SERS biosensor and its application in detection of methicillin-resistant staphylococcus aureus

Abstract

The present application relates to a SERS biosensor, which is a hairpin probe DNA modified on the surface of small-particle-size AuNPs, and a protective DNA and a Raman dye modified on the surface of large-particle-size AuNPs; an aptamer, trigger chain 1 and 2 are combined into a triple-stranded structure; then the target bacteria and exonuclease III are mixed and added for enzymatic reaction, finally a SERS hot spot is generated and a very strong SERS signal is generated, the supernatant is taken for SERS detection, and the rapid detection of the target bacteria-methicillin-resistant Staphylococcus aureus can be realized. The present application combines the traditional colorimetric analysis method and multi-stage nucleic acid amplification together, can quickly convert the trace target signal into Raman signal output through multi-stage signal amplification, and realizes the specific early rapid detection of the target bacteria.

Description

Technical Field

[0001] This invention relates to the field of bioanalytical detection, specifically to SERS biosensors and their application in the detection of methicillin-resistant Staphylococcus aureus. Background Technology

[0002] In the 20th century, as human activity expanded, bacterial infections spread widely through food, water, and medical routes. With the discovery and application of antibiotics, common sources of infection, such as Staphylococcus aureus, were significantly contained. However, due to the broad-spectrum antibacterial properties of antibiotics, excessive use of these drugs on infected individuals led to the emergence of resistant strains. Methicillin-resistant Staphylococcus aureus (MRSA), in particular, has received widespread attention since its discovery due to its multidrug resistance. MRSA was first discovered by a British researcher in the 1960s, and its occurrence became increasingly frequent in the decades following antibiotic use. Due to its multidrug resistance and highly contagious nature, MRSA poses a significant threat to postoperative patients. MRSA infections have been found in hospitals in several developed countries, with a mortality rate exceeding 50%. Further research suggests that infection rates in relatively underdeveloped regions such as Asia, Africa, and Latin America may have exceeded 80%. This poses a serious threat to public safety and food security.

[0003] Among these methods, surface-enhanced Raman spectroscopy (SERS)-based detection has attracted widespread attention due to its high sensitivity, high selectivity, and non-destructive detection capabilities. Several SERS sensors have been developed, providing new methods for bacterial removal and promoting the advancement of bacterial research. With the development of portable Raman spectrometers, SERS shows great potential in practical bacterial analysis. Since electromagnetic enhancement makes a significant contribution to the enhancement of SERS signals, nanomaterials and nanostructures that can generate strong surface plasmon resonances are now commonly considered when designing SERS biosensors. It has been reported that electromagnetic fields can be greatly enhanced at hotspots, which are the junctions of nanoparticles. Therefore, small clusters or matrices of nanoparticles can generate more hotspots in three-dimensional (3D) space, which greatly enhances the SERS signal. Furthermore, the strategy of generating three-dimensional SERS hotspots in solution is not only more stable than traditional planar matrices but also exhibits advantages in terms of improved sensitivity and stability compared to traditional SERS removal methods. Summary of the Invention

[0004] This invention designs a detection strategy based on exonuclease III (EXOIII)-assisted cyclic cleavage to generate 3D SERS hotspots, constructing a detection system for detecting methicillin-resistant Staphylococcus aureus (MRSA). This invention combines traditional colorimetric analysis with multi-stage nucleic acid amplification, rapidly converting trace target signals into Raman signals through multi-stage signal amplification, achieving early and rapid detection of specific infection by target bacteria. This detection system includes a SERS biosensor; specifically, this invention includes the following technical solutions:

[0005] The SERS biosensor of the present invention is prepared by the following method: (1) hairpin probe DNA is modified on the surface of 15-20 nm gold nanoparticles; (2) protective DNA is modified on the surface of 28-35 nm gold nanoparticles, and Raman dye is added to modify the surface of gold nanoparticles, and thiol PEG is added to prevent non-specific binding; (3) aptamer DNA, trigger chain 1 and trigger chain 2 are annealed and then naturally cooled to generate a triple-stranded structure; (4) the products obtained in steps (1), (2) and (3) are mixed and target bacteria, exonuclease III and magnesium ions are added to trigger the enzymatic digestion reaction; (5) the cyclic digestion is started to generate 3D SERS hotspots.

