Biosensor capable of enhancing Raman signal, preparation method of biosensor and application of biosensor in staphylococcus aureus detection

Abstract

The invention discloses a biosensor capable of enhancing Raman signals, a preparation method of the biosensor and application of the biosensor in staphylococcus aureus detection, and belongs to the technical field of microbiological detection. The sensor is composed of a capture probe and a signal probe. The capture probe is MXene (at) Au (at) apt, and the signal probe is Au (at) Ag (at) 4-MBA (at) apt. The capture probe can capture staphylococcus aureus and is connected with the signal probe to form a sandwich structure, and the structure can significantly enhance Raman signals and improve the detection sensitivity of the sensor. The sensor provided by the invention has the advantages of low detection limit, strong specificity, low cost, high efficiency and short time consumption, thereby having important application prospects and values in the technical field of pathogenic bacterium detection.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a biosensor capable of enhancing Raman signals, a preparation method thereof and an application thereof in Staphylococcus aureus detection. Background Art

[0002] According to the national standard GB4789.10-2010, there are three conventional detection methods for Staphylococcus aureus: qualitative test for Staphylococcus aureus, Baird-Parker (BP) plate count for Staphylococcus aureus, and most probable number count for Staphylococcus aureus. The detection methods for Staphylococcus aureus mainly include the following basic steps: pre-enrichment, selective enrichment, selective plating, biochemical screening, and serological confirmation. These methods are generally considered to be the gold standard and take about three to seven days to obtain confirmed results. Although these methods are sensitive and inexpensive, they can provide qualitative and quantitative information about the number and nature of microorganisms in food samples. However, due to the complex operation and long time consumption, they can no longer meet the needs of rapid detection of pathogens in food.

[0003] Currently, the rapid detection methods for Staphylococcus aureus include: immunoagar diffusion method, immunomagnetic bead separation method, polymerase chain reaction detection, immunofluorescence assay, enzyme-linked immunosorbent assay and other methods. The immunoagar diffusion method produces a white precipitation line through the reaction of soluble antigens and their corresponding antibodies, but the detection sensitivity of Staphylococcus aureus is low, the bacterial increase process is time-consuming, and a lot of time is required to achieve comprehensive detection through complex operations. The immunomagnetic bead separation method is a technology that uses immunomagnetic beads as capture reagents. It has been developed for the separation and identification of Staphylococcus aureus. The capture rate of Staphylococcus aureus immunomagnetic beads for target bacteria has reached a high level; however, this method has some limitations, such as high price and cross-reaction, so other technical approaches are needed to assist detection. Polymerase chain reaction is the most famous and mature nucleic acid amplification technology for detecting pathogenic microorganisms. Compared with culture-based methods, this technology has obvious advantages of rapidity, specificity, sensitivity, high efficiency and small sample size. Many PCR detection methods have been developed for detecting and verifying foodborne bacteria and viruses in food and applied to milk and dairy products. However, the limitations of PCR amplification technology include the difficulty in obtaining DNA preparation suitable for PCR amplification, and the procedure is quite complicated, requiring equipment and a clean environment. In addition, PCR cannot distinguish between living and dead cells and will provide more false negative results. Although immunofluorescence assay and enzyme-linked immunosorbent assay are simple, sensitive, rapid and specific, they are expensive when combined with antibodies, which greatly increases the cost.

[0004] As a vibrational spectroscopy technique, Raman spectroscopy has the advantages of simple pretreatment of the detection object, no influence of water, and wide coverage of detection wavenumbers compared to other spectroscopy techniques. It can realize qualitative analysis of substances and quantitative analysis within a certain range. However, ordinary Raman detection itself also has some defects. For example, when the object being tested is irradiated by laser, a fluorescent reaction will occur, and its own Raman signal is easily covered by fluorescence and other noise signals, causing interference. In addition, when performing ordinary Raman detection, the Raman signal of the substance is generally weak, the sensitivity of the measurement is not enough, and there are certain limitations in early applications. Therefore, how to improve the Raman spectral signal during the operation process is of great significance and value for the sensitive and accurate detection of Staphylococcus aureus. Summary of the invention

[0005] The present invention provides a biosensor capable of enhancing Raman signals. The sensor consists of a capture probe and a signal probe. The capture probe is MXene@Au@apt, and the signal probe is Au@Ag@4-MBA@apt.

