A method for detecting streptomycin based on a surface-enhanced Raman spectroscopy sensor embedded in a specific nuclease DNA hydrogel

Abstract

The present invention discloses a method for detecting streptomycin based on a surface enhanced Raman spectroscopy sensor embedded with a specific nuclease DNA hydrogel. The preparation method of the sensor is as follows: a gold nanorod solution is mixed with CTAB and then dripped onto a substrate to obtain a gold nanorod array Raman substrate; a DNA A chain and a DNA B chain solution are mixed with a 4-MB solution, heated and then cooled to obtain a DNA hydrogel, a lead acetate solution is mixed with a streptomycin aptamer and immersed in the DNA hydrogel to obtain a specific nuclease DNA hydrogel; the hydrogel is dripped onto a gold nanorod array Raman substrate to obtain the sensor of the present invention. The present invention combines SERS with a highly selective target analyte capture technology to achieve efficient detection of streptomycin and improve the stability and sensitivity of streptomycin detection; and the present invention has a low detection limit, good specificity and signal reproducibility, and is suitable for ultrasensitive detection of streptomycin in food.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, and in particular to a method for detecting streptomycin based on a surface enhanced Raman spectroscopy sensor embedded with a specific nuclease DNA hydrogel. Background Art

[0002] Streptomycin is an aminoglycoside antibiotic that treats bacterial infections by inhibiting the synthesis of bacterial proteins. It is widely used in animal husbandry and agriculture. However, excessive use of streptomycin will remain in foods such as meat, milk, and honey. Long-term consumption of foods with excessive streptomycin residues can cause serious damage to the ear nerves and kidneys. Due to the widespread use of streptomycin and its greater harm to the human body, establishing a sensitive strategy to selectively detect streptomycin in food is of great significance to food safety.

[0003] At present, conventional physical and chemical detection methods mainly include instrumental analysis (liquid phase, liquid chromatography-mass spectrometry, gas phase, gas chromatography-mass spectrometry, etc.) and immunoassay (enzyme-linked immunosorbent assay, immunochromatography, immunofluorescence, and radioimmunoassay, etc.). Instrumental analysis has limitations in relative error and signal reproducibility; and the cross-reaction caused by the complex matrix in the actual sample will seriously affect the credibility of immunoassay. Therefore, it is necessary to develop a highly sensitive, highly stable, and highly specific method for detecting streptomycin residues in complex food matrices.

[0004] In recent years, new nanotechnologies have received extensive attention in the fields of analytical chemistry and food safety. Among them, surface enhanced Raman spectroscopy (SERS) has attracted much attention in the detection of streptomycin residues in food due to its advantages of rapid analysis, high sensitivity, and non-destructive detection. SERS achieves the purpose of enhancing Raman signals by generating "hot spots" through the surface plasmon resonance effect of noble metal nanoparticles. Although the common noble metal nanosol SERS substrate is simple to prepare, the overall disorder between the nanoparticles in the sol leads to uneven distribution of "hot spots". In addition, it is difficult to unify the focus position during the detection process, resulting in low sensitivity, poor reproducibility and stability of the SERS signal. Therefore, more and more research is focused on establishing ordered array nanostructures to improve the stability and reproducibility of SERS signals. Ordered gold nanoarrays are not only conducive to the excitation of localized surface plasmons, but also provide a large number of molecular binding sites. Therefore, constructing ordered array gold nanorod materials as SERS substrates can improve the stability and sensitivity of SERS detection. Common methods for synthesizing high-quality SERS array substrates include template growth method (electrochemical deposition, vapor deposition, etc.) and self-assembly method (interface self-assembly, evaporation-induced self-assembly, etc.). Although the former is mature in technology, the rod spacing is usually large and cannot form good resonant coupling. The self-assembly method is simpler and less expensive than the template growth method, so it is considered to be an effective means of assembling array structure SERS substrates.

