SERS (Surface Enhanced Raman Scattering) sensor for detecting ribonuclease H activity

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) sensor for detecting ribonuclease H activity. The SERS sensor comprises a gold electrode, a single-chain RNA (Ribonucleic Acid), an initiation probe tDNA (Deoxyribose Nucleic Acid), a hairpin chain H1, a capture probe S1, Exo III and an SERS tag, the SERS tag is prepared by assembling a signal probe S2 and a signal molecule 4-MBA on the surface of a gold nano cube together. According to the SERS sensor, a target object RNase H is converted into a strong Raman signal through the assistance of Exo III and the good SERS performance of a gold nano cube; s0 with an N3 functional group and a signal probe S2 with a DBCO functional group on the SERS tag can be subjected to a click chemical reaction, and the SERS tag is assembled on the surface of a gold electrode, so that an SERS signal is detected on the surface of a substrate. The SERS sensor can efficiently and selectively detect the activity of RNase H, and the detection limit is 3.89 * 10 <-4 > U / mL (S / N = 3) and is lower than the reported value of most literatures.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a surface-enhanced Raman spectroscopy (SERS) sensor for detecting the activity of ribonuclease H (RNase H). Background Technology

[0002] RNase H is an endogenous ribonuclease whose abnormal activity or dysfunction can interfere with DNA transcription, leading to certain diseases. Therefore, there is an urgent need to develop a simple and sensitive method for detecting RNase H activity. Many methods for detecting RNase H activity have been reported, including fluorescence and colorimetric methods. However, there are no reports on the use of SERS technology for detecting RNase H activity.

[0003] Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical technique with advantages such as high sensitivity and rapid readout. SERS tags are fabricated by immobilizing Raman reporter molecules on the surface of plasmonic noble metal nanoparticles. When Raman reporter molecules are adsorbed onto the nanoparticles, the SERS intensity is significantly enhanced. This is because the sharp features of the nanocubes, with their corners and edges, provide greater SERS enhancement. Furthermore, gold nanocubes (AuNCs) exhibit good biocompatibility and long-term stability, making them particularly suitable for preparing SERS tags for biosensing applications.

[0004] In recent years, various signal amplification strategies to improve detection sensitivity have attracted much attention in the field of biosensing, such as hybridization chain reaction (HCR), catalytic hairpin self-assembly (CHA), and exonuclease-assisted signal amplification. Exonuclease III (ExoIII) is a sequence-independent enzyme that can progressively remove single nucleotides from the flattened or concave 3'-hydroxyl ends of double-stranded DNA. Due to its high efficiency, ease of operation, and wide applicability in nucleic acid detection, ExoIII shows significant application potential in bioanalysis. Summary of the Invention

[0005] The purpose of this invention is to provide a SERS sensor for detecting ribonuclease H activity. The SERS sensor is a novel "turn-on" mode SERS biosensor based on Exo III-assisted cyclic amplification combined with copper-free click chemistry and AuNCs enhancement (the response signal increases with increasing target concentration), capable of detecting RNase H activity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A SERS sensor for detecting ribonuclease H activity, the SERS sensor comprising a gold electrode, single-stranded RNA, priming probe tDNA, hairpin strand H1, capture probe S1, Exo III, and a SERS tag; the SERS tag is prepared by assembling a signal probe S2 and a signal molecule 4-MBA on the surface of a gold nanocube. The sequence of the single-stranded RNA is: 5'-SH-(CH2)6-GCGGACAUCCUACUUCGCUCUUACU-3'; The sequence of the initiating probe tDNA is: 5'-AGTAAGAGCGAAGTAGGATGTCCGC-(CH2)6-SH-3'; The sequence of the hairpin chain H1 is: 5'-N3-AGCGAAGTAGGACGACGGGGGCATCCTACTTCGCTCTTACT-3'; The sequence of the capture probe S1 is: 5'-SH-(CH2)6-TTTTTTGTCGTCCTAC-3'; The sequence of the signal probe S2 is: 5'-SH-(CH2)6-TTTTTTTTTTTTT-DBCO-3'.

