A SERS-electrochemical dual-mode sensor for miRNA detection, preparation method thereof and application thereof

Abstract

A SERS-electrochemical dual-mode sensor for miRNA detection, a preparation method thereof and an application thereof, wherein the SERS sensing mode is realized by extracting the intensity of the SERS characteristic peak of methylene blue MB, and the electrochemical sensing mode is realized by testing the MB peak current by square wave voltammetry; the sensing substrate is obtained by modifying a hairpin DNA single strand and an auxiliary strand on the surface of a silver nanorod array substrate, and when the target miR-106a is present, a stable LW / T / DNAzyme three-strand complex is formed on the substrate surface with the first reagent DNAzyme and the auxiliary single strand LW in the sensing substrate; then, under the activation of the second reagent, the first reagent is triggered to cut the hairpin DNA single strand H1 in the sensing substrate on the substrate surface, and the remaining H1 fragment after cutting further binds to a SERS / electrochemical dual-mode probe modified with a signal molecule methylene blue MB, thereby outputting SERS and electrochemical signals. The dual-mode sensor detects miR-106a in serum at attomole and femtomolar levels, respectively, meeting the requirements of high sensitivity and reliable detection.

Description

Technical Field

[0001] The invention belongs to the field of spectroscopy detection and electrochemical detection, and particularly relates to a SERS-electrochemical dual-mode sensor for miRNA detection, a preparation method thereof and an application thereof. Background Art

[0002] Reliable detection of cancer-related nucleic acid biomarkers, miRNA, is of great significance for the early diagnosis of cancer. Traditional methods for detecting miRNA include Northern blotting, real-time quantitative polymerase reaction (RT-qPCR), and microarray technology, all of which have powerful detection functions, but various complex and cumbersome operations also limit the trend of large-scale promotion. In addition, miRNA usually exists in a complex biological environment at a very low abundance, so it is urgent to develop more highly sensitive, accurate and reliable nucleic acid detection tools.

[0003] Various high-sensing nanomaterials and signal amplification strategies have been introduced into photoelectric biosensors to meet the requirements of high-sensitivity detection of miRNA, but a single detection mode often leads to deviation in results. Therefore, in order to meet the requirements of detection sensitivity, it is an inevitable trend in biological tissue research and genetic testing to develop a multi-mode joint method to output multiple signals through mutual verification of multiple sets of data, eliminate abnormal diagnostic results of a single mode, and ensure the accuracy of detection.

[0004] SERS and electrochemistry are two new photoelectric detection technologies that have attracted much attention. Both of them have shown the characteristics of fast and sensitive detection. SERS can be applied to the detection of solid-liquid interfaces, but due to the difficulty of laser focusing in the liquid environment, SERS detection takes a long time; SERS provides abundant fingerprint peaks, but for the reaction process of similar substances such as DNA hybridization, SERS cannot identify. Electrochemistry is suitable for liquid detection and takes a short time to detect, but due to the redox reaction, it cannot guarantee that the properties of the substance will not change; the impedance change of the interface can be used to determine whether the reaction is taking place, but it is impossible to obtain the overall information of the interface.

[0005] It can be seen that the sensing principles of SERS and electrochemistry are completely different. Few works have combined SERS and electrochemistry for the detection of nucleic acids, and the work using silver nanomaterials as dual-mode substrates is even scarcer. Therefore, the development of SERS-electrochemical dual-mode sensors is a very valuable work. Summary of the invention

[0006] In view of the problem that a single detection mode is easily affected by possible interferences, different instruments or human operations, leading to misdiagnosis or misdiagnosis results, in order to ensure the reliability of the detection results, the present invention proposes a SERS-electrochemical dual-mode sensor for nucleic acid detection. The combination of SERS and electrochemical dual modes realizes the complementary advantages of the two modes, avoids the diagnostic anomalies of a single mode, obtains more and richer detection information, and outputs more accurate, complete and reliable detection results. The present invention proposes a SERS-electrochemical dual-mode sensor for nucleic acids that combines the advantages of SERS and electrochemistry, greatly improving the problems of insufficient reliability and sensitivity of most existing detection methods. The sensor combining SERS and electrochemistry proposed in the present invention can achieve highly sensitive and reliable detection of cancer-related miRNA.