[0006] Preferably, in steps (1) and (2), both types of gold nanoparticles can be synthesized using the classic sodium citrate-reduction chloroauric acid method. Specifically, refer to relevant literature, or the following steps can be used: boil ultrapure water for 30 minutes, add a certain amount of 25mM chloroauric acid solution and stir vigorously, then add different amounts of 1% sodium citrate solution according to the size of the target gold nanoparticles, react for a period of time and then cool naturally to room temperature.

[0007] The hairpin probe DNA sequences, HP1 and HP2, were designed independently and are designated as SEQ ID NO.1 and SEQ ID NO.2, respectively. The sequences are as follows:

[0008] 5'-TTTTTTTTTTCGCCCAACCGCACTGTTAACAGTTGCGGTTGGGCATGATG-3';

[0009] 5'-TTTTTTTTTTCATCACAGAAACTGTGTTTGCTTTCTGTGATGCGGTTG-3'.

[0010] The protective probe DNA sequences, A1 and A2, were designed independently and are denoted as SEQ ID NO.3 and SEQ ID NO.4, respectively. The sequences are as follows:

[0011] 5'-TTTTTTTTTTTCACAGTGCGGTTGGGC-3';

[0012] 5'-TTTTTTTTTTTCACAGTTTCTGTGATG-3'.

[0013] Preferably, step (4)

[0014] The sequences of the aptamer DNA, trigger chain 1, and trigger chain 2 are as follows, denoted as SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively:

[0015] 5'-ATGCGGTTGGTTGCGGTTGGGCATGATGTATTTCTGTGATGCGGTTGTTTTT-3';

[0016] 5'-CATCATGCCCAACCGCAATTTTT-3';

[0017] 5'-CAACCGCATCACAGAAATTTTTT-3'.

[0018] Preferably, in step (5), the bacterial strain is used to determine the concentration curve by dilution plate coating method and ultraviolet absorption value, and after being cultured in LB medium for 24 hours, the absorbance is detected, and then the concentration-extracted bacterial suspension is prepared by using the concentration curve.

[0019] Preferably, in step (6) of constructing a biosensor, better sensor performance can be achieved by adjusting reaction conditions, such as reaction time, magnesium ion concentration, exonuclease III concentration, and the ratio of the two types of gold nanoparticles.

[0020] In this invention, for the triple-stranded structure formed by the aptamer and two trigger strands, the aptamer has a stronger binding ability to bacteria. When bacteria are added, the aptamer and the two trigger strands unwind their helical structure and specifically bind to the bacteria, releasing the two trigger strands. The trigger strands then bind to two types of hairpin DNA on the surface of the gold nanoparticles and trigger an enzymatic cleavage reaction to release Walker gold nanoparticles. The released Walker gold nanoparticles pair complementaryly with the protective DNA and trigger an enzymatic cleavage reaction to cut the protective DNA modified on the surface of the 28-35nm gold nanoparticles. As the reaction proceeds, the stability of the 28-35nm gold nanoparticles is disrupted, and they aggregate under the action of magnesium ions, thus amplifying the signal.

[0021] The SERS biosensor described above can be prepared using the following method:

[0022] (1) Preparation of DNA-AuNP: Mix 6 μL of 10 μM DNA with 10 μL of 10 nM AuNPs. Then incubate the mixture in a buffer containing 0.05 M sodium citrate and 0.01% Tween-20 for 3 hours, then add 20 μL of sodium citrate and incubate overnight.

[0023] (2) NaCl was continuously added over the next three days until the final NaCl concentration was 0.3M. After that, the free DNA was removed and the precipitate was dispersed in 10mM TAE buffer. Small-particle gold nanoparticles modified with hairpin DNA (hairpin DNA-AuNP) and large-particle gold nanoparticles modified with protective DNA (protective DNA-AuNP) can be obtained by using the methods in steps (1) and (2).