[0005] In recent years, many SERS array substrates synthesized by self-assembly have been applied in the field of SERS trace analysis or biological sample detection. The uniform microscopic array structure can achieve high stability and high intensity Raman signal output. However, the main disadvantage of this technology is the poor selectivity for components with similar structural properties and Raman activity. Therefore, it is necessary to overcome the problem of poor selectivity for components with similar structural properties and Raman activity based on SERS and realize efficient detection of streptomycin. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a method for detecting streptomycin based on a surface enhanced Raman spectroscopy sensor embedded in a specific nuclease DNA hydrogel. The present invention combines SERS with a highly selective target analyte capture technology to overcome the problem of poor selectivity of SERS for components with similar structural properties and Raman activity, thereby achieving efficient detection of streptomycin.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a method for preparing a surface enhanced Raman spectroscopy sensor based on a DNA hydrogel embedded with a specific nuclease, comprising the following steps:

[0009] (1) Preparation of gold nanorod array Raman substrate: The gold nanorod solution synthesized by the seed-mediated method was mixed with CTAB and then drop-coated on the pretreated substrate, and then placed in a culture dish until completely dried to obtain a gold nanorod array Raman substrate;

[0010] (2) Preparation of specific nuclease DNA hydrogel signal amplifier: DNA A chain solution and DNA B chain solution are mixed evenly with 4-MB solution, heated and then slowly cooled to obtain DNA hydrogel, and lead acetate solution and streptomycin aptamer are mixed and immersed in the DNA hydrogel to obtain specific nuclease DNA hydrogel;

[0011] (3) The specific nuclease DNA hydrogel prepared in step (2) is drop-coated on the gold nanorod array Raman substrate prepared in step (1) to obtain a surface enhanced Raman spectroscopy sensor based on embedded specific nuclease DNA hydrogel.

[0012] Preferably, in step (1), the gold nanorod solution is prepared by the following method: HAuCl4 solution and CTAB solution are mixed, then NaBH4 solution is added, and the mixture is aged at room temperature to obtain a seed solution; another HAuCl4 solution is mixed with CTAB solution, then silver nitrate solution, hydrochloric acid and ascorbic acid solution are added, the mixture is mixed evenly, then the seed solution is added, and the mixture is placed in a water bath overnight, and dispersed into pure water by centrifugation to obtain a gold nanorod solution.

[0013] Preferably, in step (1), the pretreatment method is a hydrophilic treatment method or a hydrophobic treatment method, and the hydrophilic treatment method is specifically: placing the cut substrate face up in a clean hydroxylation beaker, washing it with deionized water for 3 times, and immediately adding 5 mL of hydrogen peroxide and 15 mL of concentrated sulfuric acid after the water is poured out, slowly shaking for 30 minutes, and then washing it with deionized water for 3 times. After the washing is completed, the substrate is stored in a large amount of water;

[0014] The hydrophobic treatment method is specifically as follows: the cut substrate is dried after ultrasonic treatment with acetone for 10 minutes, the substrate is immersed in a mixed solution of concentrated sulfuric acid and hydrogen peroxide with a volume ratio of 7:3, heated at 80° C. for 10 minutes, washed with deionized water, dried, and etched in 40wt% hydrofluoric acid for 5 minutes.

[0015] Preferably, in step (1), the pre-treated substrate is a hydrophilic silicon wafer, a hydrophobic silicon wafer, a gold-plated silicon wafer and a glass wafer. When the substrate is a gold-plated silicon wafer, no pre-treatment is required.

[0016] More preferably, the pretreated substrate is a gold-plated silicon wafer.

[0017] Preferably, the DNA A chain solution in step (1) is a solution obtained by diluting the DNA A chain substrate monomer using HEPES buffer, and the DNA B chain solution is a solution obtained by diluting the DNA B chain substrate monomer using HEPES buffer.

[0018] Preferably, the sequence of the DNA A-chain substrate monomer is 5'-TCGAATCTGATATTTTTCATCTCTTCTCCGAGCCGGTCGAAATAGTGATTTTTCTGGTTACCGAC-3' (SEQ ID NO.1); the sequence of the DNA B-chain substrate monomer is 5'-TATCAGATTCGATTTTTTCACTATrAGGAAGAGATGTTTTTGTCGGTAACCAG-3' (SEQ ID NO.2), wherein TCACTATrAGGAAGAGATG is the sequence of the nuclease. The rA in the DNA B-chain substrate monomer sequence represents an RNA base.

[0019] Preferably, in step (2), the sequence of the streptomycin aptamer is 5'-AACACAAACCCCAGACCTAGGCGGACGTGCTGGGCTGTCTTGTTTCGTCTTGTGGTCTGGGG-3' (SEQ ID NO. 3).

[0020] The second aspect of the present invention provides a surface enhanced Raman spectroscopy sensor based on a DNA hydrogel embedded with specific nucleases prepared by the above preparation method.

