SERS biosensor based on crispr / cas13a system and use thereof

Abstract

Disclosed are an SERS biosensor based on a CRISPR / Cas13a system and use thereof. The SERS biosensor comprises an SERS sensing chip, a first reagent, a second reagent, a third reagent, and a fourth reagent. The SERS sensing chip is a silver nanorod array substrate with a surface modified with a hairpin-type DNA single chain H1 and a closed molecule 6-mercaptohexanol. The first reagent is an SERS probe and a buffer thereof. The second reagent is a hairpin-type nucleic acid aptamer chain MUC1-apt aqueous solution. The third reagent is a CRISPR / Cas13a system and a buffer thereof. The fourth reagent is a hairpin-type DNA single-chain H2 aqueous solution. The reagents and a gastric cancer exosome sample to be detected are mixed and then dropwise added to the surface of the SERS sensing chip, and sensitive and accurate detection of gastric cancer-related exosomes is achieved by means of the CRISPR / Cas13a system, catalytic hairpin assembly, and SERS enhancement performance of the silver nanorod array substrate.

Description

A SERS biosensor based on CRISPR / Cas13a system and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of spectroscopy detection, and particularly relates to a SERS biosensor based on CRISPR / Cas13a system and application thereof. BACKGROUND

[0002] Gastric cancer is one of the most common malignant tumors worldwide. In 2020, the global incidence of gastric cancer ranked fifth, and the mortality rate ranked fourth. Most patients have no obvious symptoms in the early stage of gastric cancer, and are often found in the late stage. The clinical diagnosis of gastric cancer mainly relies on endoscopic biopsy and medical imaging technology, and the detection rate of early tumor micro lesions is low. Therefore, early diagnosis of gastric cancer is urgent for developing more effective treatment strategies and reducing mortality.

[0003] Exosomes are extracellular vesicles involved in intercellular material transfer, information exchange, cell proliferation and differentiation, and other physiological processes, and are also related to tumor invasion and metastasis and many other pathological processes. Therefore, cancer-derived exosomes have great potential to become biomarkers for early clinical diagnosis and evaluation of cancer treatment. Traditional exosome detection methods, such as Western blotting, flow cytometry, nanoparticle tracking analysis (NTA), and enzyme-linked immunosorbent assay (ELISA), etc. However, the abundance of cancer-derived exosomes in complex body fluids is extremely low, and traditional detection methods have problems such as expensive instruments, complicated operation, time-consuming detection, insufficient sensitivity and accuracy, etc. Therefore, it is urgent to develop a sensitive and reliable exosome detection method to achieve accurate early diagnosis of gastric cancer.

[0004] Surface-enhanced Raman spectroscopy (SERS) technology has been regarded as a powerful analytical tool due to its ultra-high sensitivity and fingerprint specificity. Compared with antibody-based immunodetection, aptamers exhibit high binding affinity and specificity to targets, are easy to synthesize and store, have good stability, low immunogenicity and toxicity. In addition, single-stranded DNA aptamers can be combined with various DNA-based signal amplification reactions, which is expected to significantly improve the sensitivity and specificity of exosome detection.

[0005] The prior application of the research group, a SERS biosensor and its preparation method and application (2024101307311), provides a scheme for early gastric cancer exosome detection, which improves the detection sensitivity to a certain extent. However, the detection process requires pretreatment of the sample, which makes the overall detection efficiency not high. SUMMARY

[0006] Invention purposes: In view of the great demand for rapid, sensitive and accurate determination of exosomes at present, and the low sensitivity, poor specificity, long detection time and other shortcomings of traditional detection methods, the application discloses a SERS biosensor based on a CRISPR / Cas13a system and its application based on aptamer recognition technology, combined with a cascade signal amplification strategy of catalytic hairpin assembly (CHA). The SERS biosensor is simple to prepare, does not require amplification and professional technical personnel for detection, and has rapid detection (60 minutes), high sensitivity (detection limit as low as 10 2 particles·mL -1 horizontal), good specificity (can significantly distinguish exosomes from other cell sources), realizes rapid, sensitive and accurate determination of gastric cancer exosomes, and provides an innovative and feasible solution for early gastric cancer diagnosis.

[0007] To solve the above technical problems, the application adopts the following technical solutions:

[0008] In a first aspect, the application provides a SERS biosensor based on a CRISPR / Cas13a system, comprising: a SERS sensing chip, a first reagent, a second reagent, a third reagent and a fourth reagent.

[0009] As shown in FIG. 1A, the SERS sensing chip is a silver nanorod array substrate with a hairpin DNA single strand H1 and a blocking molecule 6-mercaptohexanol (MCH) on the surface. The hairpin DNA single strand H1 is connected to the surface of the substrate by forming an Ag-S covalent bond with the silver nanorod, and 6-mercaptohexanol (MCH) aqueous solution is added to the surface of the substrate for blocking, which can reduce non-specific adsorption.