[0007] The technical problem that the present invention also solves is to provide a method for preparing a SERS-electrochemical dual-mode combined nucleic acid sensor, which is convenient and sensitive.

[0008] The present invention introduces silver nanorod array as a dual-mode sensing substrate to prepare a sensor for detecting cancer-related miRNA-106a. The SERS and electrochemical sensing modes reach aM and fM level detection limits respectively in the complex environment of serum, achieving the purpose of highly sensitive and reliable detection.

[0009] A SERS-electrochemical dual-mode sensor for miRNA detection, comprising a sensing substrate, a first reagent, a second reagent and a third reagent probe;

[0010] The sensing substrate is a substrate modified with a hairpin-type DNA single strand H1 and an auxiliary single strand LW, wherein the substrate is a silver nanorod array substrate covered with a polydimethylsiloxane layer with small holes; the aperture of the small hole is 4 mm and the height is 1 mm;

[0011] The first reagent is DNAzyme;

[0012] The second reagent is zinc chloride Zn 2+ Solution;

[0013] The third reagent dual-mode probe is a molybdenum disulfide-based composite nanomaterial MoS2@PDA-Ag, MPA, whose surface is modified with a hairpin-type DNA single strand H2 and methylene blue MB.

[0014] Furthermore, the hairpin DNA single-strand H1 base sequence is as shown in SEQ ID NO: 1: 5'-CCA CCT CATTGA AAT ATG CAG ACG TTG AAG GAT CGT CTT TACAGG CGGAGG TGG TTT-SH-3';

[0015] The auxiliary single-stranded LW base sequence is shown in SEQ ID NO: 3: 5'-GTAAGC ACT TTTATG TGATTTT-SH-3';

[0016] The base sequence of the DNAzyme is shown in SEQ ID NO: 4: 5'-GAC GAT CTA GTT GAG CTG TCTGCA TTT TTT TTT TTT CTA CCT GCA CT-3';

[0017] The hairpin DNA single-strand H2 base sequence is shown in SEQ ID NO: 5: 5'-SH-TTT GGA TCG TCTTTA CAG GCG CCA CCT CCG CCT GTA AAGACGATC C-3';

[0018] The molybdenum disulfide-based composite nanomaterial MoS2@PDA-Ag is obtained by sequentially modifying polydopamine and silver nanoparticles AgNPs on MoS2 nanosheets MoS2 NSs.

[0019] Further, the base sequence of the target miRNA-106a is shown in SEQ ID NO: 2: 5′-AAAAGT GCTTACAGT GCA GGTAG-3′;

[0020] The specificity experiment for the detection of the target miRNA-106a base sequence corresponds to the single base mismatch SM and the complete mismatch, i.e., the nucleic acid base sequences of miRNA-21 and miRNA-155:

[0021] The base sequence of the single-base mismatch sequence SM is shown in SEQ ID NO: 6: 5′-AAA AGT GCT TACAGTGCAAGTAG-3′;

[0022] The base sequence of the complete mismatch sequence miRNA-21 is shown in SEQ ID NO: 7: 5'-UAG CUUAUCAGACUGAUG UUGA-3';

[0023] The base sequence of the complete mismatch sequence miRNA-155 is shown in SEQ ID NO: 8: 5'-UUAAUG CUAAUCGUGAUA GGG GU-3'.