[0021] The third aspect of the present invention provides the use of a surface enhanced Raman spectroscopy sensor based on a hydrogel embedded with specific nuclease DNA in the detection of streptomycin.

[0022] In a fourth aspect, the present invention provides a method for detecting streptomycin based on a surface enhanced Raman spectroscopy sensor embedded in a specific nuclease DNA hydrogel, comprising the following steps:

[0023] (1) adding a series of streptomycin standard solutions with a concentration gradient to a specific nuclease DNA hydrogel based on a surface enhanced Raman spectroscopy sensor embedded with a specific nuclease DNA hydrogel to obtain a mixture;

[0024] (2) The mixture obtained in step (1) was dropped onto a gold nanorod array Raman substrate based on a surface-enhanced Raman spectroscopy sensor embedded with a specific nuclease DNA hydrogel, and the Raman signal at 1075 cm was recorded. -1 The Raman intensity at

[0025] (3) 1075cm -1The Raman intensity at is taken as the ordinate, and the negative logarithm value of the concentration of the streptomycin standard solution is taken as the abscissa, and a working curve is drawn;

[0026] (4) Substituting the test solution for the streptomycin standard solution with a series of concentration gradients in step (1), repeating step (2), and using the working curve drawn in step (3) to detect the streptomycin content in the test solution.

[0027] Preferably, in step (1), the concentrations of the streptomycin standard solution are 0.01 nmol / L, 0.1 nmol / L, 1 nmol / L, 10 nmol / L, 100 nmol / L, and 150 nmol / L, respectively.

[0028] Preferably, in step (4), the test solution is prepared by the following method:

[0029] Place 4.00 g of sample in a 50 mL centrifuge tube, add 5% phosphoric acid solution, shake the mixture for 5-10 min, add trichloroacetic acid, vortex mix and centrifuge, repeat twice and combine the supernatants, dry with nitrogen, dissolve the residue in pure water, filter with a 0.22-μm filter membrane, and obtain the test solution.

[0030] Beneficial effects of the present invention:

[0031] (1) The present invention uses a self-assembly method to synthesize a gold nanorod array as a Raman substrate. The regular array structure of the substrate can output a stable and strong Raman signal. Combined with the specific nuclease DNA hydrogel signal amplification sensor strategy, the release of the Raman reporter factor 4-MB is precisely controlled, thereby improving the stability and sensitivity of streptomycin detection. -1 The Raman intensity at the position is linearly correlated with the concentration of the target analyte, and a new sensitive detection method is established. The linear range of streptomycin detection using the surface enhanced Raman spectroscopy sensor method based on embedded specific nuclease DNA hydrogel of the present invention is 0.01-150nmol / L, and the minimum detection limit is 4.85×10 -3 nmol / L, which can meet the needs of trace detection of streptomycin in actual samples.

[0032] (2) The surface-enhanced Raman spectroscopy sensor based on the embedded specific nuclease DNA hydrogel of the present invention has good selectivity. The stability and signal reproducibility of the gold nanorod array Raman substrate synthesized by the self-assembly method are good, and it can be widely used in the determination of streptomycin residues in various food samples.

[0033] Comparison between the present invention and other existing methods for detecting streptomycin:

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : (A) SEM image of the gold nanorod array Raman substrate; (B) FDTD simulated electric field (λ=785nm) distribution image in the XY plane; (C) FDTD simulated electric field distribution image in the XZ plane, where the gap between the gold nanorods is fixed at 3nm and the polarization direction of the incident light is parallel to the X-axis.

[0036] Depend on Figure 1 It can be seen that the gold nanorods are arranged vertically on the surface of the silicon wafer, with a maximum electromagnetic field strength of 12.2V / m and a strong signal amplification capability.

[0037] Figure 2 : (A) SERS spectra of streptomycin standard solutions with different concentrations; (B) Streptomycin working curve.

[0038] Depend on Figure 2 It can be seen that the linear range of streptomycin detection by this method is 0.01-150nmol / L.

[0039] Figure 3 : Selectivity experimental diagram of this sensor.

[0040] Depend on Figure 3 It can be seen that this method has good specificity for detecting streptomycin.

[0041] Figure 4 : Reproducibility experiment diagram of this sensor.

[0042] Depend on Figure 4 It can be seen that this method has good signal reproducibility.

[0043] Figure 5 : Schematic diagram of the method for detecting streptomycin using this sensor. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0045] Terminology Note:

[0046] The "room temperature" mentioned in the present invention refers to a temperature of 15-30°C.

