Ttago-driven autocatalytic nucleic acid circuit sers biosensor and construction method and application thereof

Abstract

Tt The Ago-driven autocatalytic nucleic acid circuit SERS biosensor and its construction method and application belong to the technical field of biological sensors and food safety rapid detection. In order to solve the technical problems that the existing salmonella typhi detection method depends on the complex pre-amplification step, lacks high-resolution multiple signal output strategy, and is easily polluted by aerosol, two Raman reporter molecules (MBA and DTNB) are embedded into Ag@Au nanostructures with gap-enhanced Raman performance, and further combined with Tt The Ago-driven autocatalytic nucleic acid circuit is combined to construct a biosensor for simultaneously detecting salmonella typhi and staphylococcus aureus, which provides a high-sensitivity, accurate and rapid method for multiple detection of pathogenic bacteria in food safety.

Description

Technical Field

[0001] This invention belongs to the field of biosensors and rapid food safety detection technology, specifically involving... Tt Ago-driven autocatalytic nucleic acid circuit SERS biosensor, its construction method and application. Background Technology

[0002] Salmonella typhimurium ( Salmonella typhimurium ) and Staphylococcus aureus ( Staphylococcus aureus Foodborne pathogens (Pseudomonas aeruginosa and Pseudomonas aeruginosa) are two common pathogens associated with bacterial infections, capable of causing rapid infection even at low concentrations. Due to their strong persistence and transmissibility, these pathogens can induce recurrent enteritis, vomiting, diarrhea, fever, and even death, posing a serious threat to human health. Therefore, accurate monitoring and early detection of foodborne pathogens are of great significance for the prevention and control of foodborne disease outbreaks.

[0003] Currently, detection technologies for foodborne pathogens mainly include traditional culture methods, immunoassays, and molecular diagnostic methods such as polymerase chain reaction (PCR). However, these methods still have many limitations in practical applications. While plate culture is considered the "gold standard," it is time-consuming (usually 36-72 hours), making it difficult to meet the needs of rapid on-site screening. Enzyme-linked immunosorbent assay (ELISA), as a typical immunoassay method, has the advantages of high throughput and ease of operation, but its sensitivity is limited, it relies on expensive antibodies, and it also has shortcomings in multiplexing. PCR-based molecular detection technologies, while possessing high sensitivity and accuracy, depend on sophisticated thermal cycling equipment and professional operators. Furthermore, aerosol contamination during amplification can lead to false positive results, limiting their widespread application in rapid on-site detection.

[0004] In recent years, programmable endonucleases, as highly efficient gene-editing tools, have shown significant application prospects in the field of nucleic acid detection. Similar to the CRISPR-Cas system, Argonaute proteins are also programmable nucleic acid-guided endonucleases deployed by eukaryotic and prokaryotic hosts to defend against viral attacks. Unlike CRISPR-Cas, prokaryotic Argonaute (pAgo) can utilize short-chain guide DNA (gDNA) to achieve precise cleavage of target nucleic acids without relying on PAM sequences, thus possessing unique advantages in multi-target detection. pAgo proteins can specifically cleave the linker between the 10th and 11th nucleotides at the 5' end of target DNA complementary to gDNA, and restart cleavage after releasing the cleaved target. Furthermore, the gDNA used by pAgo is a short-chain single-stranded DNA, offering advantages such as low synthesis cost, simple design, and high stability, further enhancing the flexibility and adaptability of the detection system. With its targeted recognition and cleavage capabilities, programmability, sequence specificity, and multiple-turnover cleavage characteristics, pAgo shows significant potential in the development of multiplex detection methods. However, to achieve higher sensitivity, existing pAgo detection methods typically require coupling with nucleic acid amplification techniques. This amplification step not only increases the types of enzymes and operational steps, prolonging detection time, but also easily leads to aerosol contamination and false positive results, severely restricting the widespread application of pAgo-based detection methods in practical scenarios. Therefore, developing a sensitive, amplification-free, pAgo-based multiplex detection strategy for rapid detection of foodborne pathogens remains a significant challenge.

[0005] Surface-enhanced Raman spectroscopy (SERS) is a powerful spectroscopic analysis technique widely used in food safety and environmental analysis due to its high sensitivity, non-destructive testing, strong anti-interference ability, and rich and narrow spectral fingerprint characteristics. When Raman reporter molecules are located on the surface of metal nanoparticles or nanostructured substrates, the local electromagnetic enhancement effect generated by plasmon resonance and the chemical enhancement effect induced by charge transfer work synergistically to significantly amplify their spectral signals. Furthermore, SERS detection allows for the simultaneous use of multiple labels and enables the simultaneous detection of multiple targets by utilizing different characteristic peaks, demonstrating great potential in multiplex analysis. However, traditional SERS nanotag probes typically physically adsorb Raman reporter molecules directly onto the nanoparticle surface. During subsequent biological modification and application, the reporter molecules are prone to detachment, resulting in poor probe signal stability and failing to meet the requirements for high-sensitivity and high-reliability target detection. Novel SERS nanotags—gap-enhanced Ramantags (GERTs)—achieve a significant improvement in signal intensity by utilizing electromagnetic field enhancement effects. GERTs form a gap ranging from sub-nanometer to several nanometers between the plasmonic shell and the core, within which Raman reporter molecules reside. Under the influence of a localized electromagnetic field, the Raman signal is amplified through enhanced hotspots. Simultaneously, the plasmonic shell provides physical protection for the adsorbed Raman reporter molecules, thus endowing the probe with excellent spectral stability. Gold nanoparticles (AuNPs), as typical SERS substrates, possess excellent stability and biocompatibility, but their SERS activity is typically several orders of magnitude lower than that of silver nanoparticles (AgNPs). While AgNPs exhibit superior SERS activity and a high molar absorption coefficient, they face challenges such as poor stability and difficulty in surface functionalization. An effective alternative is to uniformly coat an Ag core with an Au shell, forming Ag-Au core-shell GERTs. This structure combines the excellent surface chemical properties of Au with the superior optical properties of Ag. The Raman reporter molecule is confined in the nanoscale gap between the Au shell and the Ag core, achieving efficient signal amplification under the effect of enhanced hot spots. At the same time, the outer Au shell is conducive to biomolecule coupling, effectively protecting the internal Ag core and preventing the Raman reporter molecule from being affected by the external environment. This significantly improves the biocompatibility, chemical stability and signal stability of the particles, giving the probe excellent spectral stability.

[0006] Although pAgo technology and SERS / GERTs technology have made significant progress in their respective fields, no report has yet been published on the organic integration of pAgo's precise targeting and recognition capabilities with GERTs' ultra-high sensitivity signal output capabilities to construct a field detection system for foodborne pathogens that is free from nucleic acid amplification and has multiple detection capabilities. Summary of the Invention