[0024] A method for preparing a SERS-electrochemical dual-mode sensor for miRNA detection comprises the following steps:

[0025] Step 1, preparation of dual-mode sensing substrate:

[0026] The hairpin DNA H1 obtained by annealing at 90℃-95℃ and the auxiliary single-stranded LW were mixed and co-cultured with a silver nanorod array substrate. The two single-stranded strands were connected to the substrate surface through thiol groups to form Ag-S covalent bonds with the silver nanorods; in addition, mercaptohexanol (MCH) was used to block the remaining sites on the substrate surface to prepare a dual-mode sensing substrate;

[0027] Step 2, prepare the first reagent:

[0028] Design and synthesize DNAzyme based on auxiliary single-strand LW and target sequence;

[0029] Step 3, prepare the second reagent:

[0030] Prepare the second reagent as zinc chloride Zn 2+ Solution;

[0031] Step 4, prepare the third reagent:

[0032] A hairpin DNA single-strand H2 was designed and synthesized based on the hairpin DNA single-strand H1. After MPA and polyvinyl pyrrolidone (PVP) were pre-mixed, the signal molecule MB was added and mixed and co-cultured overnight. The mixture was purified by centrifugation at 8000 rpm for 20 min twice, and the supernatant was removed to obtain MPA@MB. The hairpin DNA H2 obtained by annealing at 90°C to 95°C and MPA@MB were mixed and co-cultured overnight to obtain the final product, MPA@MB-H2 dual-mode probe.

[0033] Step 5, preparation of SERS-electrochemical dual-mode sensor:

[0034] In the presence of the target miRNA, part of the target sequence hybridizes with the auxiliary single-stranded LW in the sensor substrate, and the other part hybridizes with the first reagent DNAzyme to form a LW / T / DNAzyme triple-stranded composite structure and is fixed on the surface of the sensor substrate. 2+ Under induction, the DNAzyme is triggered to cleave the hairpin DNA H1 on the surface of the sensing substrate and release itself in a continuous walker repetitive process; the remaining H1 sequence after cleavage and the H2 in the sensing probe capture and assemble the third reagent dual-mode probe MPA@MB-H2 on the substrate surface through the formed H1-H2 double chain; dual-mode detection is achieved by simultaneously outputting the SERS and electrochemical signals of MB.

[0035] Furthermore, in step 1, the concentration ratio of H1 to LW on the dual-mode sensing substrate is 5:1 to 20:1.

[0036] Furthermore, in step 5, the target sequence and DNAzyme are mixed and then incubated with the sensing substrate for 2 hours at a temperature of 37°C.

[0037] Furthermore, in step 5, the second reagent Zn 2+ The incubation concentration is 0.1 mM to 2 mM; the incubation time is 0.5 h to 2 h, and the incubation temperature is 37°C.

[0038] Furthermore, in step 5, the incubation time of the third reagent dual-mode probe is 1 hour; and the incubation temperature is 37°C.

[0039] An application of a SERS-electrochemical dual-mode sensor for miRNA detection comprises the following steps:

[0040] Step a, adding the target sequence miRNA-106a to 10% normal human serum to obtain a sample solution for later use;

[0041] Step b, replacing the target sequence in the PB buffer with the sample solution to construct a dual-mode sensor;

[0042] Step c, washing the sample obtained in step b for multiple times and then performing SERS and electrochemical tests to obtain SERS spectra and electrochemical SWV spectra of target sequences with different concentrations, using the logarithm of the target miRNA-106a concentration as the horizontal coordinate, and the SERS intensity and peak current intensity of the characteristic peak of MB as the vertical coordinates, respectively, to draw the working curves of SERS sensing and electrochemical sensing, and calculate the SERS sensing detection limit and electrochemical sensing detection limit of the sensor according to the working curves.

[0043] Furthermore, in step b, the concentration range of the target miRNA-106a detected by SERS and electrochemical sensing modes is 100aM to 1nM.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The SERS-electrochemical dual-mode sensor prepared by the present invention uses a silver nanorod array substrate with high SERS activity as a dual-mode substrate, and uses a molybdenum disulfide-based composite nanomaterial as a dual-mode probe to improve the stability of the substrate. This expands the selection range of dual-mode electrodes. In addition, the detection results of the two modes of SERS and electrochemistry are combined to verify each other, avoiding misdiagnosis results of a single mode, making the data more accurate and reliable.

[0046] (2) In addition, the highly sensitive SERS / electrochemical dual-mode sensor assisted by DNAwalker signal amplification further achieved the effect of signal amplification and achieved detection limits of 129.72aM and 1.33fM for SERS and electrochemistry in the serum environment, respectively, with high-sensitivity detection characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1It is a diagram showing the construction and sensing principle of the SERS-electrochemical dual-mode sensor involved in the present invention.