[0006] The above MXene@Au@apt was prepared by the following method:

[0007] The MXene two-dimensional material is mixed with Au nanoparticles and incubated to obtain the MXene@Au material; then the MXene@Au material is mixed with the aptamer solution and incubated to obtain the MXene@Au@apt.

[0008] In the above-mentioned preparation method of MXene@Au@apt, each raw material is selected from the following volume fractions:

[0009] 500-1000 parts of MXene two-dimensional material, 500-1000 parts of Au nanoparticles, 500-1000 parts of MXene@Au material, and 50-100 parts of aptamer solution.

[0010] In the above-mentioned preparation method of MXene@Au@apt, the usage ratio of the MXene two-dimensional material to the Au nanoparticles is 1:1; the usage ratio of the MXene@Au material to the aptamer solution is 10:1.

[0011] In a specific embodiment, each raw material is selected from the following volume parts:

[0012] 500 parts of MXene two-dimensional material, 500 parts of Au nanoparticles, 500 parts of MXene@Au material, and 50 parts of aptamer solution.

[0013] In the above-mentioned preparation method of MXene@Au@apt, the incubation conditions are selected from: incubation at 35-40°C for 10-16 hours; preferably: incubation at 37°C for 12 hours.

[0014] In the above-mentioned preparation method of MXene@Au@apt, the concentration of the aptamer solution is selected from 50 to 1000 nM, preferably 800 nM.

[0015] In the above-mentioned preparation method of MXene@Au@apt, the MXene two-dimensional material is prepared by the following method:

[0016] 40 mL of dilute hydrochloric acid solution (9 mol / L) was placed in a polytetrafluoroethylene beaker, 2 g of lithium fluoride powder was added, and the mixture was stirred at 37°C for 30 min; 2 g of Ti3AlC2 powder was then added and magnetically stirred for 24 h to etch away the Al element in Ti3AlC2; the solution was then placed in a centrifuge tube, centrifuged at 9500 rpm for 15 min, the supernatant was removed, and the multilayer Ti3C2Tx precipitate was collected, and it was washed alternately with ultrapure water and anhydrous ethanol and ultrasonically treated at the same time until the pH was close to 6; the precipitate was then dispersed in 40 mL of anhydrous ethanol and ultrasonically treated for 1 h; the solution was centrifuged at 9000 rpm for 15 min, the supernatant was removed, 40 mL of ultrapure water was added, and the ultrasonic treatment was continued for 20 min, and the solution was then centrifuged at 3500 rpm for 10 min, the supernatant was retained, and the layered Ti3C2Tx liquid, i.e., the Mxene two-dimensional material, was obtained.

[0017] In the above-mentioned preparation method of MXene@Au@apt, the Au nanoparticles are prepared by the following method:

[0018] 3.5 mL of sodium citrate (1%) was added to 100 mL of boiling HAuCl4 solution (0.01%) and stirred for 30 min, and then continued to stir for 15 min without heating to obtain Au nanoparticles.

[0019] The Au@Ag@4-MBA@apt was prepared by the following method:

[0020] The NH2OH solution and the NaOH solution were mixed, and in the dark, an AgNO3 solution was added to react; then, a HAuCl4·3H2O solution and a sodium citrate solution were added simultaneously, and the reaction was stirred to obtain an Au@AgNFs solution; the Au@AgNFs solution was centrifuged, and the Au@AgNFs precipitate was dispersed in water to prepare an Au@AgNFs suspension; then, a 4-MBA ethanol solution was added, the reaction was stirred, and the centrifugation was performed, and the obtained precipitate was an Au@AgNFs@4-MBA material; the obtained Au@AgNFs@4-MBA material was dispersed in water to prepare an Au@AgNFs@4-MBA solution; the Au@AgNFs@4-MBA solution was mixed with an aptamer solution, and incubated to obtain Au@Ag@4-MBA@apt.

[0021] In the above-mentioned preparation method of Au@Ag@4-MBA@apt, each raw material is selected from the following volume fractions:

[0022] 300-800 parts of NH2OH solution, 300-800 parts of NaOH solution, 5000-10000 parts of AgNO3 solution, 3000-8000 parts of HAuCl4·3H2O solution, 50-200 parts of sodium citrate solution, 3000-8000 parts of water for Au@AgNFs precipitation, 10-50 parts of 4-MBA ethanol solution, 3000-8000 parts of water for Au@AgNFs@4-MBA material, 300-800 parts of Au@AgNFs@4-MBA solution, and 30-80 parts of aptamer solution.