[0047] As introduced in the background technology section, the current detection methods for streptomycin residues are mainly instrumental analysis and immunoassay, which have poor selectivity, reproducibility, or low sensitivity. Therefore, it is of great significance to develop a stable, sensitive, and selective streptomycin detection method.

[0048] SERS has attracted much attention in the detection of streptomycin residues in food due to its advantages such as high sensitivity, fast response and non-destructive detection. SERS uses hot spots on the surface of noble metal nanoparticles to amplify the Raman signal of the analyte molecules. The ordered structure of the gold nanorod array SERS substrate is not only conducive to the excitation of localized surface plasmons, but also provides a large number of molecular recognition sites for the analytes, which has important application value in improving the sensitivity and stability of SERS signals. In recent years, self-assembled SERS array substrates have been applied to a series of analyte trace analysis, providing strong Raman signal intensity and good signal stability. However, the main disadvantage of this technology is the lack of selectivity for the target analyte. In order to overcome the above problems, it is necessary to develop a new SERS analysis method with specificity.

[0049] Nucleases can be used as molecular recognition elements because of their specific catalytic function. DNA hydrogels are excellent carriers of nucleases because of their good biocompatibility and large three-dimensional structure. If signal substances are added before the formation of DNA hydrogels, the signal substances will be trapped in the three-dimensional hydrogel network during the slow annealing process. DNA hydrogels embedded with nucleases can change the gel state and release signal substances according to the response of the target to achieve specific signal output. Therefore, this type of hydrogel is an excellent choice for constructing a new streptavidin SERS sensor platform.

[0050] In summary, the present invention uses a self-assembly method to synthesize a gold nanorod array Raman substrate, and combines the DNA hydrogel signal amplification strategy embedded with nuclease to construct a SERS sensor, which improves the stability and sensitivity of streptomycin detection. The DNA hydrogel is composed of the A and B chains of DNA that follow the principle of base complementary pairing, and is coated with the Raman reporter factor 4-MB during the annealing process, in which the A chain encodes the Pb 2+ Sensitive nuclease sequence. Pb 2+ It can induce the guanine-rich streptomycin nucleic acid aptamer to form a G-quadruplex DNA secondary structure; during the sensor detection process, streptomycin binds to the streptomycin aptamer and is converted into a Pb 2+ The induced G-quadruplex structure will change, releasing free Pb 2+ , and activate the nuclease sequence on the B chain of DNA, causing the DNA hydrogel to cleave on the surface of the gold nanorod array Raman substrate, releasing the Raman reporter factor 4-MB, thereby generating a strong SERS signal. At this time, the sensor is in the ON state. Otherwise, it is in the OFF state, showing a weak Raman signal ( Figure 5 The developed method for detecting streptomycin based on surface-enhanced Raman spectroscopy sensor embedded with specific nuclease DNA hydrogel has good sensitivity, reproducibility and selectivity, and has certain practical value.

[0051] In one embodiment of the present invention, a method for preparing a gold nanorod array Raman substrate is provided, comprising the following steps:

[0052] After mixing 0.10mL of 5mmol / L HAuCl4 solution with 1.9mL of 0.2mol / L CTAB, add 0.12mL of 10mmol / L freshly prepared NaBH4 solution to obtain the seed solution. After mixing 44mL of 0.5mmol / L HAuCl4 solution with 40mL of 0.2mol / L CTAB, add 0.8mL of 10mmol / L silver nitrate solution, 1.6mL of 1mol / L hydrochloric acid and 1.1mL of 0.1mol / L freshly prepared ascorbic acid solution. Add 10μL of seed solution to the mixture. Store in a water bath at 28℃ overnight and centrifuge to obtain the gold nanorod solution.

[0053] Mix 1.0mL of gold nanorod solution with 0.1mL of 2.5mmol / L CTAB, take 60μL of the mixed solution and drop it on the pretreated silicon wafer, and transfer it to a polystyrene culture dish. Place the culture dish in a programmable temperature and humidity chamber with an initial humidity of 50% and a temperature of 25℃. Then change the ambient humidity to 40-90% and the temperature to 25-65℃ until the droplets are completely dry to obtain the gold nanorod array Raman substrate.