[0048] Figure 2 This is the gel electrophoresis characterization of the DNA hybridization process involved in Example 1 of the present invention.

[0049] Figure 3 This is an optimization experiment of the sensing strategy in Example 2 of the present invention.

[0050] Figure 4 This is the SERS-electrochemical working curve and detection limit for serum sample detection in Example 3 of the present invention.

[0051] Figure 5 This is a specific characterization of the SERS-electrochemical dual-mode sensor in Example 4 of the present invention.

[0052] Figure 6 This is a characterization of the uniformity of the SERS-electrochemical dual-mode sensor in Experimental Example 5 of the present invention.

[0053] Figure 7 This is a recovery rate verification table of the SERS-electrochemical dual-mode sensor in Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings.

[0055] The 8 DNA base sequence fragments used in the present invention are all artificially synthesized by Sangon Biotech (Shanghai) Co., Ltd. The bold parts in SM are mismatched bases.

[0056] 1. The nucleic acid base sequences of the hairpin DNA single strand H1, LW, DNAzyme and miRNA-106a to be detected in the following examples are:

[0057] H1: 5'-CCA CCT CAT TGAAATATG CAGACG TTGAAG GAT CGT CTT TACAGG CGGAGGTGG TTT-SH-3';

[0058] miRNA-106a: 5'-AAAAGT GCT TACAGT GCA GGTAG-3';

[0059] LW: 5'-GTAAGCACT TTTATG TGATTT T-SH-3';

[0060] DNAzyme: 5'-GAC GAT CTA GTT GAG CTG TCT GCA TTT TTT TTT TTT CTACCT GCACT-3';

[0061] H2: 5'-SH-TTT GGA TCG TCT TTA CAG GCG CCA CCT CCG CCT GTAAAGACGATC C-3'.

[0062] 2. The nucleic acid base sequences corresponding to the single base mismatch (SM) and complete mismatch (miRNA-21 and miRNA-155) in the specific experiment for the detection of gastric cancer micronucleic acid marker miRNA-106a are:

[0063] Single base mismatch sequence (SM): 5′-AAAAGT GCT TACAGT GCAAGT AG-3′;

[0064] Complete mismatch sequence (miRNA-21): 5′-UAG CUUAUCAGA CUGAUG UUGA-3′;

[0065] Complete mismatch sequence (miRNA-155): 5'-UUAAUG CUAAUC GUGAUA GGG GU-3'.

[0066] 3. In the following examples, the silver nanorod arrays were prepared by vacuum electron beam evaporation coating technology. The silver nanorod array substrate was covered with a polydimethylsiloxane (PDMS) layer with small holes. The small holes had a diameter of 4 mm and a height of 1 mm.

[0067] Example 1, preparation of SERS-electrochemical dual-mode sensor:

[0068] (1) Preparation of dual-mode sensing substrate:

[0069] The solid silver nanorod array dual-mode substrate was prepared by multiple rinses with pure water; 2μL of 10μM hairpin DNA H1 annealed at 95°C, 2μL of 1μM auxiliary single-stranded LW and 16μL of 100mM PB buffer were mixed to form a 20μL system solution, and co-cultured with the silver nanorod array substrate in a 25°C constant temperature mixer for 3h. After washing with PB buffer solution, 20μL of 1mM mercaptohexanol (MCH) was continued to be added to the surface of the silver nanorod array substrate, and after 10min of incubation, it was rinsed multiple times to completely remove the unbound MCH to obtain a dual-mode sensing substrate.

[0070] (2) Preparation of the first reagent:

[0071] A DNAzyme having a sequence as shown in SEQ ID NO. 4 is designed and synthesized according to the auxiliary single-stranded LW and the target sequence, and its concentration can be 1 μM to 10 μM.

[0072] (3) Preparation of the second reagent:

[0073] Prepare the second reagent as zinc chloride (Zn 2+ ) solution, the concentration of which can be 0.5mM to 2mM.