[0007] To address the technical problems of existing methods for detecting Salmonella typhimurium, such as reliance on cumbersome pre-amplification steps, lack of high-resolution multiplex signal output strategies, and susceptibility to aerosol contamination, this invention constructs a... Tt An Ago-driven autocatalytic nucleic acid circuit (ANC) SERS biosensor is used for sensitive multiplex detection of foodborne pathogens (Salmonella typhimurium and Staphylococcus aureus) in food. This biosensor uses biotin-modified single-stranded DNA (ssDNA) and SERS nanotags (Ag@Ra@AuNPs) loaded with Raman reporter molecules (Ra) as signal probes, and streptavidin-modified magnetic beads (SA-MBs) as separation carriers. The detection principle of the biosensor constructed in this invention is as follows: Figure 1 As shown, the biosensor mainly consists of three functional modules: (1) Tt Ago specifically recognizes targets and its mediated ANC enables signal amplification; (2) Tt Ago-ANC-driven specific biotinylated SERS signal probe substrate cleavage; (3) magnetic separation and multi-label SERS signal transduction. This biosensor achieves ultrasensitive detection of pathogens through the following signal amplification strategy: GERTs (Ag@Ra@AuNPs) are composed of a silver core with a diameter of about 30 nm and a gold shell of 2 nm. Ra is embedded between the Ag-Au layers to form built-in narrow nano gaps and abundant high-efficiency hot spots, thereby generating excellent SERS signals. Subsequently, the reporter DNA (SH-ssDNA-biotin) modified with 3' end biotin and 5' end thiol group is mixed with the 5' end thiol group modified polyT in a specific ratio and fixed on the surface of Ag@Ra@AuNPs by freeze-thaw method to obtain biotinylated SERS signal probes (biotin-Ag@Ra@AuNPs). Using polyT to partially replace the reporter DNA can effectively adjust the grafting density of reporter DNA on the surface of the SERS probe, thereby providing Tt Ago cutting provides sufficient space, significantly improving cutting efficiency. Subsequently, biotinylated SERS signaling probes are integrated into... TtThe Ago-mediated ANC system employs four signal processors (gDNA1, gDNA2, a biotinylated SERS signal probe, and a reporter DNA complementary strand (RC)) that work synergistically. The sequences of all signal processors are carefully matched to ensure that Trigger 1 (T1) and Trigger 2 (T2) generated during the reaction also function as gDNA. Specifically, in the presence of the target, the Ago / gDNA1 complex recognizes and cleaves the target DNA through hybridization, generating ssDNA (T1) with a 5' phosphate (5'-P) end. This ssDNA then acts as the gDNA for activation. Tt Ago enables it to specifically recognize and cleave the reporter DNA on the surface of Ag@Ra@AuNPs, thereby releasing T2. The biotin at the 3' end of the reporter DNA detaches from the SERS probe surface, preventing it from being captured by SA-MBs, thus maintaining a strong SERS signal in the supernatant. The newly generated T2 and... Tt Ago binds, recognizes and cleaves RC, generates T1, and completes the ANC DNA cycle. This process establishes a self-catalytic positive feedback loop, enabling repeated cleavage reactions in the presence of target DNA and achieving exponential signal amplification through efficient cyclic cleavage. Finally, after adding SA-MBs and performing magnetic separation, a large number of SERS probes remain in the supernatant. Conversely, in the absence of a target, the reporter DNA remains intact, causing SA-MBs to bind to the SERS probes, resulting in a weakened SERS signal in the supernatant after magnetic separation. The SERS signal intensity corresponds to the target DNA concentration. By comparing the gDNA corresponding to each target with RC (RC for Salmonella Typhimurium), the signal intensity is significantly reduced. invA and RC against Staphylococcus aureus nuc By integrating biotin-modified SERS signal probes (biotin-Ag@MBA@AuNPs for Salmonella typhimurium and biotin-Ag@DTNB@AuNPs for Staphylococcus aureus) into the system, simultaneous detection of the two pathogens can be achieved.

[0008] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide Tt Ago-driven autocatalytic nucleic acid circuit SERS biosensor, the autocatalytic nucleic acid circuit SERS biosensor includes... Tt Ago protein, cleavage buffer, gDNA1, gDNA2, biotinylated SERS signal probe, and reporter DNA complementary strand; when the self-catalytic nucleic acid circuit SERS biosensor is used alone to detect Salmonella typhimurium, the gDNA1 is... invA -gDNA1, wherein gDNA2 is invA -gDNA2, the biotinylated SERS signal probe is biotin-Ag@MBA@AuNPs, and the reporter DNA complementary strand is RC. invA When the self-catalytic nucleic acid circuit SERS biosensor is used to detect Staphylococcus aureus alone, the gDNA1 is... nuc -gDNA1, wherein gDNA2 is nuc -gDNA2, the biotinylated SERS signal probe is biotin-Ag@DTNB@AuNPs, and the reporter DNA complementary strand is RC. nuc When the self-catalytic nucleic acid circuit SERS biosensor is used to simultaneously detect Salmonella typhimurium and Staphylococcus aureus, the gDNA1 is... invA -gDNA1 and nuc -gDNA1, wherein gDNA2 is invA -gDNA2 and nuc -gDNA2, the biotinylated SERS signal probes are biotin-Ag@MBA@AuNPs and biotin-Ag@DTNB@AuNPs, and the reporter DNA complementary strand is RC. invA and RC nuc The invA -gDNA1, invA -gDNA2, nuc -gDNA1 and nuc The nucleotide sequences of -gDNA2 are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.8, respectively. The RC invA and RC nuc The nucleotide sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 9, respectively. The biotinylated SERS signal probe is obtained by mixing reporter DNA modified with 3' biotin and 5' thiol groups with polyT modified with 5' thiol groups and then fixing it on the surface of Ag@Ra@AuNPs by freeze-thaw method. The Ag@Ra@AuNPs are obtained by embedding Raman reporter molecules between Ag-Au layers with silver core and gold shell. The Raman reporter molecule used to detect Salmonella typhimurium is MBA, and the Raman reporter molecule used to detect Staphylococcus aureus is DTNB. The nucleotide sequence of the reporter DNA of Salmonella typhimurium is shown in SEQ ID NO. 1, the nucleotide sequence of the reporter DNA of Staphylococcus aureus is shown in SEQ ID NO. 2, and the nucleotide sequence of polyT is shown in SEQ ID NO. 3.

[0009] A second objective of this invention is to provide a method for constructing the above-mentioned self-catalytic nucleic acid circuit SERS biosensor, the method comprising the following steps: Preparation of S1, Ag@Ra@AuNPs: 1 mL of 1% AgNO3 solution was added to 60 mL of ultrapure water. The mixture was heated to boiling with vigorous stirring and maintained for 15 min. 1 mL of 1% TSC solution and 1 mL of freshly prepared NaBH4 solution were added to the mixture sequentially. The mixture was kept boiling with vigorous stirring for 40 min, then allowed to cool naturally to room temperature. Ultrapure water was added to bring the dispersion volume to 100 mL to obtain the AgNPs seed solution. 2 mL of 1% TSC solution was added to 80 mL of ultrapure water and boiled for 15 min. 10 mL of AgNPs seed solution and 1.7 mL of 1% AgNO3 solution were added with vigorous stirring. The mixture was kept boiling for 1 h. 2 mL of 1% TSC solution and 1.7 mL of 1% AgNO3 solution were added to the reaction solution. The mixture was kept boiling for 1 h. 2 mL of 1% TSC solution and 1.7 mL of 1% AgNO3 solution were added to the reaction solution. After boiling for 1 h, 2 mL of 1% TSC solution and 1.7 mL of 1% AgNO3 solution were added to the reaction solution. 1 mL of 1% TSC solution and 1.7 mL of 1% AgNO3 solution were boiled for 1 h, then naturally cooled to room temperature. Water was added to bring the volume to 100 mL, and the mixture was aged under 254 nm UV light for 30 min. The AgNPs dispersion was collected by centrifugation and washing. 100 μL of Raman reporter molecule solution was mixed with 4 mL of AgNPs dispersion and incubated with vigorous stirring at room temperature for 4 h. Excess unbound Raman reporter molecules were removed by centrifugation. The obtained Raman reporter molecule-labeled AgNPs were redispersed in 5 mL of ultrapure water to obtain Ag@RaNPs dispersion. The Raman reporter molecule solution was prepared by dissolving MBA in ethanol, with a concentration of 0.25-4 mM, or the Raman reporter molecule solution was prepared by dissolving DTNB in ​​ethanol, with a concentration of 0.5-6 mM. 5 mL of 5% PVP solution and 50 μL of 0.50% AgNO3 solution were added sequentially to 4 mL of Ag@RaNPs dispersion. The gold growth solution was prepared by sonicating a mixture of M L-AA solution, 0.20 mL of 0.50 M NaOH solution, and 0.20 mL of 0.10 M Na2SO3 solution for 10 min, followed by the addition of 1 mL of gold growth solution. The mixture was then allowed to stand at 65°C for 1 h, and the resulting Ag@Ra@AuNPs were collected by centrifugation. After washing, the Ag@Ra@AuNPs were redispersed in 4 mL of ddH2O. The gold growth solution was prepared by adding 50 μL of HAuCl4 solution, 0.24 mL of 0.20 M NaOH solution, and 3.0 mL of 0.010 M Na2SO3 solution to 4.7 mL of ultrapure water and allowing it to stand overnight to obtain a colorless and transparent gold growth solution. The concentration of the HAuCl4 solution was 5-100 mM. S2. Preparation of biotinylated SERS signal probes: Reporter DNA modified with 3' biotin and 5' thiol groups was mixed with 5' thiol-modified polyT at a volume ratio of 1:0-1:40 and added to 1000 μL Ag@Ra@AuNPs. The mixture was frozen at -80°C for 30 min, then thawed at room temperature. 100 μL of 1% BSA was added and incubated for 1 h. After centrifugation, the precipitate was redispersed in 60 μL TN buffer. The concentrations of the 3' biotin- and 5' thiol-modified reporter DNA and the 5' thiol-modified polyT were both 100 μM, with a total volume of 250 μL. S3 Tt Preparation of Ago-driven autocatalytic nucleic acid loop detection system: When the autocatalytic nucleic acid loop SERS biosensor is used to detect Salmonella typhimurium alone, the Salmonella typhimurium detection system is prepared by: mixing 12 μL of 1×ThermoPol reaction buffer, 50-250 nM... Tt Ago, 500 nM gDNA, 100-500 nM RC invA And 10 μL of biotin-Ag@MBA@AuNPs were mixed, wherein the gDNA was invA -gDNA1 and invA -gDNA2; When the self-catalytic nucleic acid circuit SERS biosensor is used to detect Staphylococcus aureus alone, prepare the Staphylococcus aureus detection system: add 12 μL of 1×ThermoPol reaction buffer, 50-250 nM... Tt Ago, 500 nM gDNA, 100-500 nM RC nuc And 10 μL of biotin-Ag@DTNB@AuNPs were mixed, wherein the gDNA was nuc -gDNA1 and nuc -gDNA2; When the self-catalytic nucleic acid circuit SERS biosensor is used to simultaneously detect Salmonella Typhimurium and Staphylococcus aureus, a system for simultaneously detecting Salmonella Typhimurium and Staphylococcus aureus is prepared: 24 μL of 1×ThermoPol reaction buffer, 50-250 nM... Tt A mixture of AgO, 500 nM gDNA, 100-500 nM reporter DNA complementary strand, and 10 μL each of biotin-Ag@MBA@AuNPs and biotin-Ag@DTNB@AuNPs, wherein the gDNA is invA -gDNA1, invA -gDNA2, nuc -gDNA1 and nuc-gDNA2, the reporter DNA complementary strand is RC invA and RC nuc .