[0007] Furthermore, the preparation method of the above-mentioned gold nanocubes includes the following steps: S1-1: Take 0.6 mL of 10 mM NaBH4 solution and add it to 10 mL of 0.1 M CTAB solution containing 10 mM HAuCl4. Gently shake for 2 minutes and then place it in a 28°C water bath for 3 hours to prepare the gold seed solution. S1-2: Mix 2 mL of 0.2 M CTAC solution, 1.5 mL of 0.1 M ascorbic acid solution and 50 μL of gold seed solution, add 2 mL of 0.5 mM HAuCl4 solution, react at 27 °C for 15 minutes, then centrifuge, discard the supernatant, and disperse the resulting precipitate in 1 mL of 0.02 M CTAC solution to obtain gold nanoparticle solution; S1-3: Mix 125 μL of gold nanoparticle solution, 25 mL of 0.1 M CTAC solution, and 1.825 mL of 0.01 M ascorbic acid solution at 30 °C and 200 rpm under magnetic stirring for 2 minutes. Then add 25 mL of 0.01 M HAuCl4 solution and react at 30 °C and 950 rpm under magnetic stirring for 15 minutes. During this process, the solution color can be observed to gradually change from light red to dark red. After the reaction is completed, centrifuge and discard the supernatant. Disperse the obtained precipitate in 1 mL of 0.01 M CTAC solution to obtain a gold nanocube solution, which is stored at 4 °C for later use.

[0008] Furthermore, the above-mentioned method for preparing the SERS tag includes the following steps: S2-1: Add 2 μL of 100 mM TCEP solution and 2 μL of 250 μM signal probe S2 solution to 26 μL Tris buffer, and react at room temperature in the dark for 1 hour to obtain reaction solution 1; S2-2: Add SDS to the gold nanocube solution to a final concentration of 0.01wt% to obtain an SDS-containing gold nanocube solution; take 400μL of the SDS-containing gold nanocube solution, add 4μL of 2mM 4-MBA solution, and react for 30 minutes at 37℃ and 300rpm with magnetic stirring. Then add 16μL of reaction solution 1 and continue to react for 2 minutes at the same temperature and stirring speed to obtain reaction solution 2. S2-3: Add 3.85 mL of n-butanol to 420 μL of reaction solution 2, shake rapidly for 10 seconds, then add 850 μL of 5×TBE buffer, shake rapidly for 10 seconds, centrifuge, discard the supernatant, wash the resulting precipitate three times with 0.01 wt% SDS solution to obtain the SERS tag; disperse the obtained SERS tag in 320 μL of 0.01 wt% SDS solution to obtain the SERS tag solution, and store it at 4℃ for later use.