[0074] (4) Preparation of the third reagent:

[0075] A hairpin DNA single strand H2 having a sequence as shown in SEQ ID NO.6 was designed and synthesized based on the hairpin DNA single strand H1. 4 mL of 50 μg / mL MPA was pre-mixed with 400 μL of 5% PVP by mass, and then 100 μL of 2 mg / mL signal molecule MB was added and mixed and stirred overnight to obtain a mixed solution 1. The mixed solution 1 was purified twice by centrifugation at 8000 rpm for 20 min, and the supernatant was removed to obtain MPA@MB. 10 μL of 10 μM hairpin DNA H2 annealed at 90°C to 95°C and 90 μL of MPA@MB were mixed and co-cultured overnight to obtain a mixed solution 2. The mixed solution 2 was purified once by centrifugation at 6000 rpm for 20 min, and then the volume was fixed to 100 μL with PB buffer to obtain the third reagent MPA@MB-H2 dual-mode probe.

[0076] (5) Preparation of SERS-electrochemical dual-mode sensor:

[0077] First, the surface of the solid silver nanorod array dual-mode substrate was cleaned with ultrapure water, and then the AgNRs were incubated with a 20μL mixture of 2μL 10μM H1, 1μM LW and 16μL 0.1M PB buffer at 25°C for 3h. After washing with PB buffer solution, 20μL of 1mM MCH was added dropwise. After 10min, multiple rinses were performed to completely remove unbound MCH. Subsequently, 1μL 2μM first reagent DNAzyme, 2μL different concentrations of target miR-106a (T) and 17μL PB were co-incubated with AgNRs for 2h to construct a LW / T / DNAzyme triple-chain composite structure on the AgNRs substrate surface. After multiple washes, 20μL 1mM second reagent Zn 2+, and react at 37°C for 1h, DNAzyme continuously cleaves the hairpin DNA H1 on the substrate surface. After washing, an equal volume of MPA@MB-H2 probe is added and incubated for 1h. The sequence of the cleaved H1 can hybridize with H2 in the sensor probe, and the third reagent dual-mode probe MPA@MB-H2 is captured and assembled on the substrate surface through the formed H1-H2 double chain. Dual-mode detection is achieved by simultaneously outputting the SERS and electrochemical signals of MB.

[0078] The construction of DNAwalker signal amplification SERS-electrochemical dual-mode sensor was verified by 10% polyacrylamide gel electrophoresis. Figure 2 As shown. In lane 4 containing a mixture of three types of T, LW and DNAzyme, a bright band was observed, which was different from the band positions corresponding to the three single-stranded structures in lanes 1-3, and was higher than the band positions of the two possible double-stranded structures shown in lanes 8 and 9, namely the double-stranded structure of T and DNAzyme combined and the double-stranded structure of T and LW combined, indicating the efficient combination of the three-stranded complex structure of LW, T and DNAzyme. Lane 7, where H1 and H2 were added at the same time, showed two distinct bands and coincided with the positions of the single bands of lanes 5 and 6, proving that the hairpin structure of H1 and H2 can protect them from spontaneous hybridization in the natural state. Lanes 10 and 11 showed the positions of the double-stranded structures without or with Zn, respectively. 2+ The obvious band difference between the two lanes indicates that the Zn 2+ With the help of Zn, DNAzyme can be activated to catalyze the cleavage of H1 to form the remaining H1 fragment. Compared with lane 11, lane 12 with the addition of the H2 hairpin structure has a new bright band, and this band is located at the same position as the band formed by the hybridization of the remaining sequence H1-half and H2 after H1 shearing in lane 14. 2+ In the case of , no similar band was observed in lane 13. These results confirm the effectiveness of the proposed sensing mechanism.