[0010] In one embodiment of the present invention, the Raman reporter molecule solution mentioned in S1 is prepared by dissolving MBA in ethanol, wherein the concentration of MBA is 1 mM and the concentration of HAuCl4 is 50 mM.

[0011] In one embodiment of the present invention, the Raman reporter molecule solution mentioned in S1 is prepared by dissolving DTNB in ​​ethanol, wherein the concentration of DTNB is 4 mM and the concentration of HAuCl4 is 30 mM.

[0012] In one embodiment of the present invention, the 3' biotin and 5' thiol-modified reporter DNA in S2 is mixed with 5' thiol-modified polyT at a volume ratio of 1:30.

[0013] In one embodiment of the present invention, S3 is described Tt The concentration of Ago is 200 nM, and the RC invA and RC nuc The concentrations were all 400 nM.

[0014] A third objective of this invention is to provide the application of the above-described self-catalytic nucleic acid circuit SERS biosensor in detecting Salmonella typhimurium and / or Staphylococcus aureus contamination in food or food processing.

[0015] The fourth objective of this invention is to provide a kit containing the above-mentioned self-catalytic nucleic acid circuit SERS biosensor for the detection of Salmonella typhimurium and / or Staphylococcus aureus contamination in food or food processing.

[0016] A fifth objective of this invention is to provide a method for detecting contamination of Salmonella Typhimurium and / or Staphylococcus aureus in food or during food processing, the method comprising the following steps: (1) Extract genomic DNA from the sample to be tested and heat it at 95°C for 20 min to obtain single-stranded DNA, and then cool it rapidly at -20°C for 20 min. (2) When detecting Salmonella typhimurium alone, 8 μL of single-stranded DNA is mixed with the Salmonella typhimurium detection system described in claim 2, S3. When detecting Staphylococcus aureus alone, 8 μL of single-stranded DNA is mixed with the Staphylococcus aureus detection system described in claim 2, S3. When detecting both Salmonella typhimurium and Staphylococcus aureus simultaneously, 16 μL of single-stranded DNA is mixed with the system for detecting both Salmonella typhimurium and Staphylococcus aureus simultaneously. The reaction is incubated at 85°C for 10-120 min to obtain the reaction mixture. (3) Add the reaction mixture obtained in step (2) to streptavidin-functionalized magnetic beads with a concentration of 10 mg / mL, incubate at room temperature for 30 min, perform magnetic separation of the reaction product, centrifuge, collect the supernatant, spot it onto a glass slide wrapped in tin foil, and collect Raman spectra using an Optosky ATR6500 portable Raman spectrometer with a spectral acquisition range of 800-2000 cm⁻¹. -1 Each sample should be measured at least three times independently.

[0017] In one embodiment of the present invention, the incubation time in step (2) is 90 min.

[0018] In one embodiment of the present invention, when detecting Salmonella Typhimurium alone, the detection limit for Salmonella Typhimurium is 2.4 CFU / mL. For quantitative detection, the linear relationship between SERS intensity and Salmonella Typhimurium concentration is y = 613.29x + 649.33 (R²). 2 =0.99), where y is 1586 cm. -1 The fluorescence intensity at the point is given by x, where x is the logarithm of the *Salmonella typhimurium* concentration (lg CFU / mL). When *Staphylococcus aureus* is detected alone, the detection limit is 2.6 CFU / mL. For quantitative detection, the linear relationship between SERS intensity and *Staphylococcus aureus* concentration is y = 650.39x + 898.94 (R²). 2 =0.99), where y is 1304 cm. -1 The fluorescence intensity at the location, where x is the logarithm of the Staphylococcus aureus concentration (lg CFU / mL).

[0019] In a preferred embodiment of the present invention, the test samples include eggs, milk and chicken.

[0020] In one embodiment of the present invention, when simultaneously detecting *Salmonella typhimurium* and *Staphylococcus aureus*, the detection limit for *Salmonella typhimurium* is 1.7 CFU / mL. For quantitative detection, the linear relationship between SERS intensity and *Salmonella typhimurium* concentration is y = 478.29x + 1468.71 (R²). 2 =0.98), where y is 1586 cm. -1 The fluorescence intensity at the point is given by x, where x is the logarithm of the *Salmonella typhimurium* concentration (lg CFU / mL). The detection limit for *Staphylococcus aureus* is 2.7 CFU / mL. For quantitative detection, the linear relationship between SERS intensity and *Staphylococcus aureus* concentration is y = 521.56x + 284.41 (R²). 2 =0.98), where y is 1315 cm. -1The fluorescence intensity at the location, where x is the logarithm of the Staphylococcus aureus concentration (lg CFU / mL).

[0021] In a preferred embodiment of the present invention, the test samples include eggs, milk and chicken.