[0010] The first reagent is a SERS probe and its buffer solution; as shown in FIG. 1B, the SERS probe is a gold nanoparticle surface modified with Probe single strand and Raman molecule 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) composite nanoparticles; preferably, the buffer solution of the SERS probe is 0.5xTBE.

[0011] The second reagent is a hairpin nucleic acid aptamer chain MUC1-apt aqueous solution; preferably, the configuration concentration of the hairpin nucleic acid aptamer chain MUC1-apt aqueous solution is 5-20 μM.

[0012] The third reagent is a CRISPR / Cas13a system and a buffer thereof, the CRISPR / Cas13a system comprising a Cas13a protein, a crRNA, and a uracil-modified hairpin recognition single-stranded HR; preferably, the configuration concentration of the Cas13a protein and the crRNA is 0.1-2 μM, and the configuration concentration of the uracil-modified hairpin recognition single-stranded HR is 1-10 μM;

[0013] The fourth reagent is a hairpin DNA single-stranded H2 aqueous solution; preferably, the configuration concentration of the hairpin DNA single-stranded H2 is 5-20 μM.

[0014] The base sequence of the hairpin DNA single-stranded H1 is shown in SEQ ID NO. 1, the base sequence of the hairpin nucleic acid aptamer strand MUC1-apt is shown in SEQ ID NO. 2, the base sequence of the uracil-modified hairpin recognition single-stranded HR is shown in SEQ ID NO. 3, the base sequence of the crRNA is shown in SEQ ID NO. 4, the base sequence of the hairpin DNA single-stranded H2 is shown in SEQ ID NO. 5, and the base sequence of the Probe single strand is shown in SEQ ID NO. 6.

[0015] In some embodiments, the gold nanoparticles have a particle size of 15-100 nm. Further preferably, the gold nanoparticles have a particle size of 15 nm.

[0016] Preferably, the preparation method of the SERS sensing chip is as follows:

[0017] (1) Prepare a silver nanorod array, and rinse with diethyl pyrocarbonate (DEPC) water multiple times before use;

[0018] (2) Co-culture a hairpin DNA single-stranded H1 aqueous solution with a concentration in the range of 0.5-2 μM with the silver nanorod array under the culture conditions of 25-37 °C and 60-80% humidity for 3-5 hours; H1 is fixed on the surface of the silver nanorod array through covalent bonding with silver through the thiol group; further preferably, the concentration of the hairpin DNA single-stranded H1 aqueous solution is 0.5 μM;

[0019] (4) After washing the substrate with a reaction buffer with a pH of 8.3, add 10 μM of a 6-mercaptohexanol (MCH) aqueous solution to the surface of the above substrate, and place it in a 37 °C constant-temperature homogenizer for 10 minutes; wherein the volume ratio of the hairpin DNA single-stranded H1 aqueous solution to the 6-mercaptohexanol (MCH) aqueous solution is 1:1; the reaction buffer comprises 10 mM Tris-HCl, 50 mM KCl, and 1.5 M MgCl2;

[0020] Preferably, the silver nanorod array is prepared by a vacuum electron beam evaporation plating device using an oblique angle deposition technique, and the silver nanorod array substrate is covered with a polydimethylsiloxane (PDMS) film having 3x10 small holes, with a single hole diameter of 4 mm and a height of 1 mm.

[0021] Preferably, the preparation step of the first reagent comprises:

[0022] 1) Mix the Probe single strand with a solution of trithioethyl phosphine (TCEP) at a molar ratio of 1:1000, and place it in a 25℃ constant temperature shaker for 4 hours;

[0023] 2) Mix 50μM Probe single strand aqueous solution with 2.3nM AuNPs aqueous solution in PBS buffer solution at pH 7.4, and the buffer solution comprises 10mM Na2HPO4, 1.76mM KH2PO4, 137mM NaCl, 2.7mM KCl, and place it in a 25℃ constant temperature shaker for 5 hours; the volume ratio of the Probe single strand aqueous solution to the AuNPs aqueous solution is 1:50;

[0024] 3) Add 10, 20, 30 and 40μL of 2M NaCl solution to the mixture obtained in step 2) every 0.5 hours, and shake for 5 hours;

[0025] 4) Add 100μM DTNB to the mixture obtained in step 3), and place it in a 25℃ constant temperature shaker for 3 hours, centrifuge at 12000rpm for 20 minutes, and wash three times with PBS buffer solution; finally, disperse the SERS probe in PBS buffer solution and store it at 4℃ for subsequent use.

