A SERS biosensor and its preparation method and application
By using MoS2-Fe3O4-AuNSs nanocomposite as active substrate, combined with thiol-modified probe DNA and Raman microscopy system, efficient enrichment and multivariate detection of exosomes are achieved, solving the problem of expensive and difficult detection of multiple exosome subpopulations in existing biosensors, and improving the reliability and sensitivity of the detection.
Patent Information
- Application Number
- CN202210505101.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-05-10
- Publication Date
- 2025-08-29
- Estimated Expiration
- 2042-05-10
AI Technical Summary
Existing biosensors need to rely on expensive detection instruments and it is difficult to achieve simultaneous detection of multiple subpopulations of exosomes.
MoS2-Fe3O4-AuNSs nanocomposite is used as the active substrate, and signal probes are assembled through thiol-modified probe DNA to achieve high specific binding and magnetic separation of exosomes, and SERS signals are analyzed in combination with Raman microscopy.
The exosome detection steps are simplified, the dependence on expensive instruments is reduced, the reliability and sensitivity of the detection results are improved, and the simultaneous detection of multiple exosome subpopulations can be achieved.
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Figure CN114813701B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a SERS biosensor and a preparation method and application thereof, belonging to the field of biosensors. Background Art
[0002] Cancer has become a major threat to human health in today's society, with a persistently high mortality rate. Currently, surgery combined with chemotherapy and radiotherapy can effectively improve the five-year survival rate of cancer patients, but the five-year survival rate for patients with advanced cancer remains below 10%. Early-stage cancer is often asymptomatic, leading to delayed diagnosis and missed opportunities for radical surgery.
[0003] Currently, traditional cancer detection methods (such as X-rays, endoscopy, computed tomography, imaging examinations, tissue examinations, etc.) are not suitable for large-scale population screening or repeated screening due to their high prices, reliance on large instruments, and high damage to the human body. Therefore, there is an urgent need to develop more effective and less invasive screening methods to guide the early diagnosis of malignant tumors, achieve early detection, early diagnosis, and early treatment, which can effectively prolong the 5-year survival rate of cancer patients.
[0004] Cancer-related biomarkers are "molecular markers" that can provide accurate information about the occurrence and underlying mechanisms of cancer, such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), exosomes, and circulating tumor RNA (ctRNA). Therefore, highly sensitive detection of cancer-related biomarkers is one of the most promising strategies for early diagnosis of cancer.
[0005] Exosomes play a crucial role in the communication between cells and surrounding tumor cells, and are closely linked to angiogenesis, tumor development, and metastasis. They also participate in pathophysiological processes and play a fundamental role in tumor biology. Numerous experiments have demonstrated a close connection between exosomes and intercellular communication, tumor cell proliferation, and metastasis. Exosomes contain a variety of active substances from parent cells, such as proteins, nucleic acids, glycoconjugates, and lipids. Therefore, they are widely recognized as a key biomarker for liquid biopsies and have attracted significant attention from researchers. However, current methods for detecting exosomes are often time-consuming, labor-intensive, and rely on expensive instrumentation, making it difficult to simultaneously detect multiple exosome subpopulations.
[0006] In view of this, it is indeed necessary to propose a SERS biosensor and its preparation method and application to solve the above problems. Summary of the Invention
[0007] The purpose of the present invention is to provide a SERS biosensor and its preparation method and application, so as to solve the problems that existing biosensors need to rely on expensive detection instruments and are difficult to achieve simultaneous detection.
[0008] To achieve the above object, the present invention provides a method for preparing a SERS biosensor, comprising the following steps:
[0009] S1. In situ growth of star-shaped gold nanoparticles on the surface of molybdenum disulfide nanosheets to prepare MoS2-AuNSs nanocomposite materials; magnetic nanoparticles are assembled on the surface of the MoS2-AuNSs nanocomposite materials to form MoS2-Fe3O4-AuNSs composite materials, which serve as the active substrate of the SERS biosensor;
[0010] S2, thiol-modified probe DNA was assembled on the surface of MoS2-Fe3O4-AuNSs composite material through Au-S bond to obtain SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform;
[0011] S3. Immobilize the signal probe on the surface of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform to identify and capture exosomes.