[0022] The beneficial effects of this invention are: The performance improvement of the biosensor provided by this invention is mainly attributed to the following aspects: First, Tt Ag@Ra@Au nanoparticles exhibit high cleavage activity under intermediate temperature conditions (65-85°C), enabling efficient sequential cleavage of encoded SERS probes within the ANC system. Secondly, GERTs utilize electromagnetic field "hot spots" to provide a strong SERS effect, and by embedding Ra with non-overlapping spectral characteristics, signal interference is effectively avoided, thus achieving multiplexed signal output. Furthermore, the introduction of poly-T modulates the spacing of reporter DNA on the Ag@Ra@Au nanoparticle surface, promoting... Tt Ago's efficient cleavage significantly enhances the SERS signal response. These characteristics collectively give this biosensing platform great potential for high-sensitivity and multi-channel detection of foodborne pathogens. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the detection principle of the biosensor constructed in this invention; Figure 2 A schematic diagram of the design of the sequence used in the nucleic acid cycling circuit of Salmonella typhimurium; Figure 3 A schematic diagram of the design of the sequence used in the Staphylococcus aureus nucleic acid cycling circuit; Figure 4 The image shows the effect measurement results of Ag@MBANPs prepared with different MBA addition amounts in the SERS tag synthesis; where A is the Raman spectrum and B is the SERS intensity measurement result. Figure 5 The results of measuring the effect of different DTNB addition amounts in the synthesis of SERS tags are shown in the figure; where A is the Raman spectrum and B is the SERS intensity measurement result. Figure 6 Different Au values ​​in SERS tag synthesis 3+ The effect measurement results of Ag@MBA@AuNPs prepared by adding the specified amount are shown in the figure; where A is the Raman spectrum and B is the SERS intensity measurement result. Figure 7 Different Au values ​​in SERS tag synthesis 3+The effect measurement results of Ag@DTNB@AuNPs prepared by adding the specified amount are shown in the figure; where A is the Raman spectrum and B is the SERS intensity measurement result. Figure 8 Figure A shows the stability assessment results for Ag@MBA@AuNPs; where A represents the storage stability assessment results and B represents the thermal stability assessment results. Figure 9 Raman signal determination results of SERS signal probes prepared with different amounts of reporter DNA and Au@MBA@AgNPs; Figure 10 for Tt The optimization results of the Ago-driven autocatalytic nucleic acid circuit SERS biosensor are shown in the figure; where A represents the optimized loading result of reporter DNA on the SERS signal probe, and B represents the optimized loading result of reporter DNA on the SERS signal probe. Tt The optimized Ago protein concentration is shown in Figure C, where RC concentration is the optimized result, and Figure D is the optimized result. Tt The optimization results of Ago's shearing time are shown in the figure; Figure 11 To utilize Tt The sensitivity evaluation results of the Ago-driven autocatalytic nucleic acid circuit SERS biosensor for detecting Salmonella typhimurium are shown in the figure; A represents different concentrations (10) after treatment. 0 -10 8 SERS spectrum of Salmonella Typhimurium genomic DNA (CFU / mL), B is the standard curve for Salmonella Typhimurium detection; Figure 12 To utilize Tt The sensitivity evaluation results of the Ago-driven autocatalytic nucleic acid circuit SERS biosensor for detecting Staphylococcus aureus are shown in the figure; A represents different concentrations (10) after treatment. 0 -10 8 SERS spectrum of Staphylococcus aureus genomic DNA (CFU / mL), B is the standard curve for Staphylococcus aureus detection; Figure 13 To utilize Tt The sensitivity evaluation results of the Ago-driven autocatalytic nucleic acid circuit SERS biosensor for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus are shown in the figure; A represents different concentrations (10) after treatment. 0 -10 8 SERS spectra of genomic DNA of Salmonella Typhimurium and Staphylococcus aureus (CFU / mL), B is the standard curve for the detection of Salmonella Typhimurium and Staphylococcus aureus; Figure 14 for TtThe specificity and anti-interference verification results of the Ago-driven self-catalytic nucleic acid circuit SERS biosensor are shown in the figure. Among them, A is the specificity and anti-interference verification result of the biosensor for the single detection of Salmonella typhimurium, B is the specificity and anti-interference verification result of the biosensor for the single detection of Staphylococcus aureus, and C is the specificity and anti-interference verification result of the biosensor for the simultaneous detection of Salmonella typhimurium and Staphylococcus aureus. Figure 15 To utilize Tt The Ago-driven self-catalytic nucleic acid loop SERS biosensor detected Salmonella typhimurium and Staphylococcus aureus in real samples. Among them, A is a comparison of the detection results of real samples using the biosensor and qPCR, and B is a statistical result of the spiked recovery rate of Salmonella typhimurium and Staphylococcus aureus in real samples. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, any embodiments obtained by other skilled personnel without creative effort are protected by this invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The strains, materials, reagents and instruments used are all conventional strains, materials, reagents and instruments in the art, and can be obtained by those skilled in the art through commercial channels.

[0026] The reagents used in this invention are as follows: Argonaute (a thermophilic bacterium) TtAgo (amount unspecified) was purchased from New England Biolabs Technology Ltd. (Beverly, MA, USA). Trisodium citrate (TSC), L-ascorbic acid (L-AA), sodium sulfite (Na2SO3), polyvinylpyrrolidone (PVP, MW=10000), 4-mercaptobenzoic acid (MBA), and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Chloroauric acid (HAuCl4) was purchased from Maclean Biotech Co., Ltd. (Shanghai, China). AgNO3, sodium borohydride (NaBH4), NaOH, and NaCl were all analytical grade and used directly without further purification. Streptavidin-functionalized magnetic beads (SA-MBs) were purchased from Thermo Fisher Scientific (USA). The DL500 DNA marker was purchased from Tiangen Biotech (Beijing) Co., Ltd. The TIANamp bacterial DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd. 2×SGExcel Fast SYBR Mixture and 100×ROX reference dye were purchased from Sangon Biotech (Shanghai) Co., Ltd. Luria-Bertani (LB) agar and LB liquid medium were purchased from Hybio Biotechnology Co., Ltd. (Qingdao, China).

[0027] Information on the strains used in this invention is shown in Table 1.

[0028] Table 1. Strain Information

[0029] The technical principles involved in this invention are as follows: The biosensor provided by this invention uses biotin-modified ssDNA and SERS nanotags (Ag@Ra@AuNPs) loaded with Raman reporter molecules (Ra) as signal probes, and streptavidin-modified magnetic beads (SA-MBs) as separation carriers. The detection principle of the biosensor constructed in this invention is as follows: Figure 1 As shown, the biosensor mainly consists of three functional modules: (1) Tt Ago's specific target recognition and its mediated autocatalytic nucleic acid circuit (ANC) signal amplification; (2) TtAgo-ANC-driven cleavage of specific biotinylated SERS signal probe substrates; (3) magnetic separation and multi-tag SERS signal transduction. This biosensor achieves ultrasensitive detection of pathogens through the following signal amplification strategy: GERTs (Ag@Ra@AuNPs) consist of a silver core of about 30 nm and a gold shell of 2 nm. Ra is embedded between the Ag-Au layers, forming built-in narrow nano gaps and abundant high-efficiency hot spots, thereby generating excellent SERS signals. Subsequently, 3' biotin- and 5' thiol-modified reporter DNA (SH-ssDNA-biotin) is mixed with 5' thiol-modified polyT in a specific ratio and fixed on the surface of Ag@Ra@AuNPs by freeze-thaw method to obtain biotinylated SERS signal probes (biotin-Ag@Ra@AuNPs). Using polyT to partially replace reporter DNA can effectively adjust the grafting density of reporter DNA on the surface of SERS probes, thereby providing Tt Ago cutting provides ample space, significantly improving cutting efficiency. Subsequently, biotinylated SERS signaling probes are integrated into... Tt The Ago-mediated ANC system employs four signal processors (gDNA1, gDNA2, a biotinylated SERS signal probe, and a reporter DNA complementary strand (RC)) working synergistically. The sequences of all signal processors are carefully matched to ensure that Trigger 1 (T1) and Trigger 2 (T2) generated during the reaction also function as gDNA. A schematic diagram of the sequences used in this signal amplification strategy is shown below. Figure 2 and Figure 3 As shown. Specifically, in the presence of the target, the Ago / gDNA1 complex recognizes and cleaves the target DNA through hybridization, generating ssDNA (T1) with a 5' phosphate (5'-P) end. This ssDNA acts as gDNA for activation. Tt Ago, enabling it to specifically recognize and cleave the reporter DNA on the surface of Ag@Ra@AuNPs, thereby releasing T2. The biotin at the 3' end of the reporter DNA detaches from the SERS probe surface, preventing it from being captured by SA-MBs, thus retaining a strong SERS signal in the supernatant. The newly generated T2 and... TtAgo binds, recognizes and cleaves RC, generates T1, and completes the DNA cycle of ANC. This process establishes a self-catalytic positive feedback loop, enabling repeated cleavage reactions in the presence of target DNA and achieving exponential signal amplification through efficient cyclic cleavage. Finally, after adding SA-MBs and performing magnetic separation, a large number of SERS probes remain in the supernatant. Conversely, in the absence of the target, the reporter DNA remains intact, causing SA-MBs to bind to the SERS probes, and the SERS signal in the supernatant weakens after magnetic separation. The SERS signal intensity is proportional to the target DNA concentration. By adding gDNA corresponding to each target and RC (RC for Salmonella Typhimurium)... invA and RC against Staphylococcus aureus nuc The system integrates biotin-modified SERS signal probes (biotin-Ag@MBA@AuNPs for Salmonella typhimurium and biotin-Ag@DTNB@AuNPs for Staphylococcus aureus) to achieve simultaneous detection of multiple pathogens. Finally, machine learning is used to rapidly and efficiently analyze the detected SERS characteristic peaks in the mixture spectrum and establish their correlation with pathogen concentration, thereby enabling multiplex detection of pathogens.

[0030] Example 1: Tt A method for constructing an Ago-driven autocatalytic nucleic acid circuit SERS biosensor 1. Preparation and characterization of Raman reporter molecule (Ra) embedded silver-gold core-shell nanoparticles (Ag@Ra@AuNPs) (1) Synthesis of AgNPs seeds: Silver nanoparticles (AgNPs) were synthesized based on seed-mediated growth. First, AgNPs seeds with a diameter of approximately 10 nm were prepared. The specific steps were as follows: 1 mL of 1% (w / v) AgNO3 solution was added to 60 mL of ultrapure water, and the mixture was heated to boiling with vigorous stirring and maintained for 15 min. Subsequently, 1 mL of 1% (w / v) TSC solution was quickly added to the mixture, followed immediately by 1 mL of freshly prepared NaBH4 solution (0.1%, w / v). After addition, the solution turned pale yellow. The mixture was kept boiling with vigorous stirring for 40 min, and then allowed to cool naturally to room temperature. Ultrapure water was added to bring the dispersion volume to 100 mL. The obtained AgNPs were used as starting seeds.