[0026] In a second aspect, the application provides the use of the above-mentioned SERS biosensor in the preparation of a gastric cancer exosome detection kit, and the use steps comprise:

[0027] 1) Add SGC-7901 cell-derived exosomes to the PBS buffer solution to prepare a sample solution; wherein the concentration of exosomes in the obtained sample solution is in the range of 1.44x10 3 ~1.44x10 7 particles·mL -1 ;

[0028] 2) Mix the hairpin nucleic acid aptamer chain MUC1-apt, the Cas13a protein, the crRNA, the uracil-modified hairpin recognition single strand HR, the hairpin DNA single strand H2, the SERS probe and the different concentrations of gastric cancer exosome samples, and drop them onto the surface of the SERS sensing chip for co-culture; the culture condition is: 25-37 DEG C, 60-80% humidity environment, and standing for 60 minutes;

[0029] 3) After the sample obtained in step 2) is washed with DEPC water for multiple times, SERS testing is carried out, and the SERS spectra of different concentrations of target exosomes are obtained; taking the logarithm of the concentration of the target exosome as the abscissa and the SERS intensity of the characteristic peak of DTNB as the ordinate, the working curve of the SERS sensing is drawn, and the SERS sensing detection limit of the sensor is calculated according to the working curve.

[0030] The co-culture condition in step 2) is preferably 25-37 DEG C, 60-80% humidity environment, and standing for 60 minutes in a constant temperature mixing device at 300 rpm.

[0031] The detection principle of the present application is as follows:

[0032] As shown in FIG. 1C, the SERS biosensor provided by the application is used for gastric cancer exosome detection. When the target exosome exists, the recognition sequence in the hairpin type nucleic acid aptamer chain MUC1-apt specifically binds and recognizes the exosome membrane protein, so that the hairpin structure is opened, the free RNA fragment of the other part of the MUC1-apt is guided to hybridize with the spacer of the Cas13a / crRNA complex by the crRNA, and the conformational change of the ribonucleic acid protein complex is triggered, the two HEPN domains are close to form a catalytic site, and the RNA enzyme cutting activity of the Cas13a protein is successfully activated; the activated CRISPR / Cas13a system can cut any single-stranded RNA, including the uracil-modified hairpin type recognition single-stranded HR, which can be cut to release the ST fragment; then, the ST fragment can trigger the opening of the hairpin type DNA single strand H1 on the surface of the SERS sensing chip and form the H1 / ST complex, and the catalytic hairpin assembly (CHA) reaction occurs with the assistance of the hairpin type DNA single strand H2, a large number of H1-H2 double strands are formed on the surface of the SERS sensing chip, and the ST is released into the next cycle; at the same time, the free fragments still exist at the ends of the double strands formed by the combination of H1 and H2, which can be complementary to the modified Probe chain on the SERS probe; the sticky end of the H1-H2 double strand captures the SERS probe to the surface of the SERS sensing chip, so as to output the Raman signal significantly enhanced by the Raman molecules on the SERS probe, and realize the detection of the target exosome. One exosome in the sample can activate the CRISPR / Cas13a system through the MUC1-apt to produce a large number of ST fragments, and the ST fragments enter the catalytic hairpin assembly CHA, so as to realize high-sensitivity detection. Advantages:

[0033] Compared with the SERS biosensor applied by the previous application group, the SERS biosensor provided by the application is more convenient for detecting gastric cancer exosome samples, does not need sample pretreatment, can realize one-step incubation detection of the exosome sample, improves the detection efficiency, and can realize 10 2 particles·mL -1 Sensitivity detection in 60 minutes.

[0034] Compared with the immunodetection technology based on antibody recognition in the document Li J., Li Y., Chen S., et al. Highly sensitive exosome detection for early diagnosis of pancreatic cancer using immunoassay based on hierarchical surface-enhanced Raman scattering substrate. Small Methods, 2022, 6(6), 2200154, the SERS biosensor provided by the application is simple in preparation and application, and realizes 10 2 particles·mL -1 horizontal sensitive detection, has good specificity, uniformity and repeatability. The application is suitable for screening of high-risk population of gastric cancer, and can realize sensitive and accurate clinical diagnosis of early gastric cancer patients. BRIEF DESCRIPTION OF DRAWINGS

[0035] FIG. 1A is a schematic diagram of the construction of the SERS sensing chip according to the application;

[0036] FIG. 1B is a schematic diagram of the construction of the SERS probe according to the application;

[0037] FIG. 1C is a schematic diagram of the working principle of the SERS biosensor for gastric cancer exosome detection according to the application;

[0038] FIG. 2A is a SERS spectrum of the SERS biosensor for detecting gastric cancer exosomes in Example 1;

[0039] FIG. 2B is the SERS peak intensity corresponding to each spectrum in FIG. 2A at 1331 cm -1 Raman shift;

[0040] FIG. 3 is a SERS sensing chip surface hairpin type DNA single strand H1 incubation concentration optimization experiment in the specific embodiment;

[0041] FIG. 4 is a 6-mercaptohexanol (MCH) blocking time optimization experiment on the surface of the SERS sensing chip in the specific embodiment;

[0042] FIG. 5 is a detection time optimization experiment of the SERS biosensor for detecting gastric cancer exosomes in Test Example 1;

[0043] FIG. 6A is a SERS spectrum corresponding to the detection of different concentrations of gastric cancer exosomes by the SERS biosensor in Test Example 2;

[0044] Figure 6B is the SERS peak intensity corresponding to each spectral line in Figure 6A at a Raman shift of 1331 cm -1 Figure 6B is the SERS peak intensity corresponding to each spectral line in Figure 6A at a Raman shift of 1331 cm

[0045] Figure 7A is a SERS spectrum of the SERS biosensor for detecting GES-1, HepG2 and GES-1 / HepG2 / SGC-7901 mixed samples in Experimental Example 3.