[0009] The above-mentioned method for constructing a SERS sensor includes the following steps: S3-1: Add 1.1 μL of 100 μM initiator probe tDNA solution and 1 μL of 100 μM single-stranded RNA solution to 97 μL of 1×RNase H reaction buffer. Heat at 95°C for 5 minutes, then cool to 37°C. Mix with 1 μL of 100 mM TCEP solution at room temperature in the dark for 1 hour to obtain the RNA / tDNA complex solution. Immerse AuE in the RNA / tDNA complex solution, freeze at -18°C for 30 minutes, thaw at room temperature for 5 minutes, rinse the electrode surface with Tris HCl buffer, and dry with nitrogen to obtain the AuE / RNA / tDNA electrode. Immerse the AuE / RNA / tDNA electrode in 2 mM MCH solution, block at room temperature for 1 hour, rinse the electrode surface with Tris HCl buffer, and dry with nitrogen to obtain the AuE / RNA / tDNA / MCH electrode. Add different concentrations of RNase to the 1×RNase H reaction buffer. H. Immerse the AuE / RNA / tDNA / MCH electrode in the solution and perform enzymatic digestion at 37°C in a shaker for 1.5 hours, followed by inactivation at 75°C for 20 minutes to obtain a reaction solution containing tDNA. Take 30 μL of the reaction solution containing tDNA, and add 0.5 μL of 100 μM capture probe H1 solution and 4 μL of 10× NE buffer. TM1. Mix 5.5 μL of deionized water and 0.2 μL of 100 U / μL Exo III, incubate at 37 °C for 2 hours, and then inactivate at 80 °C for 20 minutes to obtain a reaction solution containing the single-stranded DNA fragment S0. S3-2: Immerse AuE in 1.5 μM capture probe S1 solution, freeze at -18℃ for 30 minutes, thaw at room temperature for 5 minutes, rinse the electrode surface with Tris HCl buffer and dry with nitrogen to obtain the AuE / S1 electrode; incubate the AuE / S1 electrode sequentially in 0.2 mL 2 mM MCH solution at room temperature for 1 hour, then incubate in 0.2 mL 1 wt% BSA at room temperature for 1.5 hours, rinse the electrode surface with Tris HCl buffer and dry with nitrogen to obtain the AuE / S1 / MCH / BSA electrode; add 7 μL of reaction solution containing single-stranded DNA fragment S0 to the surface of the AuE / S1 / MCH / BSA electrode, incubate at 37℃ for 2 hours, rinse with Tris HCl buffer and dry with nitrogen to obtain the AuE / S1 / MCH / BSA electrode. The electrode surface was rinsed with HCl buffer and dried with nitrogen to obtain the AuE / S1 / MCH / BSA / S0 electrode. The AuE / S1 / MCH / BSA / S0 electrode was immersed in SERStag solution and click-reacted at 37°C for 1.5 hours. The electrode surface was then rinsed with Tris HCl buffer and dried with nitrogen to obtain the AuE / S1 / MCH / BSA / S0 / S2 / 4-MBA / AuNCs electrode, which was then subjected to SERS detection at room temperature.

[0010] Furthermore, the gold electrodes mentioned above need to be pretreated before use. Specifically, the surface of the gold electrodes is first polished with alumina powder, and then cleaned with anhydrous ethanol and ultrapure water in sequence.

[0011] Furthermore, the sequence of the single-stranded DNA fragment S0 is: 5'-N3-AGCGAAGTAGGACGACGGGGG-3'.

[0012] The above-mentioned SERS sensor is used in the detection of ribonuclease H activity for non-disease diagnosis and treatment purposes.

[0013] The principle of the sensor in this invention: The detection principle of the SERS sensor of this invention is as follows: Figure 1As shown, a thiol-modified RNA / tDNA complex is modified onto AuE. In the presence of target RNase H, single-stranded RNA is recognized and cleaved by RNase H, releasing tDNA. The released tDNA hybridizes with hairpin strand H1 to form an H1 / DNA complex. H1 in the H1 / DNA complex is cleaved from the 3' end by Exo III to form S0, releasing tDNA. The released tDNA can further hybridize with another H1 to amplify the signal and generate a large amount of S0. A reaction solution containing S0 is dropped onto AuE / S1 / MCH / BSA, and S0 hybridizes to the capture probe S1 through base complementarity pairing to obtain AuE / S1 / MCH / BSA / S0. S0 with an N3 functional group can undergo a click chemical reaction with a signal probe S2 with a DBCO functional group on the SERS tag, assembling the SERS tag onto the AuE surface to prepare a SERS substrate, which can then be used to measure the SERS signal. In summary, this invention successfully developed a SERS sensor for detecting RNase H activity.

[0014] The present invention has the following significant advantages: Based on the combination of AuNC SERS enhancement, Exo III-assisted cyclic amplification, and copper-free click chemistry, this invention proposes a SERS biosensor for high-sensitivity detection of RNase H activity. This sensor fully utilizes the strong local electromagnetic field enhancement effect provided by the sharp corner structure of AuNC, significantly improving the SERS signal intensity. Simultaneously, AuNC exhibits good biocompatibility and long-term stability, making it suitable for constructing SERS tags for biosensing.

[0015] Regarding the preparation of SERS tags, this invention has optimized the process, reducing the preparation time of the entire SERS probe to approximately 1 hour, significantly improving the preparation efficiency. Furthermore, by employing a copper-free click chemistry strategy, utilizing the highly efficient and specific reaction between dibenzocyclooctyne (DBCO) and azide (N3) under mild conditions, stable and efficient immobilization of the SERS tag on the sensor surface was achieved.