[0031] (2) Synthesis of AgNPs: 2 mL of 1% (w / v) TSC solution was added to 80 mL of ultrapure water and boiled for 15 min. Under vigorous stirring, 10 mL of AgNPs seed solution and 1.7 mL of AgNO3 solution (1%, w / v) were added, and the mixture was kept boiling for 1 h. Then, 2 mL of TSC solution (1%, w / v) and 1.7 mL of AgNO3 solution (1%, w / v) were added to the reaction solution. After keeping the reaction boiling for 1 h, the same addition steps were repeated. The reaction solution was then allowed to cool naturally to room temperature and diluted to 100 mL with water. The resulting AgNPs dispersion was aged under 254 nm UV light for 30 min. Finally, the AgNPs dispersion was collected by centrifugation, washed three times with ultrapure water, and stored at 4°C before use.

[0032] (3) Synthesis of Ag@RaNPs: To prepare Ag@RaNPs, 100 μL of Raman reporter molecule solution (1 mM MBA or 4 mM DTNB, dissolved in ethanol) was incubated with 4 mL of AgNPs dispersion at room temperature with vigorous stirring for 4 h. The Raman reporter molecule was covalently bound to the AgNPs surface via Au-SH bonds. Subsequently, excess unbound Raman reporter molecules were removed by centrifugation (4000 rpm, 15 min). The obtained Raman reporter molecule-labeled AgNPs were redispersed in 5 mL of ultrapure water for later use.

[0033] This invention allows for the determination of the SERS intensity of Ag@MBANPs prepared with different MBA addition amounts and Ag@DTNBNPs prepared with different DTNB addition amounts. The determination results are as follows: Figure 4 and Figure 5 As shown, when the MBA concentration is 1 mM and the DTNB concentration is 4 mM, a better SERS signal can be obtained, and no obvious aggregation phenomenon is observed in the solution.

[0034] (4) Synthesis of Ag@Ra@AuNPs: 50 μL of HAuCl4 (5 mM, 10 mM, 30 mM, 50 mM or 100 mM), 0.24 mL of NaOH (0.20 M), and 3.0 mL of Na2SO3 (0.010 M) were added to 4.7 mL of ultrapure water and allowed to stand overnight to obtain a colorless and transparent gold growth solution. To synthesize Ag@Ra@AuNPs, 5 mL of PVP solution (5.0%), 50 μL of L-AA solution (0.50 M), 0.20 mL of NaOH (0.50 M), and 0.20 mL of Na2SO3 (0.10 M) were added sequentially to 4 mL of Ag@RaNPs dispersion. After sonication for 10 min, 1 mL of gold growth solution was added. The reaction was then allowed to stand at 65°C for 1 h. The obtained Ag@Ra@AuNPs were collected by centrifugation, washed three times with ultrapure water, and finally redispersed in 4 mL ddH2O.

[0035] This invention targets different HAuCl4 (Au) 3+ Ag@MBA@AuNPs and different Au values ​​were prepared by adding a certain amount of [amount missing] 3+ The SERS intensity of Ag@DTNB@AuNPs prepared by adding the specified amount can be determined, and the results are as follows: Figure 6 and Figure 7 As shown, with Au 3+ With increasing concentration, the Raman signal first strengthens and then weakens. This is because the Au concentration in the HAuCl4 solution added during the synthesis of Ag@MBA@AuNPs initially increased. 3+ The highest Raman signal intensity was observed in Ag@MBA@AuNPs prepared at a concentration of 50 mM; the Au in the HAuCl4 solution added during the synthesis of Ag@DTNB@AuNPs was also observed. 3+ The highest Raman signal intensity was obtained from the prepared Ag@DTNB@AuNPs at a concentration of 30 mM.

[0036] (5) Storage stability assessment of Ag@Ra@AuNPs Considering the similarity in the preparation methods of Ag@MBA@Au and Ag@DTNB@Au probes and the similar results observed when using the probes for detection, the storage stability of the probes was evaluated using Ag@MBA@Au probes and their corresponding target (Salmonella typhimurium) as representatives. Ag@MBA@AuNPs were prepared according to the optimized parameters described above, and then the storage stability of Ag@MBA@AuNPs was evaluated. Specifically, Ag@MBA@AuNPs were stored at 25℃ for 30 days, and the stability at 1586 cm⁻¹ was measured every 5 days. -1The storage stability was assessed by measuring changes in SERS intensity. Results are as follows: Figure 8 As shown in Figure A, after 30 days of storage at room temperature, the SERS intensity of Ag@MBA@Au remained at approximately 92% of its initial value. This indicates that the prepared Ag@MBA@Au has high storage stability.

[0037] (6) Thermal stability assessment of Ag@Ra@AuNPs Considering the similarity in the preparation methods of Ag@MBA@Au and Ag@DTNB@Au probes and the similar results observed when using the probes for detection, the thermal stability of the probes was evaluated using Ag@MBA@Au probes and their corresponding target (Salmonella typhimurium) as representatives. Ag@MBA@AuNPs were prepared according to the optimized parameters described above, and then the thermal stability of Ag@MBA@AuNPs was evaluated. Specifically, Ag@MBA@AuNPs were placed at 85℃ for 10, 30, 60, 90, and 120 min, and the thermal stability of Ag@MBA@AuNPs at 1586 cm⁻¹ was measured. -1 The thermal stability was assessed by measuring the SERS intensity change. The results are as follows: Figure 8 As shown in Figure B, no significant change in SERS intensity was observed after treatment at 85°C for 2 h. This indicates that the prepared Ag@MBA@Au possesses high thermal stability.

[0038] 2. Tt Construction of an Ago-driven autocatalytic nucleic acid circuit SERS biosensor (1) Bacterial culture and genomic DNA extraction All bacterial strains were inoculated into LB broth and incubated overnight at 37°C. Bacterial counting was performed using the spread plate method. The simplified procedure is as follows: 100 μL of each prepared bacterial suspension was spread onto LB agar plates and incubated at 37°C for 24 h. Each concentration gradient was independently replicated three times. Bacterial concentrations were obtained by calculating the corresponding colony forming units (CFU). Genomic DNA was extracted from Salmonella Typhimurium, Staphylococcus aureus, and other pathogens using the TIANamp bacterial DNA extraction kit according to the manufacturer's instructions and stored at -20°C for later use.

[0039] (2) Synthesis of biotinylated SERS signal probes (biotin-Ag@Ra@AuNPs): Reporter DNA (SH-ssDNA-biotin) was ligated to Ag@Ra@AuNPs using a freeze-thaw method. The simplified steps are as follows: 250 μL of 100 μM SH-ssDNA-biotin was added to 1000 μL of Ag@Ra@AuNPs. The mixture was then frozen at -80°C for 30 min, followed by thawing at room temperature. Afterward, 100 μL of 1% BSA was added and incubated for 1 h to block excess sites on Ag@Ra@AuNPs. Finally, the solution was centrifuged (6000 rpm, 10 min) and redispersed in 60 μL of TN buffer (10 mM Tris-HCl, 100 mM NaCl, pH 7.4).

[0040] Reporter DNA (SH-DNA) of Salmonella Typhimurium invA -biotin), Staphylococcus aureus reporter DNA (SH-DNA) nuc The nucleotide sequences of β-biotin and polyT are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0041] SEQ ID NO.1: 5'-SH-T 37 ATCGCCAGTACGATATATAAAT-Biotin-3'; SEQ ID NO.2: 5'-SH-T 37 ATAGTCTGAATGTCATTGTGTT-Biotin-3'; SEQ ID NO. 3: 5'-SH-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'.

[0042] Considering the similarity in the preparation methods of Ag@MBA@Au and Ag@DTNB@Au probes and the similar results observed when using the probes for detection, the amount of reporter DNA added was optimized, using the Ag@MBA@Au probe and its corresponding target (Salmonella typhimurium) as examples. Figure 9 As shown, when the reporter DNA was added to a final concentration of 20 μM, Ag@MBA@AuNPs maintained good dispersibility, and the SERS signal decreased by only about 10% for each. With further increases in the amount of reporter DNA, the SERS signal gradually weakened, possibly due to the formation of a dense DNA layer on the nanotag surface, which shielded part of the Raman signal.