[0024] (3) Add Raman dye to the solution of gold nanoparticles modified with protective DNA and incubate for 30 minutes. Finally, wash off the excess dye and store the solution as a reporter probe at 4°C for later use.

[0025] (4) Add the trigger strands 1, 2 and aptamer DNA to TAE buffer, anneal at 90°C for 10 minutes and then cool naturally to room temperature. Store in a 4°C refrigerator for later use.

[0026] (5) The bacteria were grown in LB medium at 37°C for 24 hours, and the bacterial concentration was then quantified by optical density. Subsequently, the bacteria were centrifuged at 8000 rpm for 10 minutes, dispersed in PBS buffer, washed three times, and stored at 4°C for later use.

[0027] (6) Quantitatively extract the bacteria from (5) and mix them with (4), (3) and gold nanoparticles modified with hairpin DNA, and add exonuclease III and Mg 2+ Incubate at 37°C for 30-50 minutes.

[0028] In the SERS biosensor described above, preferably, in step (3), Raman dye with a final concentration of 0.1 mM is added. After binding for 30 minutes, excess dye is removed by centrifugation and the mixture is dispersed in TAE solution. After modification with thiol-PEG to prevent non-specific binding, it is centrifuged and dispersed in TAE solution for later use. In this invention, DNA is pre-modified on the surface of AuNPs, which is a common modification method. Generally, the thiol-modified DNA (the customized DNA sequence is modified with thiol to facilitate the subsequent binding with Au to form Au-SH bonds), AuNPs, and citric acid are incubated together, and then NaCl is added for incubation. Gold-sulfur bonds are generated by the classic salt aging method. The preferred particle size of the gold nanoparticles modifying the hairpin DNA is 15 nm-20 nm, more preferably 15-18 nm, and most preferably 16 nm. The preferred particle size of the gold nanoparticles modifying and protecting the DNA is 28-35 nm, more preferably 28-32 nm, and most preferably 30 nm. The inventors discovered that large gold nanoparticles are more prone to aggregation, and the signal from aggregated large gold nanoparticles is significantly stronger than that from small gold nanoparticles. Conversely, small-diameter gold nanoparticles are difficult to aggregate simply through surface DNA cleavage because they exhibit better stability. Therefore, this invention employs a reporter probe with large gold nanoparticles for sensor signal output.

[0029] Preferably, in step (4) of the SERS biosensor described above, trigger chain 1 (6 μL, 100 μM), trigger chain 2 (6 μL, 100 μM), and aptamer DNA (6 μL, 100 μM) are added to 42 μL of 10 mM TAE buffer and annealed at 90°C for 10 minutes, then naturally cooled to room temperature and stored at 4°C for later use. Magnesium ions are added during annealing.

[0030] Preferably, the total enzyme digestion reaction time of the SERS biosensor described above is controlled within 40 minutes.

[0031] During bacterial culture, it is best to measure absorbance regularly to determine changes in bacterial concentration. After bacterial culture is complete, impurities in the culture medium should be removed by centrifugation and washing with TAE buffer. Before the reaction, ensure that the aptamer DNA and the two trigger strands are bound to form a triple-stranded structure to prevent a large number of free trigger strands from causing a large background signal.

[0032] In this invention, due to the use of exonuclease III-assisted multi-stage nucleic acid signal amplification, a strong signal output can be achieved with a small amount of bacteria, which is beneficial for the rapid detection of samples in the early stage of infection.

[0033] The application of the SERS biosensor of the present invention in the preparation of a detection system for detecting methicillin-resistant Staphylococcus aureus enables rapid detection of low concentrations of target analytes and can be applied to a variety of real samples.