[0054] Gold nanorods have different abilities to form arrays on different substrate materials. Experiments have confirmed that among hydrophilic silicon wafers, hydrophobic silicon wafers, gold-coated silicon wafers and glass wafers, gold-coated silicon wafers are the most ideal SERS materials. The differences between different materials are due to the different hysteresis phenomena of the contact angle during the three-dimensional assembly of the gold nanorod array structure. Compared with gold-coated silicon wafers, the other three materials have higher surface energy and are prone to adsorb impurities, resulting in poor substrate wettability, making it difficult to form a vertical gold nanorod array structure.

[0055] The gold nanorod array Raman substrate prepared by the present invention has a regular structure, strong SERS signal amplification capability and stability, and is the basis for applying surface enhanced Raman spectroscopy to trace streptomycin detection.

[0056] In another embodiment of the present invention, a specific method for preparing a specific nuclease DNA hydrogel signal amplification sensor is provided, comprising the following steps:

[0057] The DNA A chain and B chain substrate monomers were diluted to 1.5 mmol / L with HEPES buffer to obtain DNA A chain solution and DNA B chain solution, respectively. 5 μL of DNA A chain solution, 5 μL of DNA B chain solution and 5 μL of 0.05-0.5 mmol / L 4-MB were mixed, and then the temperature was increased to 70°C after ultrasonic treatment at 50°C for 1 min, and kept at this temperature for 10 min, and then slowly cooled to 10°C at a cooling rate of -0.5C / min to obtain DNA hydrogel. The DNA hydrogel was stored at room temperature for use.

[0058] 100 µL of 5-50 µmol / L lead acetate solution was mixed with 100 µL of 1 µmol / L aptamer, and after standing at room temperature for 90 min, 50 µL of streptomycin standard solution was added, and the mixture was reacted in a 20-60°C water bath for 5-55 min, and then immersed in DNA hydrogel and cultured at 25°C for 30 min to obtain a mixture.

[0059] The mixture was drop-coated on a gold nanorod array Raman substrate and the Raman spectrometer was used to record the 1075 cm -1 The negative logarithm of the concentration of the streptomycin standard solution is used as the horizontal axis, 1075cm -1 The Raman intensity at the position is used as the ordinate to draw a working curve. The drawn working curve can be used to detect the content of streptomycin in the solution to be tested.

[0060] Nuclease is a synthetic protein with globular protein characteristics, which is widely used in the field of molecular recognition and biosensing. The intramolecular cleavage that makes nuclease catalytically active is usually highly dependent on the interaction between metal ions and DNA. Due to this activation characteristic, nucleases are often used as molecular recognition elements. DNA hydrogels are good carriers for nucleases to exert their effects due to their good biocompatibility and large specific surface area. If signal substances are added before gel formation, they will be trapped in the three-dimensional network after the DNA hydrogel is formed. DNA hydrogels embedded with nucleases can change the gel state according to the response to target molecules, specifically release the stored signal substances, and achieve selective signal output. This type of hydrogel not only has the characteristics of polymer hydrogels, but also has significant advantages such as stability, flexibility, and precise programmability. It can be used as a new type of SERS detection platform.

[0061] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0062] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0063] 1. Preparation of gold nanorod array Raman substrate:

[0064] (1) Preparation of gold nanorod solution: After mixing 0.10 mL of 5 mmol / L HAuCl4 solution with 1.9 mL of 0.2 mol / L CTAB, add 0.12 mL of 10 mmol / L freshly prepared NaBH4 solution and age at room temperature for 30 min to obtain the seed solution. After mixing 44 mL of 0.5 mmol / L HAuCl4 solution with 40 mL of 0.2 mol / L CTAB, add 0.8 mL of 10 mmol / L silver nitrate solution, 1.6 mL of 1 mol / L hydrochloric acid and 1.1 mL of 0.1 mol / L freshly prepared ascorbic acid solution. After shaking the mixture for 30 s, add 10 μL of seed solution. Place in a water bath at 28 °C overnight and centrifuge at 3000 rpm for 7 min to obtain the gold nanorod solution.

[0065] (2) Self-assembly of gold nanorod solution on gold-coated silicon wafer: 1.0 mL of gold nanorod solution was mixed with 0.1 mL of 2.5 mmol / L CTAB. After ultrasonic treatment for 1 min, 60 μL of the mixed solution was drop-coated on the gold-coated silicon wafer and transferred to a polystyrene culture dish. The culture dish was placed in a programmable temperature and humidity chamber with an initial humidity of 50% and a temperature of 25°C. Then the ambient humidity was changed to 90% and the temperature was maintained at 25°C until the droplets were completely dry to obtain the gold nanorod array Raman substrate.