[0079] Example 2, SERS-electrochemical dual-mode sensor optimization experiment:

[0080] In the construction of the sensing substrate, with 1nM target sample as reference, the concentration ratio of H1 and LW was adjusted to 5:1, 10:1, 5:1, 20:1 respectively, and the H1 concentration was kept constant at 1μM in the 20μL system. The data showed that SERS( Figure 3 a) and electrochemical square wave voltammetry (SWV) test intensity value ( Figure 3 b) The best signal output was achieved when the concentration ratio was 10:1. 2+The synergistic effect of incubation time and concentration has important implications for the catalytic activity and H1 cleavage efficiency of DNAzyme. 2+ Incubation time ranges from 30 min to 120 min ( Figure 3 c and 3d), the results show that the signal intensity increases with the increase of incubation time. When the incubation time reaches 60 min, the SERS and SWV detection intensity values ​​tend to be stable, indicating that Zn 2+ The optimal incubation time was 60 min. The subsequent dual-mode sensor preparation and detection were carried out under this optimal condition.

[0081] Example 3, SERS-electrochemical working curve and detection limit for serum sample detection:

[0082] The concentration of miR-106a was diluted from 100aM to 1nM in a 10% human serum environment, and the sensor was prepared and tested according to the optimal conditions in Example 2 and the preparation steps in Example 1. The test results are as follows: Figure 4 As shown. In serum environment, SERS ( Figure 4 a) and electrochemical sensing ( Figure 4 b) The intensity also increases with increasing target concentration (in the range of 100aM-1nM). The linear calibration curves of the two modes are shown below. Figure 4 C collected 1622cm -1 SERS intensity and concentration (C miR-106a )The linear equation constructed by logarithm is I 1622 =938×logC miR-106a +17241(R 2 =0.997), the calculated detection limit LOD is 42.5fM (S / N=3). Figure 4 d According to the electrochemical peak current intensity and C miR-106a The linear equation constructed between the logarithms is ΔI = 15.63 × logC miR-106a +266.41(R 2 =0.993), LOD is 179.91aM (S / N=3).

[0083] Example 4, SERS-electrochemical specificity characterization for serum sample detection:

[0084] In PB buffer, miRNA-106a was diluted to 100 pM, and non-specific samples (i.e., single-base mismatch (SM) DNA, miR-155, and miR-21) were diluted to 1 nM. The sensor was prepared and tested according to the optimal conditions in Example 2 and the preparation steps in Example 1. Figure 5The SERS and electrochemical detection results shown in a and 5b show that the specific detection of the target miR-106a has the highest SERS / electrochemical signal, which can be clearly distinguished from blank or nonspecific miRNA, and even SM DNA. In summary, this SERS / electrochemical dual-mode sensor has good specificity and can achieve reliable detection of target miRNA.

[0085] Example 5, SERS-electrochemical homogeneity characterization for serum sample detection:

[0086] Under the same conditions, 1 nM miR-106a was reproducibly characterized by electrochemical and SERS tests on five sets of sensor electrodes. The sensor was prepared and tested according to the optimal conditions in Example 2 and the preparation steps in Example 1. The test results are shown in Figure 2. Figure 6 As shown. Figure 6 The analysis of the SERS and electrochemical detection results in 6a and 6b shows very intuitively that there is no obvious difference in the SERS and electrochemical test results of different batches, which proves that the proposed SERS / electrochemical dual-mode sensor has good repeatability and the detection results are relatively reliable.

[0087] Example 6, SERS-electrochemical dual-mode sensor recovery rate verification:

[0088] Four target samples with different concentrations (500aM, 40fM, 3pM and 200pM miR-106a) were prepared in a 10% human serum environment and the recovery rate was verified. Figure 7 As shown in the table, the recoveries of SERS sensing and electrochemical sensing are between 93.53% and 103.72% and 91.33% and 108.13%, respectively, indicating that the proposed SERS / electrochemical dual-mode sensor has good reliability for the detection of target miRNA in complex real samples.