[0046] Figure 7B is the SERS peak intensity corresponding to each spectral line in Figure 7A at a Raman shift of 1331 cm -1 Figure 7B is the SERS peak intensity corresponding to each spectral line in Figure 7A at a Raman shift of 1331 cm

[0047] Figure 8 is a uniformity characterization result of the SERS biosensor for detecting gastric cancer exosomes in Experimental Example 3.

[0048] Figure 9 is a repeatability characterization result of the SERS biosensor for detecting gastric cancer exosomes in Experimental Example 3. DETAILED DESCRIPTION

[0049] In order for those skilled in the relevant art to better understand the content of the present patent, the following detailed description of the embodiments of the present patent is given, which are implemented on the premise of the technical solutions of the present patent, and detailed implementation modes and specific operation processes are given, but the content of the present patent is not limited to the described embodiments.

[0050] The nucleic acid base sequence fragments used in the present patent are all artificially synthesized and are synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.

[0051] The specific configurations of the reagents used in the following embodiments are as follows:

[0052] The acquisition and configuration process of the gastric cancer exosome sample is as follows: the gastric cancer exosome sample is obtained by gradient ultracentrifugation from SGC-7901 cells: the intact cells in the supernatant are removed by refrigerated centrifugation (4℃, 500g, 10min), then the cell fragments and apoptotic bodies in the supernatant are removed by centrifugation (4℃, 10000g, 90min), and finally the supernatant is removed by centrifugation (4℃, 100000g, 120min) and the centrifugation product is dispersed in PBS buffer to obtain a sample solution, the concentration of the exosomes in the obtained sample solution is 1.44×10 3 ~1.44×10 7 particles·mL -1 .

[0053] The source of the second reagent, the aqueous solution of the hairpin nucleic acid aptamer chain MUC1-apt, is as follows: MUC1-apt is synthesized by Shengwo Bioengineering (Shanghai) Co., Ltd., and the base sequence of MUC1-apt is 5'-GCA GTT GAT CCT TTG GAT ACC CTG GGG ATT GGT TTT / rG / / rG / / rG / / rU / / rA / / rU / / rC / / rC / / rA / / rA / / rA / / rG / / rG / / rA / / rU / / rC / / rA / / rA / -3';

[0054] The source of the third reagent, the CRISPR / Cas13a system, is as follows: the CRISPR / Cas13a system includes a Cas13a protein, a crRNA, a hairpin recognition single-stranded HR with uracil modification, and a buffer thereof; the Cas13a protein is purchased from Guangzhou Bolais Biological Technology Co., Ltd.; the crRNA and the HR are synthesized by Shengwo Bioengineering (Shanghai) Co., Ltd.; the base sequence of the crRNA is 5'-GAC CAC CCC AAAAAU GAA GGG GAC UAAAAC UUG AUC CUU UGG AUA CCC-3'; the base sequence of the HR is 5'-TCAACA TCA C / rU / / rU / / rU / GTT AGA TCT CCA GTG ATG TTGA-3', which is a DNA sequence with uracil / rU in the middle modified; and the GTT AGA TCT CCA GTG ATG TTGA segment is an ST sequence;

[0055] The source of the fourth reagent, the aqueous solution of the hairpin DNA single strand H2, is as follows: H2 is synthesized by Shengwo Bioengineering (Shanghai) Co., Ltd.; and the base sequence of H2 is 5'-GAT CTAACA GGT ACC ATGAGT TAGATC TCCAGT TCATGG TAC CTC GAC TCAT-3';

[0056] Preparation of the first reagent, the SERS probe:

[0057] (1) The single-stranded solution of Probe is mixed with a tricarboxyethyl phosphine solution (TCEP) at a molar ratio of 1:1000, and is placed in a 25℃ constant temperature homogenizer for reaction for 4 hours; the single-stranded Probe is synthesized by Shengwo Bioengineering (Shanghai) Co., Ltd., and is composed of nucleotides and substituents with the following base sequence: 5'-SH-(CH2)6-TTT TTTATGAGT CGAG-3';

[0058] (2) 10 μL of 50 μM TCEP-treated probe single-stranded aqueous solution was mixed with 500 μL of 2.3 nM AuNPs aqueous solution in PBS buffer at pH 7.4, and placed in a constant temperature shaker at 25°C for 5 hours. The composition of the PBS buffer includes: 10 mM Na2HPO4, 1.76 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl;

[0059] (3) 10, 20, 30 and 40 μL of 2M NaCl solution were added to the mixture obtained in step 2) every 0.5 hours, and incubated for 5 hours;

[0060] (4) 50 μL of 100 μM DTNB was added to the mixture obtained in step 3), and incubated for 3 hours in a constant temperature shaker at 25°C, centrifuged (12000 rpm, 20 minutes) and washed three times with PBS buffer. Finally, the SERS probe was redispersed in 100 μL of PBS buffer 0.5xTBE and stored at 4°C for subsequent use.