[0023] In a specific embodiment, each raw material is selected from the following volume parts:

[0024] 500 parts of NH2OH solution, 500 parts of NaOH solution, 9000 parts of AgNO3 solution, 5000 parts of HAuCl4·3H2O solution, 100 parts of sodium citrate solution, 5000 parts of water for Au@AgNFs precipitation, 30 parts of 4-MBA ethanol solution, 5000 parts of water for Au@AgNFs@4-MBA material, 500 parts of Au@AgNFs@4-MBA solution, and 50 parts of aptamer solution.

[0025] In the above-mentioned preparation method of Au@Ag@4-MBA@apt, the conditions of the first stirring reaction are selected from: stirring reaction at 100-200°C for 5-60 minutes; preferably: stirring reaction at 160°C for 15 minutes.

[0026] In the above-mentioned preparation method of Au@Ag@4-MBA@apt, the conditions of the second stirring reaction are selected from: stirring the reaction at 20-40° C. for 1-5 hours; preferably: stirring the reaction at room temperature for 3 hours.

[0027] In the above-mentioned preparation method of Au@Ag@4-MBA@apt, the concentration of the NH2OH solution is 0.06M, the concentration of the NaOH solution is 0.05M; the concentration of the AgNO3 solution is 0.1mol / L; the concentration of the HAuCl4·3H2O solution is 0.8mM; the concentration of the sodium citrate solution is 1%, w / v; and the concentration of the 4-MBA ethanol solution is 102μM.

[0028] In the above-mentioned preparation method of Au@Ag@4-MBA@apt, the incubation conditions are selected from: incubation at 30-40°C for 10-20 hours; preferably: incubation at 37°C for 12 hours.

[0029] The present invention provides application of the above biosensor in detecting Staphylococcus aureus in food.

[0030] The present invention provides a method for detecting Staphylococcus aureus in food, the steps of which are as follows:

[0031] 0.1mL of the capture probe is mixed with 0.1mL of the sample solution to be tested, and incubated for 12h; 0.1mL of the mixed solution obtained by incubation is mixed with 0.1mL of the signal probe, and incubated for 12h; then the incubation system is centrifuged at 10000rpm for 10min, the supernatant is removed to leave the precipitate, and the precipitate is dispersed in 0.5mL of PBS, and the Raman signal is measured using a microscopic imaging Raman spectrometer. If Staphylococcus aureus is present in the sample to be tested, the aptamer of the capture probe will connect with Staphylococcus aureus, and connect with the signal probe through the aptamer on the signal probe, thereby generating a Raman signal; if there is no Staphylococcus aureus in the sample to be tested, the aptamer on the capture probe will not be able to recognize and capture Staphylococcus aureus, and thus cannot connect with the signal probe, and cannot generate a Raman signal.

[0032] In the above detection method, the Raman spectrum parameters are set as follows: the excitation wavelength is 532nm, the power is 6.7MW, the integration time is 0.02s, 10 accumulations, and the objective lens is 10×.

[0033] In the present invention, the food may be solid or liquid food such as milk, beverages, etc. If it is a solid food, it needs to be prepared into a solution to be tested before testing.

[0034] The beneficial effects of the present invention are:

[0035] The present invention provides a sensor, which is composed of a capture probe and a signal probe. The capture probe can capture Staphylococcus aureus and connect with the signal probe to form a sandwich structure, which can significantly enhance the Raman signal and improve the detection sensitivity of the sensor. In addition, the sensor of the present invention also has the advantages of low detection limit, strong specificity, low cost, high efficiency and short time consumption, so it has important application prospects and value in the field of pathogen detection technology.

[0036] In the present invention, the MXene two-dimensional material has a large specific surface area, which is conducive to better combination or interaction with other materials to form a more stable composite material. When combined with noble metal nanoparticles, the large specific surface area of ​​MXene can provide more attachment sites for the noble metal nanoparticles, making the combination between the two more firm, improving the stability of the composite material and the repeatability of SERS performance, which is conducive to obtaining stable and reliable detection results, thereby significantly improving the Raman scattering signal.