[0089] The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiment. Any equivalent modifications or changes made by ordinary technicians in this field based on the contents disclosed by the present invention should be included in the protection scope recorded in the claims. Sequence Listing <110> Nanjing University of Posts and Telecommunications <120> A SERS-electrochemical dual-mode sensor for miRNA detection, preparation method thereof and application thereof <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 57 <212> DNA <213> H1 (Artificial Sequence) <400> 1 ccacctcatt gaaatatgca gacgttgaag gatcgtcttt acaggcggag gtggttt 57 <210> 2 <211> 23 <212> DNA <213> miRNA-106a (Artificial Sequence) <400> 2 aaaagtgctt acagtgcagg tag 23 <210> 3 <211> 22 <212> DNA <213> LW (Artificial Sequence) <400> 3 gtaagcactt ttatgtgatt tt 22 <210> 4 <211> 47 <212> DNA <213> DNAzyme (Artificial Sequence) <400> 4 gacgatctag ttgagctgtc tgcatttttt ttttttctac ctgcact 47 <210> 5 <211> 46 <212> DNA <213> H2 (Artificial Sequence) <400> 5 tttggatcgt ctttacaggc gccacctccg cctgtaaaga cgatcc 46 <210> 6 <211> 23 <212> DNA <213> SM (Artificial Sequence) <400> 6 aaaagtgctt acagtgcaag tag 23 <210> 7 <211> 22 <212> RNA <213> miRNA-21 (Artificial Sequence) <400> 7 uagcuuauca gacugauguu ga 22 <210> 8 <211> 23 <212> RNA <213> miRNA-155 (Artificial Sequence) <400> 8 uuaaugcuaa ucgugauagg ggu 23

Claims

1. A SERS-electrochemical dual-mode sensor for miRNA detection, characterized in that: The dual-mode sensor comprises a sensing substrate, a first reagent, a second reagent and a third reagent dual-mode probe; The sensing substrate is a substrate modified with a hairpin-type DNA single strand H1 and an auxiliary single strand LW, wherein the substrate is a silver nanorod array substrate covered with a polydimethylsiloxane layer with small holes; the aperture of the small hole is 4 mm and the height is 1 mm; The first reagent is DNAzyme; The second reagent is a zinc chloride solution; The third reagent dual-mode probe is a molybdenum disulfide-based composite nanomaterial MoS2@PDA-Ag (MPA) with a hairpin-type DNA single strand H2 and methylene blue MB modified on the surface; The base sequence of the hairpin DNA single-strand H1 is shown in SEQ ID NO: 1: 5'-CCA CCT CAT TGA AAT ATGCAG ACG TTG AAG GAT CGT CTT TAC AGG CGG AGG TGG TTT-SH-3'; The auxiliary single-stranded LW base sequence is shown in SEQ ID NO: 3: 5'-GTA AGC ACT TTT ATG TGA TTT T-SH-3'; The base sequence of the DNAzyme is shown in SEQ ID NO: 4: 5'-GAC GAT CTA GTT GAG CTG TCT GCATTT TTT TTT TTT CTA CCT GCA CT-3'; The hairpin DNA single-strand H2 base sequence is shown in SEQ ID NO: 5: 5'-SH-TTT GGA TCG TCT TTACAG GCG CCA CCT CCG CCT GTA AAG ACG ATC C-3'; The molybdenum disulfide-based composite nanomaterial MoS2@PDA-Ag is obtained by sequentially modifying polydopamine and silver nanoparticles AgNPs on MoS2 nanosheets MoS2 NSs.

2. A SERS-electrochemical dual-mode sensor for miRNA detection according to claim 1, characterized in that: The base sequence of the target miRNA-106a is shown in SEQ ID NO: 2: 5′-AAA AGT GCT TAC AGT GCA GGTAG-3′; The specificity experiment for the detection of the target miRNA-106a base sequence corresponds to the single base mismatch SM and the complete mismatch, i.e., the nucleic acid base sequences of miRNA-21 and miRNA-155: The base sequence of the single-base mismatch sequence SM is shown in SEQ ID NO: 6: 5'-AAA AGT GCT TAC AGT GCAAGT AG-3'; The base sequence of the complete mismatch sequence miRNA-21 is shown in SEQ ID NO: 7: 5'-UAG CUU AUC AGA CUGAUG UUG A-3'; The base sequence of the completely mismatched sequence miRNA-155 is shown in SEQ ID NO: 8: 5'-UUA AUG CUA AUC GUGAUA GGG GU-3'.