[0043] (3) TtAgo-driven autocatalytic nucleic acid circuit SERS biosensor platform detection program for Salmonella typhimurium and Staphylococcus aureus. DNA was extracted from Salmonella Typhimurium and Staphylococcus aureus, respectively. The extracted DNA was denatured and unwound at 95°C for 20 min to form ssDNA, and then immediately placed on ice. Subsequently, the treated target DNA (DNA from Salmonella Typhimurium or Staphylococcus aureus) (8 μL) was mixed with… Tt The Ago cleavage reaction mixture (22 μL) was mixed. This reaction mixture contained: 12 μL of 1×ThermoPol reaction buffer (commercially available). Tt (Included in Ago), 200 nM Tt Ag, 500 nM gDNA (gDNA1 and gDNA2), 400 nM RC, and 10 μL SERS signal probes (biotin-Ag@Ra@AuNPs) were added. The mixture was incubated at 85°C for 90 min. Then, 5 μL of SA-MB (10 mg / mL) was added to the reaction mixture, and the mixture was incubated at room temperature for 30 min. After magnetic separation, 20 μL of the supernatant was collected and spotted onto a foil-wrapped glass slide. Raman spectra were acquired using an Optosky ATR6500 portable Raman spectrometer. The spectral acquisition range was 800–2000 cm⁻¹. -1 Each sample should be measured at least three times independently.

[0044] Salmonella typhimurium corresponding to invA -gDNA1, invA-gDNA2 and RC invA The nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0045] SEQ ID NO.4: 5'-P-AAACACCAATATCGCC-3'; SEQ ID NO.5: 5'-P-AGTACGATATTCAGTG-3'; SEQ ID NO. 6: 5'-ATTTATATATCGTACTGGCGAT-3'.

[0046] Staphylococcus aureus nuc -gDNA1, nuc-gDNA2 and RC nuc The nucleotide sequences are shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0047] SEQ ID NO.7: 5'-P-TCAACCAATAATAGTC-3'; SEQ ID NO.8: 5'-P-TGAATGTCATTGGTTG-3'; SEQ ID NO.9: 5'-AACACAATGACATTCAGACTAT-3'.

[0048] (4) Tt Operational procedures for the Ago-driven autocatalytic nucleic acid loop SERS biosensor platform to simultaneously detect two pathogens (Salmonella typhimurium and Staphylococcus aureus). DNA was extracted from Salmonella Typhimurium and Staphylococcus aureus, respectively. The extracted DNA was denatured and unwound at 95°C for 20 min to form ssDNA, and then immediately placed on ice. Subsequently, 8 μL of each of the two types of genomic DNA were mixed with... Tt The Ago cleavage reaction mixture (44 μL) was mixed. This reaction mixture contained: 24 μL of 1×ThermoPol reaction buffer and 200 nM... Tt Ago, 500 nM gDNA ( invA -gDNA1, invA -gDNA2, nuc -gDNA1 and nuc -gDNA2), 400 nM RC (RC against Salmonella typhimurium) invA RC for Staphylococcus aureus nuc The reaction mixture consisted of 10 μL of each of two SERS signal probes (biotin-Ag@MBA@AuNPs for Salmonella typhimurium and biotin-Ag@DTNB@AuNPs for Staphylococcus aureus). The reaction was incubated at 85°C for 90 min. The resulting multiple cleavage reaction products were added to 10 mL of SA-MB (10 mg / mL) for separation, and the supernatant was analyzed using an Optosky ATR6500 portable Raman spectrometer.

[0049] Example 2: Tt Optimization of Ago-driven autocatalytic nucleic acid circuit SERS biosensor Considering the similarity in the preparation methods of Ag@MBA@Au and Ag@DTNB@Au probes and the similar results observed when using the probes for detection, the Ag@MBA@Au probe and its corresponding target (Salmonella typhimurium) were used as representatives to study the loading of reporter DNA on SERS probes. Tt The concentrations of Ago, RC, and Tt Ago's cut-off time has been optimized.

[0050] 1. Optimization of reporter DNA loading on SERS probes To adjust the loading of reporter DNA on Ag@Ra@AuNPs, while keeping other modification conditions constant (250 μL, 100 μM concentration), the volume ratio of reporter DNA to polyT was varied during the modification process, set to 1:0, 1:1, 1:10, 1:20, 1:30, and 1:40 (1:0 being the control group without polyT). The optimal loading was selected by detecting the change in ΔSERS intensity in the supernatant of the reaction system. ΔSERS intensity was calculated as (SERS-SERS...) 空白 ) Calculate, where SERS and SERS 空白 The values ​​represent the SERS signals with and without the target, respectively. The results are as follows: Figure 10 As shown in Figure A, the ΔSERS intensity reaches its maximum when the volume ratio is 1:30. Further increasing the reporter DNA loading leads to a decrease in ΔSERS intensity, possibly due to the steric hindrance created by excessively high reporter DNA density on the probe surface, hindering... Tt Ago efficiently cuts reporter DNA.

[0051] 2. Tt Ago concentration optimization With other conditions remaining unchanged, set Tt The concentrations of Ago were 0 nM, 50 nM, 100 nM, 150 nM, 200 nM, and 250 nM. The results are as follows: Figure 10 As shown in B, when Tt After the Ago concentration was increased to 200 nM, the ΔSERS signal tended to plateau. Therefore, 200 nM was selected as the optimal working concentration for subsequent experiments.

[0052] 3. Optimization of RC concentration With other conditions remaining unchanged, the concentrations of RC were set to 100 nM, 200 nM, 300 nM, 400 nM, and 500 nM. The results are as follows: Figure 10 As shown in C, the ΔSERS intensity reaches its maximum at an RC concentration of 400 nM, therefore, 400 nM was selected as the optimal working concentration for subsequent experiments.

[0053] 4. Tt Optimization of Ago clipping time With other conditions remaining unchanged, set Tt The shearing times for Ago were 10 min, 30 min, 60 min, 90 min, and 120 min, respectively. The results are as follows: Figure 10As shown in D, the ΔSERS intensity tends to stabilize after 90 min, therefore 90 min is selected as the optimal reaction time.

[0054] Example 3: Tt Sensitivity Evaluation of Ago-Driven Autocatalytic Nucleic Acid Circuit SERS Biosensor (1) Utilization Tt Sensitivity Evaluation of Ago-Driven Autocatalytic Nucleic Acid Circuit SERS Biosensor for Rapid Detection of Single Pathogens The method established in step (3) of Example 1 was used to separately detect the target genomic DNA (genomic DNA of Salmonella Typhimurium and Staphylococcus aureus) after processing with gradient dilutions (10 ppm). 0 -10 8 (CFU / mL). Results are as follows: Figure 11 As shown, with increasing Salmonella typhimurium concentration, MBA at 1586 cm⁻¹... -1 The intensity of the SERS characteristic peak gradually increases at 10. 2 -10 8 Within the CFU / mL range, the logarithm of Salmonella typhimurium concentration showed a good linear relationship with SERS intensity, and the linear regression equation was y = 613.29x + 649.33 (R²). 2 =0.99). Similarly, the results for Staphylococcus aureus showed that as the bacterial concentration remained at 10... 2 -10 8 DTNB increased within the range of CFU / mL, reaching 1304 cm⁻¹. -1 The intensity of the SERS characteristic peak gradually increases at this location. Figure 12 Furthermore, it exhibits a good linear relationship with the logarithm of concentration, with the linear regression equation being y = 650.39x + 898.94 (R²). 2 =0.99). According to the IUPAC standard formula LOD=Y_blank+3×SD_blank (where Y_blank and SD_blank are the average signal intensity and standard deviation of the blank group, respectively), the detection limits of Salmonella Typhimurium and Staphylococcus aureus were calculated to be 2.4 CFU / mL and 2.6 CFU / mL, respectively.

[0055] (2) Utilization Tt Sensitivity Evaluation of Ago-Driven Autocatalytic Nucleic Acid Circuit SERS Biosensor for Detecting Dual-Target Pathogenic Bacteria The method established in step (4) of Example 1 was used to simultaneously detect the genomic DNA of two pathogens (Salmonella typhimurium and Staphylococcus aureus) in a gradient dilution buffer (10 μL / L). 0 -10 8CFU / mL). Two SERS signal probes (biotin-Ag@MBA@AuNPs and biotin-Ag@DTNB@AuNPs) were used to detect their respective targets, enabling multiple signal readout. With increasing target concentrations, MBA showed a higher concentration at 1586 cm⁻¹. -1 The Raman characteristic peak intensity at 1315 cm⁻¹ is similar to that of DTNB. -1 The intensity of the characteristic peaks at each location gradually increases. Figure 13 A in 10). 2 -10 8 CFU / mL and 10 3 -10 8 Within the CFU / mL range, the SERS intensity showed a good linear relationship with the logarithm of the concentrations of Salmonella Typhimurium and Staphylococcus aureus, respectively. Figure 13 (B in the original text). The linear regression equations for *Salmonella typhimurium* and *Staphylococcus aureus* are y = 478.29x + 1468.71 (R²). 2 =0.98) and y=521.56x+284.41(R 2 =0.98). Furthermore, the detection limits for the two pathogens were 1.7 CFU / mL and 2.7 CFU / mL, respectively.