[0016] Currently, there are no reports on SERS biosensors that combine Exo III-assisted cyclic amplification with copper-free click chemistry for the detection of RNase H activity. This invention provides a novel sensing platform for the sensitive detection of this enzyme. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the detection principle of the SERS sensor of the present invention.

[0018] Figure 2This is a graph showing the optimization of SERS sensor detection performance. In the graph, A represents the optimization of RNase H digestion reaction time, B represents the optimization of RNase H digestion reaction temperature, C represents the optimization of click reaction time, and D represents the optimization of signal probe S2 solution concentration.

[0019] Figure 3 The image shows the sensitivity detection results of the SERS sensor. In the image, A is the Raman spectrum of 4-MBA, and B is the Raman spectrum of 4-MBA at 1075 cm⁻¹. -1 The peak intensity and the logarithmic linear relationship between RNase H concentration (0.000625 U / mL~1.25 U / mL) are shown in the curve.

[0020] Figure 4 This is a graph showing the selective detection results of the SERS sensor. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below. It should be noted that the described embodiments are merely illustrative examples, intended to help understand the present invention, and not to exhaustively describe all possible implementations. Based on the concept of the present invention, any equivalent substitutions or obvious modifications obtained by those skilled in the art without creative effort fall within the scope of protection claimed by the present invention.

[0022] The gold electrode (AuE) involved in the embodiments of the present invention needs to be pretreated before use. Specifically, the electrode surface is first polished with alumina powder, and then cleaned with anhydrous ethanol and ultrapure water in sequence.

[0023] The various buffer solutions and their formulations involved in the embodiments of this invention are as follows: Tris-HCl buffer: 10 mM, pH 7.4; Tris buffer: 10mM Tris-HCl, 0.1M NaCl, pH 7.4; 1× RNaseH reaction buffer: 50mM Tris-HCl, 75mM KCl, 3mM MgCl2, 10mM DTT, pH 8.3; 10× NEBuffer TM 1: 100mM bis-tris-propane-HCl, 100mM MgCl2, 10mM DTT, pH 7.0; 5× TBE buffer: 445mM Tris, 445mM boric acid, 10mM EDTA, pH 8.0-8.6.

[0024] The nucleotide sequences involved in the embodiments of this invention are as follows: Initiation probe tDNA: 5'-AGTAAGAGCGAAGTAGGATGTCCGC-3'; Single-stranded RNA: 5'-SH-(CH2)6-GCGGACAUCCUACUUCGCUCUUACU-3'; Hairpin chain H1: 5'-N3-AGCGAAGTAGGACGACGGGGGCATCCTACTTCGCTCTTACT-3'; Capture probe S1: 5'-SH-(CH2)6-TTTTTTGTCGTCCTAC-3'; Signal probe S2: 5'-SH-(CH2)6-TTTTTTTTTTTTT-DBCO-3'; Single-stranded DNA fragment S0: 5'-N3-AGCGAAGTAGGACGACGGGGG-3'. Example 1

[0025] A method for preparing a surface-enhanced Raman spectroscopy (SERS) sensor for detecting ribonuclease H (RNase H) activity includes the following steps: S1: Preparation of gold nanocubes (AuNCs): S1-1: Take 0.6 mL of 10 mM NaBH4 solution and add it to 10 mL of 0.1 M CTAB solution containing 10 mM HAuCl4. Gently shake for 2 minutes and then place it in a 28°C water bath for 3 hours to prepare the gold seed solution. S1-2: Mix 2 mL of 0.2 M CTAC solution, 1.5 mL of 0.1 M ascorbic acid solution and 50 μL of gold seed solution, add 2 mL of 0.5 mM HAuCl4 solution, react at 27 °C for 15 minutes, then centrifuge at 14000 rpm for 30 minutes, discard the supernatant, and disperse the obtained precipitate in 1 mL of 0.02 M CTAC solution to obtain a gold nanoparticle solution with a particle size of 10 nm. S1-3: Mix 125 μL of gold nanoparticle solution, 25 mL of 0.1 M CTAC solution, and 1.825 mL of 0.01 M ascorbic acid solution at 30 °C and 200 rpm under magnetic stirring for 2 minutes. Then add 25 mL of 0.01 M HAuCl4 solution and react at 30 °C and 950 rpm under magnetic stirring for 15 minutes. During this process, the solution color can be observed to gradually change from light red to dark red. After the reaction is completed, centrifuge at 8000 rpm for 20 minutes, discard the supernatant, and disperse the obtained precipitate in 1 mL of 0.01 M CTAC solution to obtain AuNCs solution, which is stored at 4 °C for later use.