[0037] The present invention uses Au@Ag nanoflower nanoparticles as the substrate of the signal probe and combines with the signal molecule 4-MBA. The presence of 4-MBA can be used as an identification mark. In the application of biosensors, the Raman signal characteristics of 4-MBA can be used to identify specific biomolecules or chemical substances. Since the Au@Ag nanoflower nanoparticles themselves have a localized surface plasmon resonance effect, this effect can enhance the Raman scattering signal. When 4-MBA is adsorbed on the surface of the nanoparticles, its Raman signal will be significantly enhanced due to the ionic resonance effect of the nanoparticles, making the detection more sensitive.

[0038] The present invention is based on the connection of Au@Ag@4-MBA and the aptamer of Staphylococcus aureus as a signal probe. Compared with the traditional bare gold probe, the stability and specificity are greatly improved, and it can be stably stored for a long time at 4°C.

[0039] The present invention obtains corresponding results by detecting the Raman spectral signal of the solution system. Compared with the traditional method of detecting the solid phase substrate, the monitoring data of the Raman spectral signal in the solution is more stable, reliable and can be repeated in large quantities.

[0040] The detection method of the present invention has good stability and specificity, and the detection sensitivity is significantly improved, and a wide detection range (10-10 7 CFU / mL) and a low limit of detection (1.009 CFU / mL).

[0041] The present invention is simple to operate. The signal probe and capture probe of SERS can be prepared in advance. Only the capture probe needs to be placed in the sample to be detected. The aptamer can specifically identify the pathogenic bacteria to be detected in the sample. The large specific surface area of ​​MXene allows more Au nanoparticles and aptamers to be adsorbed on MXene, thereby combining with more pathogenic bacteria to be detected, and then combining with the signal probe, so that this sensor has higher sensitivity and lower detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The SEM image and element analysis of Mxene two-dimensional material; a is Ti3AlC2, b is the Mxene two-dimensional material formed after etching, c is Staphylococcus aureus obtained by biological sample preparation, d is a sandwich structure composed of all materials, with Staphylococcus aureus in the middle, and e and f are element comparison diagrams before and after Mxene etching;

[0043] Figure 2 This is the SEM image of MXene@Au material;

[0044] Figure 3The detection results of different Staphylococcus aureus concentrations; Figure a is the SERS spectrum; Figure b is the 1580cm -1 Linear fitting of signal intensity at and concentration of Staphylococcus aureus;

[0045] Figure 4 The SEM image of Staphylococcus aureus (a) and the SEM image of the sandwich structure formed after Staphylococcus aureus is captured by the sensor (b);

[0046] Figure 5 The test results of the repeatability of the biosensor; a is the SERS spectrum, and b is the RSD graph at 1580 nm;

[0047] Figure 6 The specific detection results of the biosensor; Figure a is 1580cm -1 The SERS signal values ​​at a are plotted as a bar graph, and b is the SERS spectrum. DETAILED DESCRIPTION

[0048] The Staphylococcus aureus aptamer sequence fragment used in the present invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd.; its sequence is as follows:

[0049] 5′-SH-TCGCGCGAGTCGTCTGGGGTCCCCTTTTCCTCTCCCCCCGCATCGTCCTCCC-3′ (SEQ ID NO: 1).

[0050] In the present invention, the aptamer solution refers to a PBS solution of the aptamer; the pH of the PBS buffer solution is 7.

[0051] Other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only intended to illustrate the present invention, and are not intended to limit the scope of the present invention in any way.

[0052] Example 1

[0053] In this embodiment, a 400-mesh Mxene material Ti3AlC2 is selected to prepare a layered Ti3C2Tx solution (Mxene two-dimensional material) by selectively etching the Al element component of Ti3AlC2 in the MAX phase (the general chemical formula of the MAX phase is Mn+1AXn, where M is a transition metal element, such as Ti, V, Mn, Nb, Mo, etc.; A is usually a 13th or 14th group element, such as Al, Ga, Si, Ge, etc.; X is C, N or a combination of C and N, n=1 to 4). This material has a large specific surface area and excellent performance.