[0012] As a further improvement of the present invention, S1 comprises the following steps:
[0013] S11, stirring and mixing the molybdenum disulfide nanosheet solution and the polyvinylpyrrolidone aqueous solution, and then adding a chloroauric acid precursor to prepare a MoS2-AuNPs nanocomposite material solution;
[0014] S12, adding chloroauric acid aqueous solution to the hexadecyltrimethylammonium bromide aqueous solution, then adjusting the pH value of the reaction solution; adding the MoS2-AuNPs nanocomposite material solution to react; finally, adding ascorbic acid and silver nitrate solution simultaneously to obtain MoS 2- AuNSs nanocomposite solution;
[0015] S13. Add magnetic nanoparticles to the MoS2-AuNSs nanocomposite solution and stir to mix, centrifuge, wash with anhydrous ethanol and ultrapure water, and disperse the washed product in ultrapure water to assemble the magnetic nanoparticles on the surface of the MoS2-AuNSs nanocomposite to obtain a MoS2-Fe3O4-AuNSs nanocomposite solution.
[0016] As a further improvement of the present invention, S2 is specifically: adding thiol-modified probe DNA activated by TCEP to the MoS2-Fe3O4-AuNSs nanomaterial solution and mixing them evenly, undergoing magnetic separation and phosphate buffer washing, and dissolving the washed product in phosphate buffer to obtain the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform.
[0017] As a further improvement of the present invention, the signal probe is bound to the surface of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform through base complementarity. The free signal probe in the solution is removed by magnetic separation and washed with phosphate buffer. The washed product is dissolved in phosphate buffer to obtain the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with the signal probe.
[0018] As a further improvement of the present invention, the signal probe is a nucleic acid aptamer modified with a Raman signal molecule.
[0019] To achieve the above object, the present invention further provides a SERS biosensor, which is manufactured by the above-mentioned method for preparing the SERS biosensor.
[0020] To achieve the above objectives, the present invention provides an application of the aforementioned SERS biosensor in detecting exosomes.
[0021] As a further improvement of the present invention, the following steps are included:
[0022] Exosomes were added to the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with a signal probe. The exosomes bound to the signal probe with high specificity to form a signal probe-exosome complex, which then separated from the Raman detection platform and was then removed.
[0023] The SERS signal intensity in the substrate after removal of the signal probe-exosome complex was analyzed using a Raman microscopy system.
[0024] As a further improvement of the present invention, density gradient centrifugation is performed on the exosomes to obtain high-purity exosomes.
[0025] As a further improvement of the present invention, the product from which the signal probe-exosome complex is removed is dropped onto the surface of a silicon wafer, and a Raman microscopy system is used to detect the SERS signal of the product.
[0026] The beneficial effects of the present invention are as follows: a SERS biosensor of the present invention, by using MoS2-Fe3O4-AuNSs nanomaterials as an active substrate, can amplify Raman signals, and can effectively enrich exosomes and magnetically separate the products, thereby simplifying the steps of exosome detection, reducing the dependence on expensive instruments, and improving the reliability of the detection results; at the same time, by using different types of signal probes, multiplexed detection of exosome subpopulations from different cell sources can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1Schematic diagram of the analytical principle of the SERS biosensor for multivariate detection of exosomes in the present invention.
[0028] Figure 2 This is a comparison of the Raman signal intensities of different nanomaterials used as active substrates.
[0029] Figure 3 These are Raman signal spectra of the SERS biosensor of the present invention detecting exosomes at different concentrations.
[0030] Figure 4 yes Figure 3 Medium Raman shift 1502 cm -1 The relationship between Raman signal intensity and exosome concentration.
[0031] Figure 5 This is the multiplex detection of exosomes from different cells by the SERS biosensor of the present invention. DETAILED DESCRIPTION
[0032] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.