[0026] S2: Preparation of SERS tag: S2-1: Add 2 μL of 100 mM TCEP solution and 2 μL of 100 μM signal probe S2 solution to 26 μL Tris buffer, and react at room temperature in the dark for 1 hour to obtain reaction solution 1; S2-2: Add SDS to the AuNCs solution to a final concentration of 0.01 wt% to obtain an AuNCs solution containing SDS; take 400 μL of the AuNCs solution containing SDS, add 4 μL of 2 mM 4-MBA solution, and react for 30 minutes at 37 °C and 300 rpm with magnetic stirring. Then add 16 μL of reaction solution 1 and continue to react for 2 minutes at the same temperature and stirring speed to obtain reaction solution 2. S2-3: Add 3.85 mL of n-butanol to 420 μL of reaction solution 2, shake rapidly for 10 seconds, then add 850 μL of 5×TBE buffer, shake rapidly for 10 seconds, and then centrifuge at 3500 rpm for 10 minutes. Discard the supernatant and wash the resulting precipitate three times with 0.01 wt% SDS solution to remove free signal probes S2 and 4-MBA, obtaining the SERS tag. Disperse the obtained SERS tag in 320 μL of 0.01 wt% SDS solution to obtain the SERS tag solution, and store it at 4℃ for later use.

[0027] S3: RNase H activity assay S3-1: Add 1.1 μL of 100 μM initiator probe tDNA solution and 1 μL of 100 μM single-stranded RNA solution to 97 μL of 1×RNase H reaction buffer. Heat at 95°C for 5 minutes, then cool to 37°C. Mix with 1 μL of 100 mM TCEP solution at room temperature in the dark for 1 hour to obtain the RNA / tDNA complex solution. Immerse AuE in the RNA / tDNA complex solution, freeze at -18°C for 30 minutes, thaw at room temperature for 5 minutes, rinse the electrode surface with Tris HCl buffer, and dry with nitrogen to obtain the AuE / RNA / tDNA electrode. Immerse the AuE / RNA / tDNA electrode in 2 mM MCH solution, block at room temperature for 1 hour, rinse the electrode surface with Tris HCl buffer, and dry with nitrogen to obtain the AuE / RNA / tDNA / MCH electrode. Using 1×RNase H reaction buffer as the basic system, add different concentrations of the analyte (RNase H) and react with 1×RNase H. The total volume of the system was brought up to 40 μL with H reaction buffer. The AuE / RNA / tDNA / MCH electrode was then immersed in the buffer and the enzyme digestion reaction was carried out in a shaker at 37 °C for 1.5 hours, followed by inactivation at 75 °C for 20 minutes to obtain a reaction solution containing tDNA. 30 μL of the reaction solution containing tDNA was taken and mixed with 0.5 μL of 100 μM capture probe H1 solution, 4 μL of 10× NEBuffer™ 1, 5.5 μL of deionized water and 0.2 μL of 100 U / μL ExoIII. The mixture was incubated at 37 °C for 2 hours and then inactivated at 80 °C for 20 minutes to obtain a reaction solution containing single-stranded DNA fragment S0.