[0054] The preparation steps of Mxene two-dimensional materials are as follows:

[0055] 40 mL of dilute hydrochloric acid solution (9 mol / L) was placed in a polytetrafluoroethylene beaker, 2 g of lithium fluoride powder was added, and the mixture was stirred at 37°C for 30 min; 2 g of Ti3AlC2 powder was then added and magnetically stirred for 24 h to etch away the Al element in Ti3AlC2; the solution was then placed in a centrifuge tube, centrifuged at 9500 rpm for 15 min, the supernatant was removed, and the multilayer Ti3C2Tx precipitate was collected, and it was washed alternately with ultrapure water and anhydrous ethanol and ultrasonically treated at the same time until the pH was close to 6; the precipitate was then dispersed in 40 mL of anhydrous ethanol and ultrasonically treated for 1 h; the solution was centrifuged at 9000 rpm for 15 min, the supernatant was removed, 40 mL of ultrapure water was added, and the ultrasonic treatment was continued for 20 min, and the solution was then centrifuged at 3500 rpm for 10 min, the supernatant was retained, and the layered Ti3C2Tx liquid, i.e., the Mxene two-dimensional material, was obtained.

[0056] Electron microscopy images and elemental analysis of Mxene two-dimensional materials Figure 1 As shown:

[0057] In this embodiment, Ti3AlC2, a novel two-dimensional material with a large specific surface area, is etched to remove the Al element in the MAX phase to generate Ti3C2Tx.

[0058] SEM image of Ti3AlC2 Figure 1 (a) shows the SEM image after etching. Figure 1 (b) SEM can directly observe the surface morphology and microstructure of MXene materials. MXene materials with large specific surface area usually appear in thin sheets in SEM images. This structural feature can increase the surface area of ​​the material. Its nanosheets are stacked to form abundant pores and channels, which greatly increase the specific surface area of ​​the material and enable it to fully contact with external substances. By comparing the element content, it can be found that the content of Al element in the etched compound is significantly reduced, such as Figure 1 (e) Figure 1As shown in (f). Removing the Al element helps to increase the specific surface area of ​​the material. A larger specific surface area means more active sites are exposed, which can enhance the interaction between the material and other ions.

[0059] Example 2

[0060] The preparation steps of capture probe are as follows:

[0061] 0.5 mL of MXene two-dimensional material was incubated with 0.5 mL of Au nanoparticles at 37 °C for 12 h to obtain MXene@Au material; then 0.5 mL of MXene@Au material was mixed with 50 μL of aptamer solution (800 nM) and incubated at 37 °C for 12 h to obtain MXene@Au@apt, i.e., the capture probe.

[0062] The Au nanoparticles are prepared by the following method:

[0063] Soak a three-necked round-bottom flask in aqua regia, clean it with ultrapure water, and air dry it. Take 3.5 mL of sodium citrate (1%) and add it to 100 mL of boiling HAuCl4 solution (0.01%) and stir for 30 minutes. During this period, the color of the solution changes from gray to blue to pink to wine red. After 30 minutes, continue stirring for 15 minutes (without heating) to obtain Au nanoparticles; store the obtained liquid in a dark environment at 4°C.

[0064] The SEM images of the above MXene@Au materials are shown in Figure 2. Figure 2 shown.

[0065] Example 3

[0066] The steps for preparing the signal probe are as follows:

[0067] 0.5 mL NH2OH (0.06 M) and 0.5 mL NaOH (0.05 M) were vigorously stirred at 37 ° C for 5 min; 9 mL AgNO3 solution (0.1 mol / L) was added in the dark and reacted for 5 min; then 5 mL HAuCl4·3H2O solution (0.8 mM) and 100 μL sodium citrate solution (1%, w / v) were added at the same time, stirred and reacted at 160 ° C for 15 min to obtain Au@AgNFs solution; the solution was placed in a centrifuge tube, centrifuged at 6000 rpm for 15 min, and the precipitate was dispersed in 5 mL ultrapure water to prepare Au@AgNFs suspension; then 30 μL 4-MBA ethanol solution (102 μM) was added, stirred and reacted at room temperature for 3 h, and centrifuged at 13000 rpm for 12 min to remove the unconnected 4-MBA, and the resulting precipitate was

[0068] Au@AgNFs@4-MBA materials;

[0069] The Au@AgNFs@4-MBA material was dispersed in 5 mL of ultrapure water to prepare an Au@AgNFs@4-MBA mixed solution. 0.5 mL of the Au@AgNFs@4-MBA solution was mixed with 50 μL of the aptamer solution and incubated at 37 °C for 12 h to obtain the signal probe.

[0070] The biosensor of the present invention is composed of the above-mentioned capture probe and signal probe.

