maturity. The prepared AuNP seed solution was cooled to room temperature and stored at -4°C for later use.

[0051] Preparation of Au@PB nanozymes: To prepare Au@PB nanozymes, 5 mL of AuNP seed solution was taken and treated with K3[Fe(CN)6] aqueous solution (0.5 mM, 200 μL) for 5 min, followed by treatment with CN... Etching of AuNPs surfaces. AuNP surfaces are coated with CN. Encapsulation formed Au@CN nanoparticles (Au@CNNPs). K4[Fe(CN)6] aqueous solution (0.1 mM, 1 mL) and an equal volume of FeCl3·6H2O aqueous solution were added as ligands to the Au@CNNPs solution, and the mixture was vigorously stirred at 25 °C for 3 h to prepare Au@PB nanozymes. Subsequently, the resulting solution was centrifuged at 10,000 rpm / min for 5 min to remove the supernatant, and the collected Au@PB nanozymes were washed three times with ultrapure water to remove excess ligands and stored at -4 °C.

[0052] TEM samples were prepared, and their morphology and elemental distribution were characterized. The characterization results are as follows: Figure 2 As shown, the synthesized Au@PB nanozyme has a statistically significant particle size of approximately 52 nm, and elemental distribution analysis revealed a uniform elemental distribution. The UV spectrum of Au@PB exhibits a characteristic peak at 700 nm, and the Raman spectrum shows a peak at 2156 cm⁻¹. -1 It has obvious characteristic peaks, such as Figure 3 As shown, the successful synthesis of Au@PB is demonstrated.

[0053] The POD-like catalytic activity of Au@PBNP nanozymes was investigated using a TMB-H2O2 colorimetric reaction system catalyzed by Au@PBNP nanozymes. Figure 4 As shown in Figure A, neither Au@PBNP nanozyme nor H2O2 can effectively catalyze the oxidation of TMB to a blue color on their own. However, when Au@PBNP nanozyme and H2O2 are added to the TMB solution simultaneously, the mixed solution turns a distinct blue color and exhibits the highest UV-vis absorbance at 652 nm, confirming that Au@PBNP nanozyme possesses POD-like catalytic activity.

[0054] Au@PB nanozyme at 2156 cm⁻¹ 1 The Au@PB nanoparticles exhibit a single tensile vibration Raman peak, but their Raman intensity is relatively weak. The surface-enhanced Raman scattering activity of the Au@PB nanoparticles was found to be very strong. First, 4-NTP was used as a Raman reporter to verify the SERS properties of the Au@PB nanoparticles. Figure 4 As shown in Figure B, in the presence of Au@PB, the 4-NTP is at 1081.9 cm⁻¹. 1 1336.1cm 1 1571.7cm 1The presence of a characteristic spectral band with significantly increased intensity indicates that Au@PB possesses high SERS activity. These results demonstrate the successful preparation of a bifunctional Au@PB nanozyme exhibiting both high SERS activity and peroxidase-like activity.

[0055] Example 2 Preparation of recognition probes and capture probes Preparation of Au@PB-Ab2 (recognition probe): 1 mL of Au@PB nanozyme was mixed with 1 mL of PBS (0.01 M, pH 7.4) and 50 µL of streptavidin-modified specific antibody Ab2 (PBS diluted to 8 μg / mL). After incubation for 1 h, 100 µL of LBSA aqueous solution (1%, w / v) was added and incubation continued for 30 min. The final product was collected by centrifugation, washed twice with PBST, and then dispersed in PBST for later use. Preparation of magnetic beads-Ab1 (capture probe): The magnetic microsphere mother liquor was sonicated for 10 minutes to fully disperse it. Transfer 400 μL of magnetic microspheres (5 mg / mL) to a centrifuge tube, perform magnetic separation, wash twice with MES (0.015 M, pH=5.5), and bring the volume to 5 mg / mL (0.4 mL). Vortex and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (20 µL each, 10 mg / mL), vortex until mixed, and incubate at 37°C for 30 min. Magnetic separation removes the supernatant, wash once with MES, resuspend to 0.4 mL, vortex and add 12.6 µL of antibody, and incubate at 37°C for 4 h. Wash twice with PBST, perform magnetic separation, add 0.2 mL of 1% BSA (prepared with PBST), and incubate at 37°C for 1 h for blocking. Magnetic separation removes the supernatant, bring the volume to 0.4 mL with PBST, and store at -20°C.