[0033] See also Figure 1 As shown, the present invention discloses a SERS biosensor and its preparation method and application. Figure 1 As shown in Figure A, first, molybdenum disulfide (MoS2) nanosheets were prepared by chemical synthesis, and then star-shaped gold nanoparticles (AuNSs) and magnetic nanoparticles (Fe3O4) were functionalized on the surface of the MoS2 material as a SERS active substrate. Then, the probe DNA was assembled on the surface of the MoS2-Fe3O4-AuNSs nanocomposite material through Au-S bonds. Finally, the nucleic acid aptamer labeled with a Raman signal molecule (ROX) was fixed on the surface of the SERS active substrate through DNA base complementation as a signal probe, that is, in this embodiment, the signal probe is a nucleic acid aptamer labeled with a Raman signal molecule (ROX). As shown in Figure A, first, molybdenum disulfide (MoS2) nanosheets were prepared by chemical synthesis, and then star-shaped gold nanoparticles (AuNSs) and magnetic nanoparticles (Fe3O4) were functionalized on the surface of the MoS2 material as a SERS active substrate. Figure 1 As shown in Figure B, when exosomes are absent, a large number of SERS signal probes are immobilized on the SERS-active substrate, resulting in a strong SERS signal. When exosomes are added, the signal probes specifically bind to the exosome surface proteins, and the resulting signal probe-exosome complex detaches from the SERS-active substrate surface, resulting in a decrease in the SERS signal and enabling highly sensitive detection of exosomes.
[0034] In this example, the SERS signal molecule used was 6-carboxy-X-rhodamine (ROX), and exosomes secreted by human gastric cancer cells (SGC-7901) were used as the exosomes to be detected. The method of the present invention was used to perform an exosome detection experiment. Of course, in other examples, the types of SERS signal molecules and exosomes can be selected based on actual conditions and are not limited here.
[0035] Specifically, the preparation method of the SERS biosensor of the present invention comprises the following steps:
[0036] S1. In situ growth of star-shaped gold nanoparticles on the surface of molybdenum disulfide nanosheets to prepare MoS2-AuNSs nanocomposite materials; assembly of magnetic nanoparticles on the surface of MoS2-AuNSs nanocomposite materials to form MoS2-Fe3O4-AuNSs composite materials, which serve as the active substrate of SERS biosensors.
[0037] S11. Prepare MoS2-AuNPs solution.
[0038] Star-shaped gold nanoparticles (AuNPs) were in situ grown on the surface of molybdenum disulfide nanosheets (MoS2) to prepare MoS2-AuNPs nanocomposite materials.
[0039] Specifically, a molybdenum disulfide nanosheet solution with a mass concentration of 0.025 mg / mL and a polyvinylpyrrolidone aqueous solution with a mass fraction of 5% were stirred and mixed, and a chloroauric acid precursor with a molar concentration of 10 mM was added to prepare a molybdenum disulfide nanocomposite material (MoS2-AuNPs) solution functionalized with star-shaped gold nanoparticles.
[0040] Preparation of S12, MoS2-AuNSs nanocomposite solutions.
[0041] A 10 mM aqueous chloroauric acid solution was added to the hexadecyltrimethylammonium bromide solution. A 1 M hydrochloric acid solution was then added to adjust the pH of the reaction solution to 5.5. The MoS2-AuNPs solution obtained in S11 was then added and allowed to react for 2 minutes. Finally, 100 mM ascorbic acid and 1 mM silver nitrate solution were added simultaneously and allowed to react for 2 minutes to obtain a MoS2-AuNSs nanocomposite solution.
[0042] Preparation of S13, MoS2-Fe3O4-AuNSs nanocomposite solution.
[0043] Magnetic nanoparticles (Fe3O4) are assembled on the surface of MoS2-AuNSs nanocomposite through electrostatic interaction to form MoS2-Fe3O4-AuNSs nanocomposite, which serves as the active substrate of SERS biosensor. The specific steps are as follows:
[0044] The MoS2-AuNSs nanocomposite solution was ultrasonically dispersed in ultrapure water, and magnetic nanoparticles (Fe3O4) were added. The mixture was stirred at 60°C for 2 hours to mix thoroughly. The mixture was subjected to gradient centrifugation to obtain a product. The product was washed with anhydrous ethanol and then ultrapure water, and then dispersed in ultrapure water to obtain a MoS2-Fe3O4-AuNSs nanocomposite solution, which served as the active substrate for the SERS biosensor.