[0066] 2. Establishment of a method for detecting streptomycin based on a surface-enhanced Raman spectroscopy sensor embedded with specific nuclease DNA hydrogel:

[0067] (1) Preparation of DNA hydrogel encapsulating 4-MB: Dilute DNA A-chain and B-chain substrate monomers to 1.5 mmol / L respectively with HEPES buffer. Mix 5 μL of DNA A-chain solution, 5 μL of DNA B-chain solution and 5 μL of 0.1 mmol / L 4-MB. After ultrasonication at 50°C for 1 min, raise the temperature to 70°C and keep it at this temperature for 10 min. Then, slowly cool it to 10°C at a cooling rate of -0.5°C / min. The prepared DNA hydrogel is stored at room temperature for later use.

[0068] (2) Construction of sensor detection program: Mix 100µL of 20µmol / L lead acetate solution with 100µL of 1µmol / L aptamer, let stand at room temperature for 90 minutes, then add 50µL of 0.01nmol / L, 0.1nmol / L, 1nmol / L, 10nmol / L, 100nmol / L, and 150nmol / L streptomycin standard solutions respectively. Mix the solutions evenly and react in a 40°C water bath for 45 minutes. Immerse the mixed solution in the DNA hydrogel prepared above and incubate at 25°C for 30 minutes. Apply the mixture dropwise on the gold nanorod array Raman substrate and use a Raman spectrometer to record the 1075cm -1 The negative logarithm of the concentration of the streptomycin standard solution is used as the horizontal axis, 1075cm -1 The working curve is drawn with the Raman intensity at as the ordinate.

[0069] Embodiment 2:

[0070] The streptomycin in chicken and milk powder samples was detected using the analytical method for detecting streptomycin based on the surface enhanced Raman spectroscopy sensor embedded in the specific nuclease DNA hydrogel established in Example 1, as follows:

[0071] (1) Accurately weigh 4.00 g of minced chicken and 4.00 g of milk powder and place them in 50 mL centrifuge tubes, then perform the following operations: Add 15 mL of 5% phosphoric acid solution. After shaking the mixture for 10 min, add 1.5 g of trichloroacetic acid. After vortex mixing, centrifuge at 4000 rpm for 10 min. Repeat the extraction step twice, collect and combine the supernatant. After nitrogen drying, dissolve the residue in 2 mL of pure water and filter it with a 0.22-μm filter membrane to obtain the test solution.

[0072] (2) Mix 100µL of 20µmol / L lead acetate solution with 100µL of 1µmol / L aptamer, let stand at room temperature for 90 minutes, then add 50µL of the test solution. Mix the solution evenly and react in a 40℃ water bath for 45 minutes. Immerse the prepared DNA hydrogel in the above solution and incubate at 25℃ for 30 minutes. Apply the mixture dropwise on the gold nanorod array Raman substrate and use a Raman spectrometer to record 1075cm -1 The Raman signal at .

[0073] According to the standard curve drawn in Example 1, the concentrations of streptomycin in the chicken and milk powder samples were 708.08 nmol / L and 53.97 nmol / L, respectively.

[0074] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. SEQUENCE LISTING <110> Shandong Agricultural University <120> A surface-enhanced Raman spectroscopy sensor based on embedded specific nuclease DNA hydrogel for the detection of Streptavidin Vegetarian Method <130> 2022 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 65 <212> DNA <213> Artificial sequence <400> 1 tcgaatctga tatttttcat ctcttctccg agccggtcga aatagtgatt tttctggtta 60 ccgac 65 <210> 2 <211> 52 <212> DNA <213> Artificial sequence <400> 2 tatcagattc gattttttca ctataggaag agatgttttt gtcggtaacc ag 52 <210> 3 <211> 62 <212> DNA <213> Artificial sequence <400> 3 aacacaaacc ccagacctag gcggacgtgc tgggctgtct tgtttcgtct tgtggtctgg 60 gg 62