[0061] Regarding the preparation of the SERS sensing chip:

[0062] (1) Silver nanorod arrays were prepared, and the specific preparation is described in the literature Song C., Chen J., Zhao Y., et al. Gold-modified silver nanorod arrays for SERS-based immunoassays with improved sensitivity. Journal of Materials Chemistry B, 2014, 2(43): 7488-7494. The silver nanorod array substrate was prepared by vacuum electron beam evaporation coating technology, and the silver nanorod array substrate was covered with a polydimethylsiloxane (PDMS) film with 3x10 holes, with a single hole diameter of 4 mm and a height of 1 mm. Before use, it was rinsed several times with diethyl pyrocarbonate (DEPC) water;

[0063] (2) 20 μL of 500 nM hairpin DNA single-stranded H1 aqueous solution was co-cultured with silver nanorod arrays, and H1 was covalently bonded to silver through a thiol group to be fixed on the surface of the silver nanorod array; Incubation conditions: 37°C, 60% humidity environment for 3 hours; H1 is synthesized by GenScript Biotech (Shanghai) Co., Ltd., and is composed of nucleotides with the following base sequence and substituent group: 5'-TCAACATCACTGGAGATCTAACCTCATGGTACCTGTTAGATCTCGTTTTTTT-SH-(CH2)6-3'.

[0064] (3) After washing the substrate with reaction buffer (10 mM Tris-HCl, 50 mM KC1, 1.5 M MgCl2, pH 8.3), 20 μL of 10 μM 6-mercaptohexanol (MCH) aqueous solution was added to the surface of the above substrate, and placed in a constant temperature shaker at 37°C for 10 minutes.

[0065] Optimization of incubation concentration of hairpin type DNA single strand H1 on the surface of SERS sensing chip

[0066] In the preparation process of SERS sensing chip, the silver nanorod array substrate was co-cultured with 20 μL of 10 nM, 50 nM, 100 nM, 200 nM, 300 nM, 500 nM, 1 μM, 3 μM and 5 μM hairpin type DNA single strand H1 solution respectively, and placed in a constant temperature shaker at 25°C for 3 hours. Then the substrate was washed with reaction buffer and DEPC water multiple times to obtain the SERS sensing chip for detecting gastric cancer exosomes. Then, the hairpin nucleic acid aptamer chain MUC1-apt, Cas13a protein, crRNA, uracil modified hairpin recognition single strand HR, hairpin DNA single strand H2, SERS probe and 10 6 particles·mL -1 Gastric cancer exosome sample mixing; the above mixed solution was added to the surface of the SERS sensing chip, and placed in a constant temperature shaker at 25°C and 300 rpm for 60 minutes. Then the small holes were washed with reaction buffer and ultrapure water and SERS detection was performed to obtain the SERS spectrum, as shown in Figure 3. It can be seen that when the concentration of H1 is less than 500 nM, the SERS intensity monotonically increases; when the concentration of H1 is greater than 500 nM, the SERS intensity reaches a saturated state, indicating that the optimal incubation concentration of H1 is 500 nM.

[0067] Optimization of blocking time of 6-mercaptohexanol (MCH) on the surface of SERS sensing chip

[0068] In the preparation process of SERS sensing chip, the silver nanorod array substrate was co-cultured with 20 μL of 500 nM hairpin type DNA single strand H1 for 3 hours. After washing the substrate with reaction buffer, 20 μL of 10 μM 6-mercaptohexanol (MCH) aqueous solution was added to the surface of the above substrate and placed in a constant temperature shaker at 37°C for 0, 5, 10, 20, 40 and 60 minutes respectively. The substrate was washed with reaction buffer and DEPC water multiple times to obtain the SERS sensing chip, and then SERS detection was performed on the blank sample and 10 6 particles·mL -1 Exosome, the ratio of SERS intensity of target exosome to blank sample (I T / I blank), see Fig. 4. It can be seen that the ratio of SERS intensity is the largest when the MCH blocking time is 10 minutes T / I blank ), indicating that the optimal blocking time of MCH aqueous solution is 10 min.