[0045] S2, thiol-modified probe DNA was assembled on the surface of MoS2-Fe3O4-AuNSs composite material through Au-S bond to obtain SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform.
[0046] To a 900 μL, 10 mg / mL MoS2-Fe3O4-AuNSs nanomaterial solution, 100 μL of thiol-modified probe DNA (SH-DNA) activated with TCEP (1 mM molar concentration of TCEP) was added. The probe DNA was then mixed thoroughly and allowed to react at room temperature for 14 hours. After magnetic separation, the product was washed three times with 1 mL of phosphate buffer to remove unreacted probe DNA. The washed product was then dissolved in 900 μL of phosphate buffer (10 mM, pH 7.0) to obtain the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform.
[0047] S3. The nucleic acid aptamer modified with the Raman signal molecule ROX (ROX-Apt) was immobilized on the surface of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform to identify and capture exosomes.
[0048] Specifically, the Raman signal molecule ROX-modified nucleic acid aptamer (ROX-Apt) is bound to the surface of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform through base complementarity, and then the Raman signal molecule ROX-modified nucleic acid aptamer free in the solution is removed by magnetic separation to obtain the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with a signal probe.
[0049] A nucleic acid aptamer modified with the Raman signal molecule ROX was added to the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform, mixed thoroughly, and incubated at room temperature for 14 hours. The reaction solution was magnetically separated, the supernatant removed, and the product washed three times with 1 mL of phosphate buffer to remove unreacted signal probe. The washed product was then redissolved in phosphate buffer to obtain the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with the signal probe. The phosphate buffer had a molar concentration of 10 mM and a pH of 7.0.
[0050] Target exosomes were added to the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with a signal probe. The exosomes bound to the signal probe with high specificity, thereby detaching from the Raman detection platform. The signal probe-exosome complex was then removed by magnetic separation.
[0051] Exosome culture was performed by culturing human gastric cancer cell line SGC-7901 cells in a medium containing 10% exosome-free fetal bovine serum in a cell culture incubator at 37°C and 5% CO2 for 48 hours. The supernatant was collected in a 50mL centrifuge tube and centrifuged at 500g for 10 minutes to separate intact cells. The supernatant was then centrifuged at 10,000g for 90 minutes to separate cell debris and apoptotic bodies. Finally, the supernatant was centrifuged at 100,000g for 120 minutes, and the supernatant was removed to obtain exosomes. The exosomes were then dispersed in phosphate buffer (PBS) with a molar concentration of 10mM and a pH of 7.0.
[0052] Among them, the separation of exosomes uses density gradient centrifugation, which can effectively separate exosome subpopulations to obtain exosomes with higher purity.
[0053] The detection of target exosomes is specifically as follows: the signal probe is fixed on the surface of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform through base complementary pairing, which is used to identify and capture exosomes.
[0054] All the following solutions were prepared with ultrapure water sterilized by high temperature and high pressure. All experimental consumables were sterilized by high temperature and high pressure, and all operations were performed in a biological safety cabinet.
[0055] The concentration was 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 65 μL of each of the five target exosomes (particles / μL) was added to a corresponding solution of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with a signal probe. The reaction was incubated at 37°C in a thermoshaker in the dark for 2 hours with gentle shaking. The signal probe-exosome complexes were then removed from the supernatant by magnetic separation. The volume of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with a signal probe in each set was 45 μL.
[0056] The substrate obtained by magnetic separation was dropped onto the surface of a silicon wafer and dried, and then the SERS signal intensity in the substrate was analyzed using a Raman microscope system.
[0057] The surface treatment of the silicon wafer was as follows: ultrasonic cleaning of the silicon wafer was performed with ethanol and ultrapure water for 10 min in sequence, and then drying with nitrogen.