Claims

1. A method for preparing a surface enhanced Raman spectroscopy sensor based on a DNA hydrogel embedded with specific nuclease, characterized in that: The following steps are involved: (1) Mixing the gold nanorod solution synthesized by the seed-mediated method with CTAB and then dropping it onto the pretreated substrate, and then placing it in a culture dish until it is completely dry, thereby obtaining a gold nanorod array Raman substrate; (2) Mix the complementary paired DNA A chain solution and DNA B chain solution with the 4-MB solution, heat and then slowly cool to obtain a DNA hydrogel, mix the lead acetate solution with the streptomycin aptamer and immerse it in the DNA hydrogel to obtain a specific nuclease DNA hydrogel; The DNA A chain solution is a solution obtained by diluting the DNA A chain substrate monomer with HEPES buffer, and the DNA B chain solution is a solution obtained by diluting the DNA B chain substrate monomer with HEPES buffer; the sequence of the DNA A chain substrate monomer is 5'-TCGAATCTGATATTTTTCATCTCTTCTCCGAGCCGGTCGAAATAGTGATTTTTCTGGTTACCGAC-3'; the sequence of the DNA B chain substrate monomer is 5'-TATCAGATTCGATTTTTTCACTATrAGGAAGAGATGTTTTTGTCGGTAACCAG-3', wherein TCACTATrAGGAAGAGATG is the sequence of the nuclease, and rA in the DNA B chain substrate monomer sequence represents an RNA base; the sequence of the streptomycin aptamer is 5'-AACACAAACCCCAGACCTAGGCGGACGTGCTGGGCTGTCTTGTTTCGTCTTGTGGTCTGGGG-3'; (3) The specific nuclease DNA hydrogel prepared in step (2) is drop-coated on the gold nanorod array Raman substrate prepared in step (1) to obtain a surface enhanced Raman spectroscopy sensor based on embedded specific nuclease DNA hydrogel.

2. The preparation method according to claim 1, characterized in that: In step (1), the gold nanorod solution is prepared by the following method: HAuCl4 solution and CTAB solution are mixed, then NaBH4 solution is added, and the mixture is aged at room temperature to obtain a seed solution; another HAuCl4 solution is mixed with CTAB solution, then silver nitrate solution, hydrochloric acid and ascorbic acid solution are added, the mixture is mixed evenly, then the seed solution is added, and the mixture is placed in a water bath overnight, and dispersed into pure water by centrifugation to obtain a gold nanorod solution.

3. The preparation method according to claim 1, characterized in that: In step (1), the pretreated substrate is a hydrophilic silicon wafer, a hydrophobic silicon wafer, a gold-coated silicon wafer and a glass wafer.

4. A surface enhanced Raman spectroscopy sensor based on a DNA hydrogel embedded with a specific nuclease prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the surface enhanced Raman spectroscopy sensor based on the embedded specific nuclease DNA hydrogel according to claim 4 in the detection of streptomycin.

6. The method for detecting streptomycin using the surface enhanced Raman spectroscopy sensor based on embedded specific nuclease DNA hydrogel according to claim 4, characterized in that: The following steps are involved: (1) adding a series of streptomycin standard solutions with a concentration gradient to the specific nuclease DNA hydrogel of the surface enhanced Raman spectroscopy sensor based on the embedded specific nuclease DNA hydrogel according to claim 4 to obtain a mixture; (2) Dropping the mixture obtained in step (1) onto the gold nanorod array Raman substrate of the surface enhanced Raman spectroscopy sensor based on embedded specific nuclease DNA hydrogel as described in claim 4, recording the 1075 cm -1 The Raman intensity at (3) 1075cm -1 The Raman intensity at is taken as the ordinate, and the negative logarithm value of the concentration of the streptomycin standard solution is taken as the abscissa, and a working curve is drawn; (4) Substituting the test solution for the streptomycin standard solution with a series of concentration gradients in step (1), repeating step (2), and using the working curve drawn in step (3) to detect the streptomycin content in the test solution.

7. The method according to claim 6, characterized in that In step (1), the concentrations of the streptomycin standard solution are 0.01 nmol / L, 0.1 nmol / L, 1 nmol / L, 10 nmol / L, 100 nmol / L, and 150 nmol / L, respectively.

8. The method according to claim 6, characterized in that In step (4), the test solution is prepared by the following method: Place 4.00 g of sample in a 50 mL centrifuge tube, add 5% phosphoric acid solution, shake the mixture for 5-10 min, add trichloroacetic acid, vortex mix and centrifuge, repeat twice and combine the supernatants, dry with nitrogen, dissolve the residue in pure water, filter with a 0.22-μm filter membrane, and obtain the test solution.

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