[0069] Example 1 SERS biosensor for detection of gastric cancer exosomes

[0070] 2 μL of 10 μM MUC1-apt, 2 μL of 0.25 μM Cas13a protein, 2 μL of 0.25 μM crRNA, 2 μL of 2.5 μM HR, 2 μL of 10 μM H2, 2 μL of gastric cancer exosomes with different concentrations (10 3 ~ 10 7 particles·mL -1 ) and 5 μL of SERS probe in 20 μL of reaction buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 M MgCl2, pH 8.3).

[0071] The mixed solution without the addition of gastric cancer exosomes was used as a blank sample. The mixed solution containing different concentrations of gastric cancer exosomes and the blank sample were respectively dropped onto the surface of the SERS sensing chip, incubated in a constant temperature shaker at 37°C and 300 rpm for 60 minutes, and then washed with the above-mentioned reaction buffer and DEPC water in sequence. After natural air drying, SERS testing was performed on the SERS sensing chip to obtain the SERS spectra and characteristic signal intensity values of different concentrations of gastric cancer exosomes and the blank sample.

[0072] Based on 10 6 particles·mL -1 The complete solution of exosomes was used as a sample to prepare an incomplete mixed solution missing a reagent (such as gastric cancer exosomes, crRNA or HR) as a control, as well as a complete mixed solution containing all reagents and a complete mixed solution of different concentrations of gastric cancer exosomes (10 3 ~ 10 7 particles·mL -1 ).

[0073] Referring to Figs. 2A and 2B, the SERS intensity measured by the incomplete detection system is consistent with the signal intensity of the blank sample. In contrast, the complete mixed solution containing all reagents can effectively perform a cascade signal amplification reaction, obtain a significantly enhanced SERS signal, and the SERS signal gradually increases with the increase of the concentration of the target gastric cancer exosomes.

[0074] Test Example 1: Detection time optimization test of SERS biosensor for detection of gastric cancer exosomes

[0075] The SERS sensing chip prepared above was added with 2 μL of 10 μM MUC1-apt, 2 μL of 0.25 μM Cas13a protein, 2 μL of 0.25 μM crRNA, 2 μL of 2.5 μM HR, 2 μL of 10 μM H2, 2 μL of 10 6 particles·mL -1 The gastric cancer exosomes and 5 μL of SERS probes were placed in 20 μL of reaction buffer and incubated in a constant temperature shaker at 37 °C and 300 rpm for 10, 20, 30, 40, 60, 80, 100 and 120 minutes, respectively. Then the wells were washed with reaction buffer and DEPC water and subjected to SERS detection. The SERS spectra were obtained, as shown in Fig. 5. It can be seen that by testing the SERS signals of the detection chips incubated for different time, it was observed that the SERS intensity gradually increased from 0 to 60 minutes, and reached saturation state at 60 minutes of incubation, indicating that the optimal detection time of the SERS biosensor for detecting gastric cancer exosomes was 60 minutes.

[0076] Test Example 2: Working curve and detection limit of SERS biosensor for detecting gastric cancer exosomes

[0077] According to the SERS sensing chip optimized above and the detection time optimized in Test Example 1, the working curve and detection limit of the SERS biosensor for detecting gastric cancer exosomes were determined according to the detection process in Example 1: The gastric cancer exosomes were diluted with PBS buffer to different concentrations of 10 3 ~10 7 particles·mL -1 The SERS sensing chip prepared above was added with 2 μL of 10 μM MUC1-apt, 2 μL of 0.25 μM Cas13a protein, 2 μL of 0.25 μM crRNA, 2 μL of 2.5 μM HR, 2 μL of 10 μM H2, 2 μL of 10 3 ~10 7 particles·mL -1The gastric cancer exosome and 5 μL SERS probe are placed in 20 μL reaction buffer at 37 °C 300 rpm constant temperature homogenizer for 60 minutes; then the reaction buffer and DEPC water are used to clean the small hole and perform SERS detection, and the SERS spectrum and its characteristic signal intensity value are obtained. The logarithm of the target exosome concentration is taken as the abscissa, and the SERS characteristic peak intensity value is taken as the ordinate, to draw the working curve of the SERS biosensor for detecting gastric cancer exosomes, and the detection limit of the SERS sensor for detecting gastric cancer exosomes is calculated according to the working curve. As can be seen, Fig. 6A is the SERS spectrum obtained by detecting gastric cancer exosomes of different concentrations, and the SERS intensity monotonously increases with the increase of the target exosome concentration; Fig. 6B is the SERS peak intensity of each spectrum in Fig. 6A at 1331 cm -1 Raman shift, the SERS peak intensity corresponding to the SERS peak intensity of each spectrum in Fig. 6A at 1331 cm 1331 = 1499.52 × LgC exo -1309.76 (R 2 = 0.983), and the detection limit LOD is as low as 1.26 x 10 2 particles·mL -1 .