[0058] The product after removing the signal probe-exosome complex was dropped onto the surface of a silicon wafer, and the SERS signal of the product was detected using a Raman microscope system. Then, the working curves of the exosome sensor were drawn for the target exosome concentration and the corresponding SERS signal intensity, and the linear detection range and detection limit of exosomes were calculated to construct the detection sensitivity of the SERS biosensor of the present invention to the target exosomes. After testing, the concentration detection range of the SERS biosensor of the present invention was 1×10 1 to 1×10 6 particles / μl, and the detection limit was 3 particles / μl.
[0059] See also Figure 2 As shown in the figure, the Raman signal intensity comparison curves on the SERS active substrate composed of different nanomaterials are shown, with 4-mercaptobenzoic acid (4-MBA) as the SERS signal indicator, specifically: 4-MBA (0.1M), 4-MBA (10 -3 M)-MoS2、4-MBA(10 -3 M)-AuNPs, 4-MBA(10 -3 M)-MoS2-Fe3O4-AuNPs, 4-MBA(10 -3 M)-MoS2-Fe3O4-AuNSs Raman signal intensity comparison curve, among which 4-MBA(10 -3 M)-MoS2、4-MBA(10 -3 M)-AuNPs, 4-MBA(10 -3 M)-MoS2-Fe3O4-AuNPs and 4-MBA(10 -3M)-MoS2-Fe3O4-AuNSs refers to the use of MoS2, AuNPs, MoS2-Fe3O4-AuNPs, and MoS2-Fe3O4-AuNSs nanomaterials as active substrates, respectively. -3 M's 4-MBA was used as a SERS signal indicator.
[0060] Among them, 0.1M 4-MBA on a clean silicon wafer is at 1074cm -1 and 1585cm -1 The SERS signals are weak. -3 The SERS intensity of 0.1M 4-MBA on the MoS2 active substrate is higher than that on the silicon wafer, indicating that the active substrate with MoS2 nanosheets has a Raman signal enhancement effect. Further experiments show that 10 -3 The SERS signal intensity of the SERS active substrates prepared by M's 4-MBA with AuNPs, MoS2-Fe3O4-AuNPs, and MoS2-Fe3O4-AuNSs was significantly higher than that of the active substrate prepared by M's 4-MBA with MoS2 nanosheets. Among them, the active substrate of MoS2-Fe3O4-AuNSs nanomaterials showed excellent signal enhancement ability.
[0061] See also Figure 3 As shown in FIG, as the concentration of exosomes increases, the signal intensity of the SERS biosensor of the present invention decreases, thereby achieving highly sensitive detection of exosomes.
[0062] See also Figure 4 As shown, the Raman shift is 1502cm -1 The relationship between the Raman signal intensity and the exosome concentration can be seen from the figure: the detection range of the biosensor of the present invention for exosomes is 1×10 1 -1×10 6 particles / μL, the linear equation is y = -595.37x + 4095.79, and the detection limit of the biosensor is estimated to be 3 particles / μL. Specifically, D = 3N / S, where D is the detection limit and N is the exosome concentration of 1×10 1 The standard deviation of particles / μl, S is the slope.
[0063] The detection comparison between the SERS biosensor based on MoS2-Fe3O4-AuNSs nanomaterials of the present invention and the existing detection method is as follows:
[0064]
[0065] It can be seen from the table that the detection range of the SERS biosensor based on MoS2-Fe3O4-AuNSs nanomaterials of the present invention is 1×10 1 -1×10 6 particles / μl, and the detection limit is 3 particles / μl. Compared with the prior art, the SERS biosensor of the present invention has a wider detection range and a lower detection limit.
[0066] The MoS2-Fe3O4-AuNSs nanomaterial-based SERS biosensor of the present invention can not only expand the concentration detection range of exosomes and lower the detection limit of exosomes, but also detect exosomes of multiple cells at the same time.