[0028] S3-2: Immerse AuE in 1.5 μM capture probe S1 solution, freeze at -18℃ for 30 minutes, thaw at room temperature for 5 minutes, rinse the electrode surface with Tris HCl buffer and dry with nitrogen to obtain the AuE / S1 electrode; incubate the AuE / S1 electrode sequentially in 0.2 mL 2 mM MCH solution at room temperature for 1 hour, then incubate in 0.2 mL 1 wt% BSA at room temperature for 1.5 hours, rinse the electrode surface with Tris HCl buffer and dry with nitrogen to obtain the AuE / S1 / MCH / BSA electrode; add 7 μL of reaction solution containing single-stranded DNA fragment S0 to the surface of the AuE / S1 / MCH / BSA electrode, incubate at 37℃ for 2 hours, rinse with Tris HCl buffer and dry with nitrogen to obtain the AuE / S1 / MCH / BSA electrode. The electrode surface was rinsed with HCl buffer and dried with nitrogen to obtain the AuE / S1 / MCH / BSA / S0 electrode. The AuE / S1 / MCH / BSA / S0 electrode was immersed in SERStag solution and reacted at 37°C for 1.5 hours. The electrode surface was then rinsed with Tris HCl buffer and dried with nitrogen to obtain the AuE / S1 / MCH / BSA / S0 / S2 / 4-MBA / AuNCs electrode. SERS detection was performed at room temperature, and signals from 7 points were randomly collected each time, and the average value was taken. Example 2

[0029] To achieve optimal detection performance of the SERS sensor in Example 1, four experimental parameters were optimized in this example: the time of the enzyme digestion reaction in step S3-1 (0.5, 1, 1.5, 2, 2.5 hours), the temperature of the enzyme digestion reaction in step S3-1 (27, 32, 37, 40, 45℃), the time of the click reaction in step S3-2 (0.5, 1, 1.5, 2, 2.5 hours), and the concentration of the signal probe S2 solution in step S2-1 (124, 186, 250, 311, 373 nM).

[0030] The results are as follows Figure 2 As shown. Figure 2 Figure A shows the optimization of the RNase H digestion reaction time. As can be seen from the figure, the Raman intensity increases with the extension of the digestion reaction time, reaching its maximum when the reaction time is 1.5 hours. Therefore, 1.5 hours was selected as the optimal time for subsequent digestion reactions. Figure 2 Figure B shows the optimization of the RNase H enzymatic digestion reaction temperature. As can be seen from the figure, the Raman intensity continued to increase as the digestion reaction temperature increased from 27℃ to 45℃. However, when the digestion reaction temperature exceeded 37℃, the Raman intensity decreased. The structure of RNase H may be damaged by high temperatures, resulting in a corresponding decrease in activity. Therefore, 37℃ was selected as the optimal temperature for subsequent enzymatic digestion reactions. Figure 2C represents the optimization of the click response time. As shown in the figure, the Raman intensity increases with the extension of the click response time, reaching its maximum when the click response time is 1.5 hours. Therefore, 1.5 hours was chosen as the optimal click response time for subsequent experiments. Figure 2 D represents the optimization of the concentration of the signal probe S2 solution. As shown in the figure, the Raman intensity increases with the increase of the concentration of the signal probe S2 solution, reaching its maximum when the concentration of the signal probe S2 solution is 250 nM. Therefore, 250 nM is selected as the optimal concentration of the signal probe S2 solution for subsequent experiments. Example 3

[0031] Under the optimized experimental parameters of Example 2, the sensitivity of the SERS sensor of the present invention was explored by using different concentrations of RNase H.

[0032] like Figure 3 As shown, with the increase of RNase H concentration, at 1075 cm⁻¹... -1 The Raman intensity gradually increased at the specified location. A good linear relationship was found between the logarithm of RNase H concentration (from 0.000625 U / mL to 1.25 U / mL) and the Raman intensity, with the corresponding regression equation being ISERS = 18550.70 + 2801.41logC. RNase H (U / mL, R) 2 =0.996). The limit of detection (LOD) is calculated to be 3.89 × 10⁻⁶. -4 U / mL (S / N=3). This indicates that the SERS sensor of the present invention can achieve highly sensitive detection of RNase H activity. Example 4

[0033] Under the optimized experimental parameters of Example 2, the selectivity of the SERS sensor of the present invention was evaluated by using bovine serum albumin, DNA methyltransferase, Nb.BbvCI enzyme, Nt.BstNBI enzyme, and APE 1 enzyme as interfering agents. The concentrations of the interfering agents were all higher than the concentration of RNase H (3.89 × 10⁻⁶). -4 (U / mL) 100 times higher. Select 1075cm. -1 The Raman peak intensity at a certain point is used as an indicator for evaluating the effect.