[0034] The principle of this invention is as follows:

[0035] 1. Aptamer recognition of bacteria and multi-level nucleic acid amplification

[0036] When target bacteria are introduced, carefully selected aptamers specifically bind to the bacteria and release two complementary strands. This ensures the selectivity of the biosensor, avoids false positive signals, and achieves the conversion of bacteria into DNA strands, while simultaneously amplifying the nucleic acid signal. The released trigger strand then opens the hairpin DNA and, with the help of exonucleases, cleaves it to release the molecular machinery, thus achieving multi-stage nucleic acid amplification. Specifically, small gold nanoparticles (approximately 18 nm) are modified with hairpin DNA1 and hairpin DNA2, while larger gold nanoparticles (approximately 30 nm) are modified with protective DNA1 and protective DNA2. Trigger DNA1 and trigger DNA2 then open and cleave hairpin DNA1 and DNA2, respectively, generating two residual strands—hairpin DNA residual 1 and hairpin DNA residual 2. These two residual strands bind to and cleave protective DNA1 and protective DNA2, respectively, generating two more residual strands—protective DNA residual 1 and protective DNA residual 2. Finally, the large gold nanoparticles aggregate, achieving multi-stage nucleic acid amplification and signal amplification.

[0037] 2. Basis for SERS detection strategy based on exonuclease III cyclic cleavage to destroy gold nanoparticles and form 3D hotspots

[0038] The Walker chain on the surface of the molecular machine generated in the reaction binds to the DNA chain on the reporter probe and, with the help of enzymes, cyclically cleaves along the gold nanoparticles. During the cleavage process, the charge structure on the surface of the reporter probe is altered. At the same time, a large number of magnesium ions are present in the solution, which ultimately leads to the aggregation of the reporter probe. Thus, a 3D SERS hotspot is formed, which greatly enhances the signal intensity of the Raman dye modified on the reporter probe. Because this process is very rapid and the signal amplification is huge, it can achieve rapid detection of target bacteria at ultra-low concentrations.

[0039] This invention, based on the specific recognition of target bacteria by aptamers and a carefully designed enzymatic cleavage reaction of DNA, incorporates Raman dyes and gold nanoparticles. It utilizes traditional colorimetric analysis methods while pioneering a bioanalytical method with higher signal intensity and lower detection limits, developing a biosensor capable of high-speed analysis of ultra-low concentrations of target bacteria. Specifically, after converting the bacterial signal into a nucleic acid signal output, enzymatic multilevel amplification of nucleic acid and molecular machinery are used to rapidly disrupt the stability of gold nanoparticles and induce their aggregation, ultimately generating SERS hotspots and a strong SERS signal, achieving rapid detection of methicillin-resistant Staphylococcus aureus (MRSA). Attached Figure Description

[0040] Figure 1 A schematic diagram illustrating the principle of SERS method for detecting MRSA;

[0041] Figure 2 The process of multi-stage nucleic acid amplification and 3DSERS hotspot generation was verified by polyacrylamide gel chromatography and agarose gel electrophoresis.

[0042] Figure 3 Transmission electron microscopy image of the 3D SERS hotspot generation process;

[0043] Figure 4 This is a graph showing the relationship between MRSA concentration and Raman intensity, as well as the linear relationship, in Example 1;

[0044] Figure 5 This is an optimization of the conditions for the MRSA detection process in Example 1;

[0045] Figure 6 This refers to the stability and selectivity of MRSA in Example 1;

[0046] Figure 7 This is a comparison of the detection effects of various bacteria in various real samples in Example 1. Detailed Implementation

[0047] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

[0048] Salmonella enteritidis (CMCC(B)50335) was provided by the China Center for Type Culture Collection. Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300), Salmonella typhi (ATCC 14028), Bacillus subtilis (ATCC 6633), and Escherichia coli (E. coli, ATCC 8739) were purchased from the China General Microbiological Culture Collection Center.