3. A method for preparing a SERS-electrochemical dual-mode sensor for miRNA detection as claimed in any one of claims 1 to 2, characterized in that: The steps of the preparation method are as follows: Step 1, preparation of dual-mode sensing substrate: A solid silver nanorod array dual-mode substrate was prepared by multiple washing with pure water; the hairpin DNA H1 and the auxiliary single-strand LW obtained by annealing at 90°C-95°C were mixed and co-cultured with the silver nanorod array substrate, and the two single-strands were connected to the substrate surface by forming an Ag-S covalent bond with the silver nanorods AgNRs through the thiol group; in addition, the remaining sites on the substrate surface were blocked with mercaptohexanol MCH, and after washing with PB buffer solution, mercaptohexanol MCH was continuously added dropwise to the surface of the silver nanorod array substrate, and after incubation, it was washed multiple times to completely remove the unbound MCH, so as to prepare a dual-mode sensing substrate; Step 2, prepare the first reagent: Design and synthesize DNAzyme based on auxiliary single-strand LW and target sequence; Step 3, prepare the second reagent: Prepare the second reagent as zinc chloride solution; Step 4, prepare the third reagent: According to the hairpin DNA single strand H1, the hairpin DNA single strand H2 was designed and synthesized. After MPA and polyvinyl pyrrolidone PVP were pre-mixed, the signal molecule MB was added and mixed and co-cultured overnight. The mixture was centrifuged and purified twice at 8000 rpm for 20 min, and the supernatant was removed to obtain MPA@MB. The hairpin DNA H2 obtained by annealing at 90℃~95℃ and MPA@MB were mixed and co-cultured overnight. The mixture was centrifuged and purified once at 6000 rpm for 20 min, and then the volume was fixed with PB buffer to obtain the final product MPA@MB-H2 dual-mode probe; Step 5, preparation of SERS-electrochemical dual-mode sensor: First, the surface of the solid silver nanorod array dual-mode substrate was cleaned with ultrapure water, and then the AgNRs were incubated with a 20 μL mixture of 2 μL 10 μM H1, 1 μM LW, and 16 μL 0.1 M PB buffer at 25°C for 3 h; after washing with PB buffer solution, 20 μL of 1 mM MCH was added dropwise; After 10 min, the reaction mixture was rinsed several times to completely remove the unbound MCH. Subsequently, a 20 μL mixture of 1 μL 2 μM first reagent DNAzyme, 2 μL different concentrations of target miR-106a, and 17 μL PB was incubated with AgNRs for 2 h to construct a LW / T / DNAzyme triple-chain composite structure on the surface of the AgNRs substrate. After multiple washings, 20 μL 1 mM second reagent Zn 2+ , and reacted at 37°C for 1 h. The DNAzyme continuously cleaved the hairpin DNA H1 on the substrate surface. The remaining H1 sequence after cleavage hybridized with H2 in the sensor probe, and the third reagent dual-mode probe MPA@MB-H2 was captured and assembled on the substrate surface through the formed H1-H2 double chain. Dual-mode detection was achieved by simultaneously outputting the SERS and electrochemical signals of MB.

4. The method for preparing a SERS-electrochemical dual-mode sensor for miRNA detection according to claim 3, characterized in that: In the step 1, the concentration ratio of H1 to LW on the dual-mode sensing substrate is 5:1-20:

1.

5. The method for preparing a SERS-electrochemical dual-mode sensor for miRNA detection according to claim 3, characterized in that: In step 5, the target sequence and DNAzyme are mixed and then incubated with the sensing substrate for 2 h at a temperature of 37°C.

6. The method for preparing a SERS-electrochemical dual-mode sensor for miRNA detection according to claim 3, characterized in that: In step 5, the second reagent Zn 2+ The incubation concentration is 0.1 mM~2 mM; the incubation time is 0.5 h~2 h, and the incubation temperature is 37°C.

7. The method for preparing a SERS-electrochemical dual-mode sensor for miRNA detection according to claim 3, characterized in that: In step 5, the incubation time of the third reagent dual-mode probe is 1 h; and the incubation temperature is 37°C.

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