[0056] The following analysis confirms the successful modification of antibodies onto the surfaces of Pt@AB nanozymes and magnetic beads: Because the potential increases due to potential neutralization after antibody modification, analysis of the particle surface potential confirms successful antibody modification. Figure 5 As shown in Figure A, because the carboxyl group on MB carries a negative charge, it forms an amide bond with the amino group on the antibody, neutralizing the charge. This results in a decrease in the Zeta potential of MB-Ab1 compared to MBNPs, confirming the successful synthesis of MB-Ab1. Figure 5 As shown in Figure B, the potential of the antibody increased after modification with Au@PB. The key is that the weak positive charge (amino group) of the antibody molecule neutralized part of the negative charge (the -CN group has a negative charge), which confirms the successful synthesis of Au@PB-Ab2.

[0057] Example 3 Preparation of specific Raman tags 50 µL of PS nanospheres were mixed with 950 µL of deionized water and vortexed for 1 min. 250 µL of 2 mM 4-nitrobenzenethiophenol (4-NTP) was added, and the mixture was vortexed for 5 min at room temperature. The mixture was then centrifuged and washed three times (10000 rpm, 20 min) to obtain PS@4-NTP, which was then suspended in deionized water. The PS@4-NTP was centrifuged (10000 rpm, 20 min), and incubated with a mixture of EDC, NHS, and MES to activate the carboxyl groups. After centrifugation, washing, and resuspending, ssDNA was added, and the mixture was incubated at 37 °C. Washing yielded PS@4-NTP@ssDNA, which was then suspended in PBS and stored at 4 °C.

[0058] Figure 6 Images A, B, and C show SEM images of the PS microspheres, indicating that the PS microspheres exhibit uniformly dispersed monodisperse spheres. The diameter of the PS microspheres was 200 nm. After loading with the Raman reporter, the diameter of the PS microspheres increased to approximately 230 nm. The size distribution of the microspheres was uneven, indicating that the THF solution caused the PS microspheres to swell. Figure C shows that after modifying the PS microspheres with ssDNA in a PBS buffer system, the diameter of the PS@4-NTP@ssDNA microspheres was slightly larger than that of PS@4-NTP. After ssDNA modification, the surface morphology became rough, which is presumably due to the EDC / NHS activation of the carboxyl group modification of ssDNA, thus confirming the successful modification of ssDNA on the surface of the PS microspheres. The Raman spectra of PS@4-NTP before and after depolymerization also confirmed the successful synthesis of the Raman tag. Figure 6 D).

[0059] Example 4 Sensitivity detection Under optimal detection conditions, the concentration detected using the constructed sensor was 1×10⁻⁶. 1 -1×10 7 CFU / mL of Bacillus cyclophosphamide (CFU / mL) A.acidoterrestris The obtained ultraviolet spectrum is as follows: Figure 7 As shown in A. From Figure 7 A shows that, as A.acidoterrestris As the concentration increases, the intensity of the ultraviolet signal increases accordingly. Figure 7 B shows A.acidoterrestris Concentration at 1×10 1 -1×10 7 Within the CFU / mL range, the colorimetric signal intensity and A.acidoterrestris The logarithm of the concentration showed a good linear relationship, with the linear regression equation being y = 0.18911x - 0.14858 and the correlation coefficient (R²) being [missing value]. 2 The value is 0.98. The calculated LOD is 13.5 CFU / mL. The Raman spectrum is as follows: Figure 7 As shown in C, it can be seen that, with A.acidoterrestrisAs the concentration increases, the intensity of the ultraviolet signal increases accordingly. Figure 7 D shows A.acidoterrestris Concentration at 1×10 1 -1×10 7 Within the CFU / mL range, the Raman signal intensity and A.acidoterrestris The logarithm of the concentration showed a good linear relationship, with the linear regression equation being y = 1789.0714x + 911.6832, and the correlation coefficient (R²) being [missing value]. 2 The value was 0.98. The calculated LOD was 1.9 CFU / mL.

[0060] The sensitivity results indicate that this sensor is sensitive to... A.acidoterrestris It exhibits extremely high detection sensitivity, enabling effective detection of even very low concentrations of pathogenic bacteria in food. This high sensitivity is primarily attributed to the efficient release mechanism of the Raman reporter molecule in the PS@4-NTP@ssDNA complex and the optimized SERS signal enhancement effect. This sensitivity level is suitable for detecting pathogenic bacteria in food. A.acidoterrestris It provides reliable technical support for rapid and accurate detection, and has important application value in food safety.