[0078] Test Example 3: Performance characterization of SERS biosensor for detecting gastric cancer exosomes

[0079] 1) Specific characterization of SERS biosensor for detecting exosomes of different cell sources

[0080] Prepare 10 6 particles·mL -1 of exosomes from human normal gastric mucosa cells GES-1, human hepatocellular carcinoma cells HepG2, and GES-1 / HepG2 / SGC-7901 mixed samples, respectively, 2 μL of 10 μM MUC1-apt, 2 μL of 0.25 μM Cas13a protein, 2 μL of 0.25 μM crRNA, 2 μL of 2.5 μM HR, 2 μL of 10 μM H2, 2 μL of exosomes of different cell sources, and 5 μL of SERS probe are placed in 20 μL of reaction buffer at 37 °C 300 rpm constant temperature homogenizer for 60 minutes, and then the reaction buffer and DEPC water are used to clean the small hole and perform SERS detection. The SERS spectrum and its characteristic signal intensity value for detecting exosomes of different sources are obtained. As can be seen, Fig. 7A is the SERS spectrum of the SERS biosensor for detecting GES-1, HepG2, and GES-1 / HepG2 / SGC-7901 mixed samples; Fig. 7B is the SERS peak intensity of each spectrum in Fig. 7A at 1331 cm -1SERS peak intensity corresponding to the Raman shift. The prepared SERS biosensor can well distinguish the gastric cancer-derived exosomes from other cell-derived exosomes, which indicates that the SERS biosensor has good specificity.

[0081] 2) Uniformity characterization of SERS biosensor for detecting gastric cancer exosomes

[0082] In the above-prepared SERS sensing chip surface, 2 μL of 10 μM MUC1-apt, 2 μL of 0.25 μM Cas13a protein, 2 μL of 0.25 μM crRNA, 2 μL of 2.5 μM HR, 2 μL of 10 μM H2, 2 μL of different concentrations of gastric cancer exosomes (10 3 , 10 5 , and 10 7 particles·mL -1 ) and 5 μL of SERS probe were added to 20 μL of reaction buffer, and were placed in a 37°C 300 rpm constant temperature shaker for 60 minutes. Subsequently, the wells were washed with reaction buffer and DEPC water, and the SERS signals of 20 random points on the SERS sensing chip were recorded to study the uniformity of the silver nanorod array substrate modified with hairpin-shaped single-stranded DNA H1 on the surface. Figure 8 is a record of the SERS signals of 20 random points on the SERS sensing chip for detecting 10 3 , 10 5 , and 10 7 particles·mL -1 gastric cancer exosomes, showing small signal fluctuations, and the relative standard deviation (RSD) of SERS intensity is less than 8.04%, indicating that the proposed SERS biosensor for detecting gastric cancer exosomes has good uniformity.

[0083] 3) Reproducibility characterization of SERS biosensor for detecting gastric cancer exosomes

[0084] In the above-prepared 6 groups of SERS sensing chips of Example 1, 2 μL of 10 μM MUC1-apt, 2 μL of 0.25 μM Cas13a protein, 2 μL of 0.25 μM crRNA, 2 μL of 2.5 μM HR, 2 μL of 10 μM H2, 2 μL of 10 7 particles·mL -1 gastric cancer exosomes and 5 μL of SERS probe were added to 20 μL of reaction buffer, and were placed in a 37°C 300 rpm constant temperature shaker for 60 minutes. Subsequently, the wells were washed with reaction buffer and DEPC water, and the average SERS signal intensity values were obtained by recording the SERS spectra of 10 random points on different detection chips. Figure 9 is a record of the SERS signals of 107 particles mL -1 The SERS intensity of gastric cancer exosomes shows a small relative standard deviation (RSD = 5.45%), indicating that the proposed SERS biosensor for detecting gastric cancer exosomes has good reproducibility.

Claims

1. A SERS biosensor based on CRISPR / Casl3a system, characterized in that, The SERS biosensor comprises a SERS sensing chip, a first reagent, a second reagent, a third reagent and a fourth reagent. The SERS sensing chip is a silver nanorod array substrate which is modified with a hairpin type DNA single strand H1 and a blocking molecule 6-mercaptohexanol MCH on the surface. The first reagent is a SERS probe and a buffer solution thereof; the SERS probe is a gold nanoparticle surface modified with a Probe single strand and a Raman molecule DTNB composite nanoparticle. The second reagent is a hairpin type nucleic acid aptamer chain MUC1-apt aqueous solution. The third reagent is a CRISPR / Cas13a system and a buffer solution thereof, and the CRISPR / Cas13a system comprises a Cas13a protein, a crRNA and a hairpin type recognition single strand HR modified with uracil. The fourth reagent is a hairpin type DNA single strand H2 aqueous solution. The base sequence of the hairpin type DNA single strand H1 is shown as SEQ ID NO. 1, the base sequence of the hairpin type nucleic acid aptamer chain MUC1-apt is shown as SEQ ID NO. 2, the base sequence of the hairpin type recognition single strand HR modified with uracil is shown as SEQ ID NO. 3, the base sequence of the crRNA is shown as SEQ ID NO. 4, the base sequence of the hairpin type DNA single strand H2 is shown as SEQ ID NO. 5, and the base sequence of the Probe single strand is shown as SEQ ID NO.