[0049] Example 1

[0050] The multi-stage nucleic acid amplification process was confirmed by 10% polyacrylamide gel chromatography. The triple-stranded structure formed by the complementarity of aptamer DNA and two trigger strands of DNA was disrupted by bacteria, generating individual trigger strand bands. The trigger strands then opened the hairpin DNA, forming a double-stranded structure. Exonuclease III cleaved the hairpin DNA in the double-stranded structure, generating a Walker strand. Finally, the Walker strand bound to the protective DNA and was cleaved by exonuclease III. Electrophoresis was then performed at 96V for 12 hours in 1×TAE buffer. The final electrophoresis results were displayed using a gel imaging system, as shown below. Figure 2 A.

[0051] The 3D SERS hotspot generation process was confirmed on a 1.5% agarose gel. Hairpin DNA was modified onto gold nanoparticles of approximately 16 nm, and then protective DNA was modified onto gold nanoparticles of approximately 30 nm. After the hairpin DNA-gold nanoparticle cleavage was completed, the protective DNA-gold nanoparticles bound to each other but did not cleave. The DNA cleavage reaction here is multi-stage; that is, the next stage cannot occur without the previous stage, which is specifically controlled by DNA sequence design. The trigger DNA can only open the hairpin DNA but cannot bind to the protective DNA. Only when the hairpin DNA is opened by the trigger DNA and cleaved by the enzyme to produce hairpin DNA remnants can the next stage reaction occur, i.e., the remnant DNA binds to the protective DNA and cleaves the protective DNA with the assistance of the enzyme.

[0052] Finally, the reaction was completed: after adding bacteria, the triple-stranded structure, and two types of gold nanoparticles, the reaction was carried out at 5 mM MgAc2 for 40 minutes to generate 3D SERS hotspots. Electrophoresis was then performed in 0.5×TBE buffer at 8 V / cm³. The final electrophoresis results were captured by a smartphone, such as... Figure 2 B.

[0053] Specifically, the SERS sensor was prepared as follows: 6 μL of 10 μM DNA was mixed with 10 μL of 10 nM AuNPs. The mixture was then incubated for 3 hours in a buffer containing 0.05 M sodium citrate and 0.01% Tween-20. 20 μL of sodium citrate was then added and the mixture was incubated overnight. Over the next three days, NaCl was continuously added until the final NaCl concentration reached 0.3 M. Afterward, the free DNA was removed, and the precipitate was dispersed in 10 mM TAE buffer. Following this method, 16 nm gold nanoparticles modified with hairpin DNA and 30 nm gold nanoparticles modified with protective DNA were obtained. Raman dye was added to the solution of gold nanoparticles modified with protective DNA and incubated for 30 minutes. Finally, excess dye was washed off to obtain the reporter probe, which was stored at 4°C for later use. Trigger strand 1, trigger strand 2, aptamer DNA, and magnesium ions were added to TAE buffer and annealed at 90°C for 10 minutes, then allowed to cool naturally to room temperature to form a triple-stranded structure. This triple-stranded structure was stored at 4°C for later use. Bacteria were grown in LB medium at 37°C for 24 hours, and bacterial concentration was then quantified by optical density. Subsequently, the bacteria were centrifuged at 8000 rpm for 10 minutes, dispersed in PBS buffer, washed three times, and stored at 4°C for later use. After incubation, the target bacteria were quantified and diluted to gradient concentrations. Finally, hairpin DNA-AuNP and the reporter probe were mixed in TAE buffer, and triple-stranded DNA, exonuclease III, and 5 mM MgAc2 were added. The mixture was incubated at 37°C for 40 minutes. This resulted in the formation of SERS hotspots and a strong SERS signal. The supernatant was used for SERS detection, enabling rapid detection of the target bacterium—methicillin-resistant Staphylococcus aureus.