[0034] like Figure 4 As shown, RNase H exhibits a significant Raman intensity, while the SERS intensity of interfering substances is essentially the same as that of the blank. This indicates that the SERS sensor of this invention has good selectivity in the detection of RNase H.

[0035] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A SERS sensor for detecting ribonuclease H activity, characterized in that: The SERS sensor includes a gold electrode, single-stranded RNA, a priming probe tDNA, a hairpin strand H1, a capture probe S1, Exo III, and a SERS tag. The SERStag was prepared by assembling the signal probe S2 and the signal molecule 4-MBA on the surface of a gold nanocube. The sequence of the single-stranded RNA is: 5'-SH-(CH2)6-GCGGACAUCCUACUUCGCUCUUACU-3'; The sequence of the initiating probe tDNA is: 5'-AGTAAGAGCGAAGTAGGATGTCCGC-(CH2)6-SH-3'; The sequence of the hairpin chain H1 is: 5'-N3-AGCGAAGTAGGACGACGGGGGCATCCTACTTCGCTCTTACT-3'; The sequence of the capture probe S1 is: 5'-SH-(CH2)6-TTTTTTGTCGTCCTAC-3'; The sequence of the signal probe S2 is: 5'-SH-(CH2)6-TTTTTTTTTTTTT-DBCO-3'.

2. The SERS sensor according to claim 1, characterized in that: The preparation method of the gold nanocube includes the following steps: S1-1: Take 0.6 mL of 10 mM NaBH4 solution and add it to 10 mL of 0.1 M CTAB solution containing 10 mM HAuCl4. Gently shake for 2 minutes and then place it in a 28°C water bath for 3 hours to prepare the gold seed solution. S1-2: Mix 2 mL of 0.2 M CTAC solution, 1.5 mL of 0.1 M ascorbic acid solution and 50 μL of gold seed solution, add 2 mL of 0.5 mM HAuCl4 solution, react at 27 °C for 15 minutes, then centrifuge, discard the supernatant, and disperse the resulting precipitate in 1 mL of 0.02 M CTAC solution to obtain gold nanoparticle solution; S1-3: Mix 125 μL of gold nanoparticle solution, 25 mL of 0.1 M CTAC solution, and 1.825 mL of 0.01 M ascorbic acid solution at 30 °C and 200 rpm under magnetic stirring for 2 minutes. Then add 25 mL of 0.01 M HAuCl4 solution and react at 30 °C and 950 rpm under magnetic stirring for 15 minutes. During this process, the solution color can be observed to gradually change from light red to dark red. After the reaction is completed, centrifuge and discard the supernatant. Disperse the obtained precipitate in 1 mL of 0.01 M CTAC solution to obtain a gold nanocube solution, which is stored at 4 °C for later use.

3. The SERS sensor according to claim 1, characterized in that: The method for preparing the SERS tag includes the following steps: S2-1: Add 2 μL of 100 mM TCEP solution and 2 μL of 250 μM signal probe S2 solution to 26 μL Tris buffer, and react at room temperature in the dark for 1 hour to obtain reaction solution 1; S2-2: Add SDS to the gold nanocube solution to a final concentration of 0.01wt% to obtain an SDS-containing gold nanocube solution; take 400μL of the SDS-containing gold nanocube solution, add 4μL of 2mM 4-MBA solution, and react for 30 minutes at 37℃ and 300rpm with magnetic stirring. Then add 16μL of reaction solution 1 and continue to react for 2 minutes at the same temperature and stirring speed to obtain reaction solution 2. S2-3: Add 3.85 mL of n-butanol to 420 μL of reaction solution 2, shake rapidly for 10 seconds, then add 850 μL of 5× TBE buffer, shake rapidly for 10 seconds, centrifuge, discard the supernatant, wash the resulting precipitate three times with 0.01 wt% SDS solution to obtain the SERS tag; disperse the obtained SERS tag in 320 μL of 0.01 wt% SDS solution to obtain the SERS tag solution, and store it at 4℃ for later use.