[0056] Example 4: Tt Ago-driven autocatalytic nucleic acid circuit SERS biosensor was used to verify the specificity and anti-interference properties of Salmonella Typhimurium and Staphylococcus aureus. To verify the specificity of the biosensor provided in Example 1 in detecting Salmonella Typhimurium alone (see step (3) in Example 1), this example tested samples containing only Salmonella Typhimurium, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, or Cronobacter sakazakii genomic DNA after processing. To verify the anti-interference ability of the biosensor in detecting Salmonella Typhimurium alone, this example tested samples containing Salmonella Typhimurium, Escherichia coli, Listeria monocytogenes, and Cronobacter sakazakii genomic DNA after processing (mixed samples), and a blank control was set up. The detection results are as follows. Figure 14 As shown in A, the corresponding SERS signal changes significantly only when the target bacteria (Salmonella typhimurium) are present, indicating that the biosensor provided by this invention has excellent specificity when detecting Salmonella typhimurium alone. In addition, the detection of mixed samples also causes significant changes in the corresponding SERS signal, indicating that the biosensor provided by this invention has excellent anti-interference ability when detecting Salmonella typhimurium alone.

[0057] To verify the specificity of the biosensor provided in Example 1 in detecting Staphylococcus aureus alone (see step (3) in Example 1), this example tested samples containing only Salmonella Typhimurium, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, or Cronobacter sakazakii genomic DNA after processing. To verify the anti-interference ability of the biosensor in detecting Staphylococcus aureus alone, this example tested samples containing Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Cronobacter sakazakii genomic DNA after processing (mixed samples), and a blank control was set up. The detection results are as follows. Figure 14 As shown in B, the corresponding SERS signal changes significantly only when the target bacteria (Staphylococcus aureus) are present, indicating that the biosensor provided by this invention has excellent specificity when detecting Staphylococcus aureus alone. In addition, the detection of mixed samples also causes significant changes in the corresponding SERS signal, indicating that the biosensor provided by this invention has excellent anti-interference ability when detecting Staphylococcus aureus alone.

[0058] To verify the specificity of the biosensor provided in Example 1 in simultaneously detecting Salmonella Typhimurium and Staphylococcus aureus (method described in step (4) of Example 1), this example tested samples containing only Salmonella Typhimurium + Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, or Cronobacter sakazakii genomic DNA after processing. To verify the anti-interference ability of the biosensor in simultaneously detecting Salmonella Typhimurium and Staphylococcus aureus, this example tested samples (mixed samples) containing genomic DNA of Salmonella Typhimurium, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Cronobacter sakazakii after processing, with a blank control set up. The detection results are as follows: Figure 14 As shown in C, the corresponding SERS signal changes significantly only when the target bacteria (Salmonella Typhimurium and Staphylococcus aureus) are present, indicating that the biosensor provided by this invention has excellent specificity for simultaneously detecting Salmonella Typhimurium and Staphylococcus aureus. In addition, the detection of mixed samples also causes significant changes in the corresponding SERS signal, indicating that the biosensor provided by this invention has excellent anti-interference ability for simultaneously detecting Salmonella Typhimurium and Staphylococcus aureus.

[0059] Example 5: Utilizing Tt Accuracy Evaluation of Ago-Driven Autocatalytic Nucleic Acid Circuit SERS Biosensor in Detecting Real Samples To evaluate the applicability of the biosensor and its detection method provided by this invention in complex samples, the biosensor was used to detect Salmonella Typhimurium and Staphylococcus aureus in artificially contaminated milk, egg, and chicken samples. A total of 24 samples were analyzed, including 8 samples each of milk, chicken, and eggs. Selective plate coating confirmed that the samples before artificial contamination were free of Salmonella Typhimurium and Staphylococcus aureus. Actual samples were pretreated according to national standards (GB4789.4-2024, GB 4789.30-2016). Food sample pretreatment was as follows: For chicken samples, chopped chicken (2 g) was mixed with 18 mL of sterile water and homogenized; after centrifugation, the supernatant was collected as the pretreated chicken sample. For egg samples, egg yolks and egg whites were mixed, diluted 10-fold with sterile water, and then centrifuged to collect the supernatant as the pretreated egg sample. For milk samples, milk was diluted 10-fold, centrifuged, and the supernatant was collected as the pretreated milk sample. Then, 2 mL of the pretreated food sample was taken, and subsequently, *Salmonella typhimurium* and *Staphylococcus aureus* were artificially added to the food sample to create a blind sample. For direct spiked analysis, 10 mL of the sample was added to each of the three samples where the target bacteria were not detected. 5 CFU / mL, 10 6 CFU / mL and 10 7 CFU / mL of foodborne pathogen targets were extracted. After DNA extraction, the extracted DNA was heated at 98°C for 15 min to obtain ssDNA, and then rapidly cooled at -20°C for 20 min. Finally, these processed DNAs were analyzed using a biosensor, and the recovery rate was calculated.

[0060] use Tt The Ago-driven autocatalytic nucleic acid circuit SERS biosensor detected 3 positive samples of Salmonella typhimurium and 2 positive samples of Staphylococcus aureus. Figure 15 (A) This method showed good consistency with qPCR detection results. These results demonstrate that the biosensor and detection method constructed in this invention have high reliability in detecting foodborne pathogens in different food samples.

[0061] To verify the practical application performance of this method in multiplex detection, different concentrations of two foodborne pathogens (Salmonella typhimurium and Staphylococcus aureus, with concentrations of 10 and 10, respectively) were added to actual samples where the target was not detected. 5 10 6 and 10 7 (CFU / mL) was used to assess its recovery rate. See Table 2 and... Figure 15As shown in Figure B, the average recoveries of Salmonella Typhimurium and Staphylococcus aureus ranged from 89% to 110%, with relative standard deviations (RSDs) of the coefficients of variation ranging from 1.55% to 7.25%. These results demonstrate that this method enables multiplex detection and exhibits excellent detection performance in complex samples.

[0062] Table 2 Tt The Ago-driven autocatalytic nucleic acid circuit SERS biosensor simultaneously detected the recovery rates of Salmonella typhimurium and Staphylococcus aureus added to real samples.

[0063] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A kind Tt Ago-driven autocatalytic nucleic acid circuit SERS biosensor, characterized in that, The self-catalytic nucleic acid circuit SERS biosensor includes Tt Ago protein, cleavage buffer, gDNA1, gDNA2, biotinylated SERS signal probe, and reporter DNA complementary strand; when the self-catalytic nucleic acid circuit SERS biosensor is used alone to detect Salmonella typhimurium, the gDNA1 is... invA -gDNA1, wherein gDNA2 is invA -gDNA2, the biotinylated SERS signal probe is biotin-Ag@MBA@AuNPs, and the reporter DNA complementary strand is RC. invA When the self-catalytic nucleic acid circuit SERS biosensor is used to detect Staphylococcus aureus alone, the gDNA1 is... nuc -gDNA1, wherein gDNA2 is nuc -gDNA2, the biotinylated SERS signal probe is biotin-Ag@DTNB@AuNPs, and the reporter DNA complementary strand is RC. nuc When the self-catalytic nucleic acid circuit SERS biosensor is used to simultaneously detect Salmonella typhimurium and Staphylococcus aureus, the gDNA1 is... invA -gDNA1 and nuc -gDNA1, wherein gDNA2 is invA -gDNA2 and nuc -gDNA2, the biotinylated SERS signal probes are biotin-Ag@MBA@AuNPs and biotin-Ag@DTNB@AuNPs, and the reporter DNA complementary strand is RC. invA and RC nuc The invA -gDNA1, invA -gDNA2, nuc -gDNA1 and nuc The nucleotide sequences of -gDNA2 are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.8, respectively. The RC invA and RC nuc The nucleotide sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 9, respectively. The biotinylated SERS signal probe is obtained by mixing reporter DNA modified with 3' biotin and 5' thiol groups with polyT modified with 5' thiol groups and then fixing it on the surface of Ag@Ra@AuNPs by freeze-thaw method. The Ag@Ra@AuNPs are obtained by embedding Raman reporter molecules between Ag-Au layers with silver core and gold shell. The Raman reporter molecule used to detect Salmonella typhimurium is MBA, and the Raman reporter molecule used to detect Staphylococcus aureus is DTNB. The nucleotide sequence of the reporter DNA of Salmonella typhimurium is shown in SEQ ID NO. 1, the nucleotide sequence of the reporter DNA of Staphylococcus aureus is shown in SEQ ID NO. 2, and the nucleotide sequence of polyT is shown in SEQ ID NO.