[0054] like Figure 2 Figure A illustrates a multi-stage nucleic acid amplification strategy using polyacrylamide gel electrophoresis. The trigger strand binds to hairpin DNA (well 1) to form a double-stranded structure (well 2). The slower migration speed causes the band to shift upwards. Subsequently, exonuclease III cleaves the hairpin DNA within the double-stranded structure, causing the double-stranded band to disappear and a new band to form (well 3). The cleavage product (well 4) mixes with exonuclease III and shifts to form a new band (well 5). This shift may be because, although no cleavage reaction occurs, the enzyme alters the charge of the DNA strand. The positions of wells 5 and 3 are essentially identical, indicating the formation of the target product. Binding the cleavage product to protective DNA (well 6) forms a new band (well 7), which disappears upon the addition of exonuclease III (well 8). These results demonstrate that hairpin opening and two-stage cleavage can proceed normally.

[0055] like Figure 2Figure B shows the agarose gel electrophoresis analysis of the formation process of 3D SERS hotspots from AuNPs. Naked 16nm AuNPs (well 1) and hairpin DNA-modified 16nm AuNPs (well 2) were used as references. Naked 30nm AuNPs (well 3) and protected DNA-modified 30nm AuNPs (well 4) were compared, showing that the DNA-modified gold nanoparticle bands shifted upwards, indicating an increase in particle size. After the first round of enzymatic digestion, the molecular machinery was released and complementary paired with the dye probe (well 5), further increasing the particle size. Subsequent further cleavage generated 3D SERS hotspots (well 6), forming a large number of gold nanoparticle aggregates with a lower band migration rate. These results demonstrate the successful assembly of the designed biosensor.

[0056] like Figure 3 The image shown is a TEM image, and image AF illustrates the changes in gold nanoparticles during the reaction from 0 to 50 minutes. Figure A shows two types of gold nanoparticles: 16nm gold nanoparticles modified with hairpin DNA and 30nm gold nanoparticles modified with protective DNA. As the reaction proceeds, we can see that the aggregation degree of the gold nanoparticles increases, and the initially existing intermediate states (complementary to the dye probe DNA) gradually disappear, eventually forming aggregated states. This is the 3D SERS hotspot, resulting in a very strong SERS signal.

[0057] like Figure 4 The diagram shows the detection performance of this strategy for gradient concentrations of target bacterial MRSA in TAE buffer. Specifically, it investigates the detection effect from 10... 0 -10 7 The correlation between CFU / mL bacterial concentration and SERS signal shows a good linear relationship, indicating good analytical performance.

[0058] like Figure 5 The diagram shows optimized experimental conditions, primarily investigating which conditions yielded the best signal under varying conditions such as the gold-to-size ratio, magnesium ion concentration, exonuclease concentration, and the size of the dye probe gold nanoparticles. Before actual detection, it is necessary to optimize the key factors affecting the reaction process to obtain the most suitable reaction conditions. Without changing other conditions: such as... Figure 5 A. By controlling the ratio of the two types of gold nanoparticles to 1, a better signal intensity can be obtained; for example... Figure 5 B, controlling the magnesium ion concentration at 5mM can obtain a better signal strength; such as Figure 5 C. Maintaining the concentration of exonuclease III (EXOIII) at 0.25 U / μL can yield a better signal intensity; such as Figure 5D. Choosing 30nm gold nanoparticles as DNA orbiters yields good signal intensity; however, to achieve better stability and higher signal intensity, larger gold nanoparticles were not used because larger particle sizes are more prone to precipitation during the reaction, leading to reduced signal and stability. With the addition of Mg... 2+ Increased concentration makes gold nanoparticles more prone to aggregation, causing changes in the SERS signal and affecting experimental stability.

[0059] like Figure 6 Figures A and B show the changes obtained by measuring a sample 30 times consecutively, indicating that the sample does not undergo significant signal changes under multiple detections, demonstrating a certain degree of detection stability. Figure 6 Figures C and 6D show the data obtained by continuously detecting five samples for seven days. Although the signal decreased to some extent over time, the overall decrease was small, indicating that the strategy has good time stability.

[0060] like Figure 7 The diagram illustrates the detection of various bacteria and target bacteria in multiple real samples. Detection was performed in six real samples: TAE buffer, serum, sewage, urine, milk, and saliva. Five target bacteria—MRSA, Salmonella typhimurium, Escherichia coli, Streptococcus enteritidis, and Bacillus subtilis—were also detected. The results demonstrate that the sensor of this invention exhibits good selectivity and the ability to detect bacteria in real samples, showing potential for further development and application in clinical testing and real-time field monitoring.