4. The method for constructing a SERS sensor as described in claim 1, characterized in that: Includes the following steps: S3-1: Add 1.1 μL of 100 μM initiator probe tDNA solution and 1 μL of 100 μM single-stranded RNA solution to 97 μL of 1×RNase H reaction buffer. Heat at 95°C for 5 minutes, then cool to 37°C. Mix with 1 μL of 100 mM TCEP solution at room temperature in the dark for 1 hour to obtain the RNA / tDNA complex solution. Immerse AuE in the RNA / tDNA complex solution, freeze at -18°C for 30 minutes, thaw at room temperature for 5 minutes, rinse the electrode surface with Tris HCl buffer, and dry with nitrogen to obtain the AuE / RNA / tDNA electrode. Immerse the AuE / RNA / tDNA electrode in 2 mM MCH solution, block at room temperature for 1 hour, rinse the electrode surface with Tris HCl buffer, and dry with nitrogen to obtain the AuE / RNA / tDNA / MCH electrode. Add different concentrations of RNase to the 1×RNase H reaction buffer. H. Immerse the AuE / RNA / tDNA / MCH electrode in the solution and perform enzymatic digestion at 37°C in a shaker for 1.5 hours, followed by inactivation at 75°C for 20 minutes to obtain a reaction solution containing tDNA. Take 30 μL of the reaction solution containing tDNA, and add 0.5 μL of 100 μM capture probe H1 solution and 4 μL of 10× NE buffer. TM 1. Mix 5.5 μL of deionized water and 0.2 μL of 100 U / μL ExoIII, incubate at 37 °C for 2 hours, and then inactivate at 80 °C for 20 minutes to obtain a reaction solution containing the single-stranded DNA fragment S0. S3-2: Immerse AuE in 1.5 μM capture probe S1 solution, freeze at -18℃ for 30 minutes, thaw at room temperature for 5 minutes, rinse the electrode surface with Tris HCl buffer and dry with nitrogen to obtain the AuE / S1 electrode; incubate the AuE / S1 electrode sequentially in 0.2 mL 2 mM MCH solution at room temperature for 1 hour, then incubate in 0.2 mL 1 wt% BSA at room temperature for 1.5 hours, rinse the electrode surface with Tris HCl buffer and dry with nitrogen to obtain the AuE / S1 / MCH / BSA electrode; add 7 μL of reaction solution containing single-stranded DNA fragment S0 to the surface of the AuE / S1 / MCH / BSA electrode, incubate at 37℃ for 2 hours, rinse with Tris HCl buffer and dry with nitrogen to obtain the AuE / S1 / MCH / BSA electrode. The electrode surface was rinsed with HCl buffer and dried with nitrogen to obtain the AuE / S1 / MCH / BSA / S0 electrode. The AuE / S1 / MCH / BSA / S0 electrode was immersed in SERStag solution and click-reacted at 37°C for 1.5 hours. The electrode surface was then rinsed with Tris HCl buffer and dried with nitrogen to obtain the AuE / S1 / MCH / BSA / S0 / S2 / 4-MBA / AuNCs electrode, which was then subjected to SERS detection at room temperature.

5. The construction method according to claim 4, characterized in that: The gold electrode needs to be pretreated before use. Specifically, the surface of the gold electrode is first polished with alumina powder, and then cleaned with anhydrous ethanol and ultrapure water in sequence.

6. The construction method according to claim 4, characterized in that: The sequence of the single-stranded DNA fragment S0 is: 5'-N3-AGCGAAGTAGGACGACGGGGG-3'.

7. The application of the SERS sensor as described in claim 1 in detecting ribonuclease H activity for non-disease diagnosis and treatment purposes.

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