[0071] Example 4

[0072] Detection of Staphylococcus aureus:

[0073] Staphylococcus aureus was inoculated into milk and fermented at 37°C to obtain Staphylococcus aureus liquid. The biosensor (capture probe + signal probe) was used to measure the concentration of 10 1 cfu / mL, 1×10 2 cfu / mL, 1×10 3 cfu / mL, 1×10 4 cfu / mL, 1×10 5 cfu / mL, 1×10 6 cfu / mL, 1×10 7 cfu / mL of Staphylococcus aureus liquid was tested.

[0074] The detection method is as follows:

[0075] Mix 0.1 mL of the capture probe with 0.1 mL of Staphylococcus aureus solution and incubate for 12 hours to ensure that the capture probe is connected to the Staphylococcus aureus; take 0.1 mL of the mixed solution obtained by incubation and mix it with 0.1 mL of the signal probe, incubate for 12 hours to allow them to connect with each other; then centrifuge the incubation system at 10,000 rpm for 10 minutes, remove the supernatant and leave the precipitate, and disperse the precipitate in 0.5 mL of PBS, and use a microscopic imaging Raman spectrometer to measure the Raman signal.

[0076] The Raman spectroscopy parameters were set as follows: the excitation wavelength was 532 nm, the power was 6.7 MW, the integration time was 0.02 s, 10 accumulations, and the objective lens was 10×.

[0077] Test results such as Figure 3 and Figure 4 As shown:

[0078] like Figure 3 As shown in (a), it can be seen that as the concentration of Staphylococcus aureus decreases, the Raman signal intensity of 4-MBA also gradually decreases, but when the concentration is 10 1cfu / mL still has a certain signal intensity. According to the different concentrations of Staphylococcus aureus SERS spectrum 1580cm -1 The signal intensity at the position was linearly fitted with the concentration of Staphylococcus aureus, as shown in Figure 3 (b) shows that the working curve equation is y = 4216x-3163, R 2 =0.99855, indicating that from 1×10 1 cfu / mL to 1×10 7 cfu / mL showed a good linear relationship. The detection limit was defined by the ratio of three times the standard deviation of the SERS signal intensity value of the blank sample to the slope of the fitting working curve, and the detection limit LOD of Staphylococcus aureus was calculated to be 1.009 cfu / mL. This shows that the sensor of the present invention has excellent sensitivity.

[0079] Figure 4 The SEM image of Staphylococcus aureus (a) and the SEM image of the sandwich structure formed after Staphylococcus aureus is captured by the sensor (b). Figure 4 (b) It can be seen that the bottom layer is MXene with a large specific surface area, on which Au nanoparticles and aptamers are combined to form a MXene@Au@apt structure. The upper layer is a combination of Au@Ag nanoflowers, 4-MBA and aptamers, forming a Au@Ag@4-MBA@apt structure, with Staphylococcus aureus sandwiched in the middle to form a sandwich structure. This structure can significantly enhance the Raman intensity.

[0080] Example 5

[0081] Recovery rate of biosensor:

[0082] The concentration was 1×10 4 cfu / mL, 1×10 5 cfu / mL, 1×10 6 cfu / mL of Staphylococcus aureus were mixed with milk samples of the same volume but different brands for recovery test. The experimental results are shown in Table 1.

[0083] Table 1 Recovery rate of Staphylococcus aureus detection

[0084]

[0085] As can be seen from Table 1, the detection concentration of Staphylococcus aureus in different milk samples 1, 2, and 3 is almost close to the added concentration, and the sensor's detection recovery rate for Staphylococcus aureus is 91.6-109.8%. Figure 5 As shown, the relative standard deviation (RSD) value of the detection signal was calculated to be less than 5.83%.

[0086] Example 6

[0087] Specificity of Biosensors:

[0088] The concentration was selected to be 1×10 7 cfu / m of Escherichia coli O:157, Salmonella, Shigella and Vibrio Parahaemolyticus were used as the control group, and samples without bacteria were selected as blank controls to verify the specificity of the sensor for the detection of Staphylococcus aureus.

[0089] The test results are as follows Figure 6 As shown:

[0090] Figure 6 (b) shows the SERS detection spectra of five bacteria and a blank control. It is obvious that except for the Staphylococcus aureus group, 4-MBA is located at 1580cm -1 The Raman characteristic peak at 1580cm -1 The SERS signal values ​​at the locations are plotted as a bar graph, such as Figure 6 As shown in (a), the SERS signal value of the Staphylococcus aureus group is significantly higher than that of the other groups. Therefore, this sensing detection strategy successfully detected only Staphylococcus aureus, proving that the prepared SERS sensor has good specificity for Staphylococcus aureus detection.