[0061] Example 5 Specificity analysis To comprehensively evaluate the constructed colorimetric-SERS dual-mode immunobiosensor against Bacillus cyclophosphamide (Cyclocarya pallida) A.acidoterrestris The specificity of ) will A.acidoterrestris Compared with 7 non-target pathogenic bacteria (including Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, and Pseudomonas aeruginosa) at the same concentration (1×10⁻⁶) 7 Parallel assays were performed under conditions of (CFU / mL). Specificity experimental results (e.g.) Figure 8 A and B represent colorimetric and Raman methods, respectively. The constructed colorimetric-SERS dual-mode immunobiosensor is used for... A.acidoterrestris It exhibits excellent specificity, effectively distinguishing target bacteria from other non-target pathogenic bacteria, thus providing a basis for the prevention and treatment of foodborne pathogens. A.acidoterrestris This provides reliable technical support for rapid and accurate detection. This specific advantage is crucial for food quality and safety testing, especially in complex food matrices, as it avoids false positive results caused by interference from other microorganisms, ensuring the accuracy of test results.

[0062] Example 6 Real sample testing To evaluate the application potential of this dual-mode immunobiosensor in actual food testing, this invention uses 100 μL of different concentrations of... A.acidoterrestris It was added to 0.9 ml of juice to simulate a real, complex sample for testing. The experiment used "MB-Ab1- A.acidoterrestris The Ab2-Au@PB sandwich immune complex strategy uses magnetic microspheres to specifically capture target bacteria, followed by colorimetric detection using the peroxidase-like activity of Au@PB nanozymes, and SERS detection using PS@4-NTP@ssDNA Raman tag.

[0063] Test results as follows Figure 9 As shown in Table 1, when A.acidoterrestris The added concentrations were 10 3 10 5 and 10 7 At CFU / mL, the dual-mode detection system effectively detected the target bacteria, yielding positive results. By comparing the spiked concentration and the detected concentration, the recoveries of the Raman and colorimetric methods were calculated to be 99.28%–100.34% and 99.40%–101.30%, respectively, with relative standard deviations (RSDs) ranging from 1.37% to 9.88% (as shown in Table 1). These data indicate that this detection method maintains high accuracy and precision in complex fruit juice matrices, with a recovery rate of nearly 100% and a low RSD value, demonstrating the reliability and stability of the dual-mode detection platform in practical applications.

[0064] Table 1. Juice A.acidoterrestris Test results

[0065] Example 7 Sensor storage stability analysis To evaluate the practicality of the constructed SERS dual-mode detection platform, this invention systematically investigated the storage stability of the recognition probe (Au@PB-Ab2) and the Raman tag (PS@4-NTP@ssDNA). The experimental results showed that the storage stability of the recognition probe (Au@PB-Ab2) was satisfactory. Figure 10 A) After being stored at 4°C for 15 days, its effect on... A.acidoterrestris The detection performance decreased by only 4.61%; after being stored at 4°C for 25 days, the Raman tag showed a lower detection performance at 1081.9 cm⁻¹. -1 1336.1cm -1 and 1571.7cm -1 High SERS signal intensity was still present at the three characteristic Raman shifts, and the average rate of change of Raman signal intensity was less than 10%, indicating that the Raman tag maintained good structural integrity and signal stability during long-term storage. Figure 10 B).

[0066] The above stability results fully demonstrate that both the Raman tag and the recognition probe can maintain high stability under normal storage conditions, effectively solving the stability problem during reagent storage and transportation.

Claims

1. A dual-function recognition probe for detecting foodborne pathogens, characterized in that, This includes Au@PB nanozymes with peroxidase catalysis and Raman-enhanced substrate functions, and Ab2, a streptavidin-modified antibody against foodborne pathogens to be detected, wherein: The Au@PB nanozyme comprises a gold nanoparticle core and a Prussian blue shell.

2. The dual-function recognition probe for detecting foodborne pathogens according to claim 1, characterized in that, The Au@PB nanozyme was prepared in the following specific manner: Gold nanoparticles with a particle size of 50 nm were obtained by redox reaction of tetrachloroauric acid and sodium citrate. The core of gold nanoparticles was etched with potassium ferricyanide to obtain an Au@CN nanoparticle solution; Equal volumes of potassium ferrocyanide aqueous solution and ferric chloride hexahydrate aqueous solution were added to the Au@CN nanoparticle solution, and the mixture was stirred to react. Prussian blue was generated in situ on the surface of the Au@CN nanoparticles, yielding Au@PB nanozyme.