6.

2. The SERS biosensor based on CRISPR / Casl3a system of claim 1, wherein, The configuration concentration of the hairpin type nucleic acid aptamer chain MUC1-apt aqueous solution is 5-20 μM.

3. The SERS biosensor based on CRISPR / Casl3a system of claim 1, wherein, The configuration concentrations of the Cas13a protein and the crRNA are both 0.1-2 μM, and the configuration concentration of the hairpin type recognition single strand HR modified with uracil is 1-10 μM.

4. The SERS biosensor based on CRISPR / Casl3a system of claim 1, wherein, The configuration concentration of the hairpin type DNA single strand H2 is 5-20 μM.

5. The SERS biosensor based on CRISPR / Casl3a system of claim 1, wherein, The particle size of the gold nanoparticles is 15-100 nm.

6. The SERS biosensor based on CRISPR / Casl3a system of claim 1, wherein, The preparation method of the SERS sensing chip is as follows: (1) preparing a silver nanorod array, and washing the silver nanorod array with diethyl pyrocarbonate DEPC water multiple times before use; (2) co-culturing a hairpin type DNA single strand H1 aqueous solution with a concentration in the range of 0.5-2 μM with the silver nanorod array, and placing the mixture in a 25-37℃, 60-80% humidity environment for 3-5 hours; H1 is fixed on the surface of the silver nanorod array by forming a covalent bond with silver through a hydrophobic group; (3) after washing the substrate with a reaction buffer solution with a pH of 8.3, adding 10 μM of an MCH aqueous solution to the surface of the substrate, and placing the substrate in a 37℃ constant temperature homogenizer for 10 minutes; the volume ratio of the hairpin type DNA single strand H1 aqueous solution to the MCH aqueous solution is 1:1; the reaction buffer solution comprises 10 mM Tris-HCl, 50 mM KCl and 1.5 M MgCl2.

7. The SERS biosensor based on CRISPR / Casl3a system according to claim 1, wherein, The silver nanorod array is prepared by using an inclined angle deposition technology through a vacuum electron beam evaporation coating equipment; the silver nanorod array substrate is covered with a polydimethylsiloxane (PDMS) film with 3×10 holes, and the aperture of each hole is 4 mm and the height is 1 mm.

8. The SERS biosensor based on CRISPR / Casl3a system of claim 1, wherein, The preparation steps of the first reagent include: 1) Mix the Probe single strand with a solution of trithioethyl phosphine (TCEP) at a molar ratio of 1:1000, and place it in a constant temperature shaker at 25°C for 4 hours; 2) Mix 50 μM of the Probe single strand aqueous solution with 2.3 nM of the AuNPs aqueous solution in a PBS buffer at pH 7.4, which includes 10 mM Na2HPO4, 1.76 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, and place it in a constant temperature shaker at 25°C for 5 hours of oscillation incubation; the volume ratio of the Probe single strand aqueous solution to the AuNPs aqueous solution is 1:50; 3) Add 10, 20, 30, and 40 μL of 2M NaCl solution to the mixture obtained in step 2) every 0.5 hours, and incubate for 5 hours of oscillation; 4) Add 100 μM of DTNB to the mixture obtained in step 3), and place it in a constant temperature shaker at 25°C for 3 hours of oscillation incubation, centrifuge at a speed of 12000 rpm for 20 minutes, and wash it with PBS buffer three times; finally, re-disperse the SERS probe in PBS buffer, and store it at 4°C for subsequent use.

9. The use of the SERS biosensor according to any one of claims 1-8 in the preparation of a gastric cancer exosome detection kit, wherein the use steps include: 1) adding SGC-7901 cell-derived exosomes in a PBS buffer for preparing a sample solution; wherein the concentration of the exosomes in the obtained sample solution ranges from 1.44 x 10 3 ~1.44 x 10 7 particles·mL -1 ; 2) Mix the hairpin aptamer chain MUC1-apt, the Cas13a protein, the crRNA, the uracil-modified hairpin recognition single strand HR, the hairpin DNA single strand H2, the SERS probe, and different concentrations of gastric cancer exosome samples, and drop them onto the surface of the SERS sensing chip for co-culture; the culture conditions are 25-37°C and 60-80% humidity for 60 minutes; 3) Wash the sample obtained in step 2) with DEPC water multiple times, and then perform SERS testing to obtain the SERS spectra of different concentrations of target exosomes; take the logarithm of the target exosome concentration as the abscissa, and take the SERS intensity of the DTNB characteristic peak as the ordinate, respectively, to draw the working curve of the SERS sensor; and calculate the SERS sensor detection limit of the sensor according to the working curve; The co-culture conditions in step 2) are 25-37°C and 60-80% humidity in a constant temperature shaker at 300 rpm for 60 minutes.

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