[0091] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A biosensor capable of enhancing Raman signals, characterized in that: The sensor consists of a capture probe and a signal probe; the capture probe is MXene@Au@apt, and the signal probe is Au@Ag@4-MBA@apt.

2. The biosensor according to claim 1, characterized in that The MXene@Au@apt is prepared by the following method: The MXene two-dimensional material is mixed with Au nanoparticles and incubated to obtain the MXene@Au material; then the MXene@Au material is mixed with the aptamer solution and incubated to obtain the MXene@Au@apt.

3. The biosensor according to claim 2, characterized in that The raw materials are selected from the following volume parts: 500-1000 parts of MXene two-dimensional material, 500-1000 parts of Au nanoparticles, 500-1000 parts of MXene@Au material, and 50-100 parts of aptamer solution.

4. The biosensor according to claim 2, characterized in that The incubation conditions are selected from: incubation at 35-40° C. for 10-16 hours; and / or the concentration of the aptamer solution is selected from 50-1000 nM.

5. The biosensor according to claim 1, characterized in that The Au@Ag@4-MBA@apt was prepared by the following method: The NH2OH solution and the NaOH solution were mixed, and in the dark, the AgNO3 solution was added to react; then the HAuCl4·3H2O solution and the sodium citrate solution were added at the same time, and the reaction was stirred to obtain the Au@AgNFs solution; The Au@AgNFs solution was centrifuged, and the Au@AgNFs precipitate was dispersed in water to prepare an Au@AgNFs suspension; Then, 4-MBA ethanol solution was added, the reaction was stirred, and centrifuged to obtain the precipitate of Au@AgNFs@4-MBA material; the obtained Au@AgNFs@4-MBA material was dispersed in water to prepare Au@AgNFs@4-MBA solution; the Au@AgNFs@4-MBA solution was mixed with the aptamer solution, incubated, and Au@Ag@4-MBA@apt was obtained.

6. The biosensor according to claim 5, characterized in that The raw materials are selected from the following volume parts: 300-800 parts of NH2OH solution, 300-800 parts of NaOH solution, 5000-10000 parts of AgNO3 solution, 3000-8000 parts of HAuCl4·3H2O solution, 50-200 parts of sodium citrate solution, 3000-8000 parts of water for Au@AgNFs precipitation, 10-50 parts of 4-MBA ethanol solution, 3000-8000 parts of water for Au@AgNFs@4-MBA material, 300-800 parts of Au@AgNFs@4-MBA solution, and 30-80 parts of aptamer solution.

7. The biosensor according to claim 5, characterized in that The conditions for the first stirring reaction are selected from: stirring the reaction at 100-200°C for 5-60 minutes; and / or, the conditions for the second stirring reaction are selected from: stirring the reaction at 20-40°C for 1-5 hours; and / or, the conditions for incubation are selected from: incubating at 30-40°C for 10-20 hours.

8. Use of the biosensor according to any one of claims 1 to 7 in detecting Staphylococcus aureus in food.

9. A method for detecting Staphylococcus aureus in food, characterized in that: Here are the steps: 0.1mL of the capture probe is mixed with 0.1mL of the sample solution to be tested, and incubated for 12h; 0.1mL of the mixed solution obtained by incubation is mixed with 0.1mL of the signal probe, and incubated for 12h; then the incubation system is centrifuged at 10000rpm for 10min, the supernatant is removed to leave the precipitate, and the precipitate is dispersed in 0.5mL of PBS, and the Raman signal is measured using a microscopic imaging Raman spectrometer. If Staphylococcus aureus is present in the sample to be tested, the aptamer of the capture probe will connect with Staphylococcus aureus, and connect with the signal probe through the aptamer on the signal probe, thereby generating a Raman signal; if there is no Staphylococcus aureus in the sample to be tested, the aptamer on the capture probe will not be able to recognize and capture Staphylococcus aureus, and thus cannot connect with the signal probe, and cannot generate a Raman signal.

10. The detection method according to claim 9, characterized in that: The Raman spectrum parameters were set as follows: the excitation wavelength was 532 nm, the power was 6.7 MW, the integration time was 0.02 s, 10 accumulations, and the objective lens was 10×.

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