[0061] It should be noted that the above-described technical content of this invention is merely an explanation and clarification to enable those skilled in the art to understand the technical essence of this invention, and therefore is not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the claims. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the essential spirit of this invention should be within the scope of protection of this invention.

Claims

1. The SERS biosensor was prepared using the following method: (1) Hairpin probe DNA is modified on the surface of 15-20 nm gold nanoparticles; the hairpin probe DNA sequence includes HP1 and HP2, which are SEQ ID NO.1 and SEQ ID NO.2, respectively; (2) The protective probe DNA was modified on the surface of 28-35 nm gold nanoparticles, and then Raman dye was added for modification; the protective probe DNA sequence includes A1 and A2, which are SEQ ID NO. 3 and SEQ ID NO. 4, respectively; (3) The aptamer DNA and two trigger strand DNAs are complementary and paired to form a triple-stranded structure; the sequence of the aptamer DNA is SEQ ID NO. 5, and the two trigger strand DNAs include trigger strand 1 and trigger strand 2, whose sequences are SEQ ID NO. 6 and SEQ ID NO. 7, respectively; (4) Mix the products obtained in steps (1), (2) and (3) and add the target bacteria, exonuclease III and magnesium ions to trigger the enzymatic digestion reaction, and cyclically digest to generate 3D SERS hotspots.

2. The SERS biosensor as described in claim 1, characterized in that, The gold nanoparticles in steps (1) and (2) were obtained by sodium citrate reduction.

3. The SERS biosensor as described in claim 1, characterized in that, Step (1) Gold nanoparticles 15-18 nm; Step (2) Gold nanoparticles 28-32 nm.

4. The SERS biosensor as described in claim 1, characterized in that, Step (3) involves annealing the three DNA strands simultaneously at 90°C and cooling them to room temperature to form a triple-stranded structure.

5. The SERS biosensor as described in claim 1, characterized in that, The target bacteria in step (4) are prepared into bacterial suspensions with different concentration gradients.

6. The SERS biosensor according to any one of claims 1-5, characterized in that, It was prepared using the following method: (1) Hairpin probe DNA was modified on the surface of 15-20 nm gold nanoparticles to obtain gold nanoparticles modified with hairpin DNA; (2) The protective probe DNA was modified on the surface of 28-35 nm gold nanoparticles to obtain gold nanoparticles modified with protective DNA; (3) Add Raman dye to the gold nanoparticle solution that has been modified to protect the probe DNA in step (2) and incubate for 30 minutes. Finally, remove excess dye and store in a 4°C refrigerator for later use. (4) Add the two trigger strand DNAs and aptamer DNA to TAE buffer, anneal at 90°C for 10 minutes, cool naturally to room temperature, and then store in a 4°C refrigerator for later use. (5) The target bacteria were grown in LB medium at 37°C for 24 hours. The bacterial concentration was then quantified by optical density. Subsequently, the bacteria were centrifuged at 8000 rpm for 10 minutes, dispersed in PBS buffer, washed three times, and stored in a refrigerator at 4°C for later use. (6) Quantitatively extract the target bacteria after treatment in step (5) and mix them with the storage solution in step (3), the storage solution in step (4), and the gold nanoparticles modified with hairpin DNA in step (1) in TAE buffer, and add exonuclease III and Mg 2+ Incubate at 37°C for 30-50 minutes.

7. The SERS biosensor as described in claim 6, characterized in that, Step (3) The final concentration of Raman dye is 0.1 mM. After binding for 25-35 minutes, excess dye is removed by centrifugation and washing and dispersed in TAE solution.

8. The use of the SERS biosensor according to any one of claims 1-7 in the preparation of a detection system for detecting methicillin-resistant Staphylococcus aureus.

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