3.

2. The method for constructing the self-catalytic nucleic acid circuit SERS biosensor according to claim 1, characterized in that, Includes the following steps: Preparation of S1, Ag@Ra@AuNPs: 1 mL of 1% AgNO3 solution was added to 60 mL of ultrapure water. The mixture was heated to boiling with vigorous stirring and maintained for 15 min. 1 mL of 1% trisodium citrate solution and 1 mL of 0.1% freshly prepared NaBH4 solution were added to the mixture sequentially. The mixture was kept boiling with vigorous stirring for 40 min, then allowed to cool naturally to room temperature. Ultrapure water was added to bring the dispersion volume to 100 mL to obtain the AgNPs seed solution. 2 mL of 1% TSC solution was added to 80 mL of ultrapure water and boiled for 15 min. 10 mL of AgNPs seed solution and 1.7 mL of 1% AgNO3 solution were added with vigorous stirring. The mixture was kept boiling for 1 h. 2 mL of 1% TSC solution and 1.7 mL of 1% AgNO3 solution were added to the reaction solution. The mixture was kept boiling for 1 h. 2 mL of 1% TSC solution and 1.7 mL of 1% AgNO3 solution were added to the reaction solution. After boiling for 1 h, 2 mL of 1% AgNO3 solution was added to the reaction solution. 1 mL of 1% TSC solution and 1.7 mL of 1% AgNO3 solution were boiled for 1 h, then naturally cooled to room temperature. Water was added to bring the volume to 100 mL, and the mixture was aged under 254 nm UV light for 30 min. The AgNPs dispersion was collected by centrifugation and washing. 100 μL of Raman reporter molecule solution was mixed with 4 mL of AgNPs dispersion and incubated with vigorous stirring at room temperature for 4 h. Excess unbound Raman reporter molecules were removed by centrifugation. The obtained Raman reporter molecule-labeled AgNPs were redispersed in 5 mL of ultrapure water to obtain Ag@RaNPs dispersion. The Raman reporter molecule solution was prepared by dissolving MBA in ethanol, with a concentration of 0.25-4 mM, or the Raman reporter molecule solution was prepared by dissolving DTNB in ​​ethanol, with a concentration of 0.5-6 mM. 5 mL of 5% PVP solution and 50 μL of 0.50% AgNO3 solution were added sequentially to 4 mL of Ag@RaNPs dispersion. The gold growth solution was prepared by sonicating a mixture of M L-AA solution, 0.20 mL of 0.50 M NaOH solution, and 0.20 mL of 0.10 M Na2SO3 solution for 10 min, followed by the addition of 1 mL of gold growth solution. The mixture was then allowed to stand at 65°C for 1 h, and the resulting Ag@Ra@AuNPs were collected by centrifugation. After washing, the Ag@Ra@AuNPs were redispersed in 4 mL of ddH2O. The gold growth solution was prepared by adding 50 μL of HAuCl4 solution, 0.24 mL of 0.20 M NaOH solution, and 3.0 mL of 0.010 M Na2SO3 solution to 4.7 mL of ultrapure water and allowing it to stand overnight to obtain a colorless and transparent gold growth solution. The concentration of the HAuCl4 solution was 5-100 mM. S2. Preparation of biotinylated SERS signal probes: Reporter DNA modified with 3' biotin and 5' thiol at the end was mixed with 5' thiol-modified polyT at a volume ratio of 1:0-1:40 and added to 1000 μL Ag@Ra@AuNPs. The mixture was frozen at -80°C for 30 min, then thawed at room temperature. 100 μL of 1% BSA was added and incubated for 1 h. The mixture was centrifuged and the precipitate was redispersed in 60 μL TN buffer. The concentrations of the 3' biotin-modified and 5' thiol-modified reporter DNA and the 5' thiol-modified polyT were both 100 μM, with a total volume of 250 μL. S3 Tt Preparation of Ago-driven autocatalytic nucleic acid loop detection system: When the autocatalytic nucleic acid loop SERS biosensor is used to detect Salmonella typhimurium alone, the Salmonella typhimurium detection system is prepared by: mixing 12 μL of 1×ThermoPol reaction buffer, 50-250 nM... Tt Ago, 500 nM gDNA, 100-500 nM RC invA And 10 μL of biotin-Ag@MBA@AuNPs were mixed, wherein the gDNA was invA -gDNA1 and invA -gDNA2; When the self-catalytic nucleic acid circuit SERS biosensor is used to detect Staphylococcus aureus alone, prepare the Staphylococcus aureus detection system: add 12 μL of 1×ThermoPol reaction buffer, 50-250 nM... Tt Ago, 500 nM gDNA, 100-500 nM RC nuc And 10 μL of biotin-Ag@DTNB@AuNPs were mixed, wherein the gDNA was nuc -gDNA1 and nuc -gDNA2; When the self-catalytic nucleic acid circuit SERS biosensor is used to simultaneously detect Salmonella Typhimurium and Staphylococcus aureus, a system for simultaneously detecting Salmonella Typhimurium and Staphylococcus aureus is prepared: 24 μL of 1×ThermoPol reaction buffer, 50-250 nM... Tt A mixture of AgO, 500 nM gDNA, 100-500 nM reporter DNA complementary strand, and 10 μL each of biotin-Ag@MBA@AuNPs and biotin-Ag@DTNB@AuNPs, wherein the gDNA is invA -gDNA1, invA -gDNA2, nuc -gDNA1 and nuc -gDNA2, the reporter DNA complementary strand is RC invA and RC nuc .

3. The construction method according to claim 2, characterized in that, The Raman reporter molecule solution described in S1 is prepared by dissolving MBA in ethanol, with an MBA concentration of 1 mM and a HAuCl4 concentration of 50 mM.

4. The construction method according to claim 2, characterized in that, The Raman reporter molecule solution described in S1 is a solution in which DTNB is dissolved in ethanol at a concentration of 4 mM and a concentration of HAuCl4 of 30 mM.

5. The construction method according to claim 2, characterized in that, The reporter DNA modified with 3' biotin and 5' thiol in S2 is mixed with polyT modified with 5' thiol at a volume ratio of 1:

30.

6. The construction method according to claim 2, characterized in that, S3 as described Tt The concentration of Ago is 200 nM, and the RC invA and RC nuc The concentrations were all 400 nM.

7. The application of the self-catalytic nucleic acid circuit SERS biosensor of claim 1 in detecting Salmonella typhimurium and / or Staphylococcus aureus contamination in food or food processing.

8. The application of a kit containing the self-catalytic nucleic acid circuit SERS biosensor of claim 1 in the detection of Salmonella typhimurium and / or Staphylococcus aureus contamination in food or food processing.

9. A method for detecting contamination of food with Salmonella typhimurium and / or Staphylococcus aureus during food or food processing, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested and heat it at 95°C for 20 min to obtain single-stranded DNA, and then cool it rapidly at -20°C for 20 min. (2) When detecting Salmonella typhimurium alone, 8 μL of single-stranded DNA is mixed with the Salmonella typhimurium detection system described in claim 2, S3. When detecting Staphylococcus aureus alone, 8 μL of single-stranded DNA is mixed with the Staphylococcus aureus detection system described in claim 2, S3. When detecting both Salmonella typhimurium and Staphylococcus aureus simultaneously, 16 μL of single-stranded DNA is mixed with the system for detecting both Salmonella typhimurium and Staphylococcus aureus simultaneously. The reaction is incubated at 85°C for 10-120 min to obtain the reaction mixture. (3) Add the reaction mixture obtained in step (2) to streptavidin-functionalized magnetic beads with a concentration of 10 mg / mL, incubate at room temperature for 30 min, perform magnetic separation of the reaction product, centrifuge, collect the supernatant, spot it onto a glass slide wrapped in tin foil, and collect Raman spectra using an Optosky ATR6500 portable Raman spectrometer with a spectral acquisition range of 800-2000 cm⁻¹. -1 Each sample should be measured at least three times independently.

10. The method according to claim 9, characterized in that, The incubation time in step (2) is 90 min.

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