3. The dual-function recognition probe for detecting foodborne pathogens according to claim 2, characterized in that, The specific preparation of the gold nanoparticle core is as follows: By mass-volume percentage, 300 μL of 1% tetrachloroauric acid solution and 30 mL of ultrapure water were taken, stirred and mixed, and heated to boiling. Immediately after boiling, 180 μL of 1% sodium citrate solution was added. After the reaction was completed, the solution was cooled to room temperature to obtain the gold nanoparticle core solution. The preparation of Au@PB nanozymes is as follows: 5 mL of AuNP seed solution and 200 μL of 0.5 mM K4[Fe(CN)6] aqueous solution were mixed and treated for 5 min to form Au@CN nanoparticles; Subsequently, 1 mL of 0.1 mM K₄[Fe(CN)₆] aqueous solution and 1 mL of 0.1 mM FeCl₃ were added to the Au@CN nanoparticle solution. The product was collected by stirring vigorously at 25°C for 3 hours in an aqueous solution of 6H2O, followed by centrifugation at 10000 rpm / min for 5 minutes, and the product was washed to obtain Au@PB nanozyme.

4. A dual-mode detection kit for foodborne pathogens, characterized in that, This includes capture probes, recognition probes, Raman tags, colorimetric detection reagents, and Raman detection reagents, among which: The identification probe is the identification probe according to any one of claims 1 to 3; The capture probe is a magnetic microsphere modified with a specific antibody Ab1 against the foodborne pathogen to be detected; The Raman tag is a polystyrene microsphere loaded with a Raman reporter, and the surface of the polystyrene microsphere is modified with single-stranded DNA; the single-stranded DNA is a single-stranded DNA modified with both amino and biotin. The colorimetric detection reagents include: buffer system reagents and colorimetric reaction reagents; The Raman detection-related reagents include: buffer system reagents and microsphere dissociation reagents.

5. The dual-mode detection kit for foodborne pathogens according to claim 4, characterized in that, Among the colorimetric detection reagents: the buffer system reagent is PBST solution, and the colorimetric reaction reagents are BR buffer solution, TMB solution, and H2O2 solution at pH=4.35; In the Raman detection reagents: the buffer system reagent is PBS solution, and the microsphere dissociation reagent is THF reagent.

6. The dual-mode detection kit for foodborne pathogens according to claim 4, characterized in that, The linear range of this kit is 1×10⁻⁶. 1 -1×10 7 The minimum detection concentration for Raman spectroscopy is 1.9 CFU / mL, and the minimum detection concentration for colorimetric spectroscopy is 13.5 CFU / mL.

7. A dual-mode detection method for foodborne pathogens, characterized in that, The detection was performed using a dual-mode detection kit for foodborne pathogens as described in any one of claims 4 to 6.

8. The dual-mode detection method for foodborne pathogens according to claim 7, characterized in that, When performing colorimetric testing: The capture probe was added to the buffer system and vortexed for 30 seconds. The sample solution and recognition probe were added and vortexed for 30 seconds. After incubation at 37°C for 45 minutes, the sample was adsorbed by a magnet, washed, and resuspended in the buffer system to obtain the immune complex solution. The immune complex solution and colorimetric reagent were mixed and incubated for 15 min. The supernatant was then taken and its absorbance was measured at 652 nm. The ratio of the identification probe to the capture probe is 4:1, and the concentration of the capture probe is 5 mg / mL. The volume of the sample solution used is 50 μL; The ratio of the immune complex solution to the colorimetric reagent is 1:

10.

9. The dual-mode detection method for foodborne pathogens according to claim 8, characterized in that, Raman spectroscopy was performed after colorimetric analysis. The immune complex solution was mixed with the Raman tag, incubated at 37°C with shaking for 25 min, and then magnetically separated and washed before being resuspended in a buffer system to obtain mixture 1. Add the microsphere dissociation reagent to mixture 1, and react to obtain mixture 2; Two drops of the mixture were placed on a glass slide wrapped in aluminum foil, and Raman spectroscopy was performed. The ratio of the immune complex solution, Raman tag, and microsphere dissociation reagent is 50:3:

30. The amount of the mixture 2 is 2 μL, and the laser power for Raman detection is 10 mW.

10. The dual-mode detection method for foodborne pathogens according to claim 7, characterized in that, The foodborne pathogen is Bacillus cyclophosphamide. A.acidoterrestris .

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