[0067] In this embodiment, the simultaneous detection of exosomes of gastric cancer cell line SGC-7901, colon cancer cell line SW480 and leukemia tumor CEM based on MoS2-Fe3O4-AuNSs nanomaterial SERS biosensor is taken as an example, but it should not be limited to this.
[0068] Specifically, nucleic acid aptamers labeled with different Raman signal molecules are used for the simultaneous detection of exosomes from multiple cells. Among them, the nucleic acid aptamer sequence of the CD63 protein in the exosomes of the gastric cancer cell line SGC-7901 cells is: 5'-ROX-CACCCTCGCGTGACTAATGCTA-3', the nucleic acid aptamer sequence of the EpCAM protein in the exosomes of the colon cancer cell line SW480 cells is: 5'-FAM-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTT GGCCTG-3', and the nucleic acid aptamer sequence of the PTK7 protein in the exosomes of the leukemia tumor CEM cells is: 5'-Cy5-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'.
[0069] The nucleic acid aptamers targeting proteins highly expressed on the surface of exosomes of gastric cancer cell line SGC-7901 cells, colon cancer cell line SW480 cells, and leukemia tumor CEM cells were incubated with the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform, so as to combine different types of targeting protein nucleic acid aptamers on the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform, that is, different types of signal probes were set on the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform to bind to different cell exosomes.
[0070] The exosomes secreted by gastric cancer cell line SGC-7901, colon cancer cell line SW480 and leukemia tumor CEM cells were captured and detected using the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with different types of signal probes.
[0071] Finally, by using a Raman microscopy system to analyze the SERS signals of nucleic acid aptamers of different cell exosomes in the magnetic separation products, the simultaneous detection of multiple cell exosome subtypes can be achieved.
[0072] Since the preparation method of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform and the steps of capturing and detecting exosomes with the signal probe have been described in detail in the above specification, they will not be described in detail here. Specifically, SERS in the present invention is interpreted as surface enhanced Raman.
[0073] See also Figure 5 As shown in the figure, it can be seen that the signal probe formed by nucleic acid aptamers labeled with different Raman signal molecules is used to simultaneously detect multiple cell exosomes.
[0074] As shown in curve a, when no exosomes secreted by cells are added, that is, when exosomes do not exist, the Raman spectrum obtained by the test will simultaneously show multiple Raman characteristic peaks (923 cm -1 、1138cm -1 、1171cm -1 、1321cm -1 、1366cm -1 、1465cm -1 、1502cm -1 、1606cm -1 and 1645cm -1 ), which enables the simultaneous detection of exosomes secreted by multiple cells.
[0075] As shown in curve b, the aptamer modified with the Raman signal molecule azadibenzocyclooctyne-Cy5 (Cy5) was used as the signal probe. When the exosomes secreted by CEM cells were detected, the Raman spectrum obtained by the test was at 923 cm -1 、1138cm -1 、1366cm -1 、1465cm -1 and 1606cm -1 Multiple Raman characteristic peaks of Cy5 will appear in other places and disappear with the addition of CEM cell exosomes.
[0076] As shown in curve c, the Raman signal molecule 6-carboxyl-X-rhodamine (ROX) modified nucleic acid aptamer is used as a signal probe. When detecting exosomes secreted by SGC-7901 cells, the Raman spectrum obtained by the test is at 1502 cm -1 and 1645cm -1 The two Raman characteristic peaks of ROX that appeared at the 40 nm spectroscopy site disappeared with the addition of SGC-7901 cell exosomes.
[0077] As shown in curve d, the aptamer modified with Raman signal molecule 6-carboxyfluorescein dye (FAM) was used as the signal probe. When the exosomes secreted by SW480 cells were detected, the Raman spectrum obtained by the test was at 1171 cm -1 and 1321cm -1 The two FAM Raman characteristic peaks appearing at the pores will disappear with the addition of SW480 cell exosomes, thus achieving the simultaneous detection of exosomes secreted by multiple cells.
[0078] In summary, the SERS biosensor of the present invention can amplify the Raman signal by using MoS2-Fe3O4-AuNSs nanomaterials as the active substrate, and can effectively enrich exosomes and magnetically separate the products, simplifying the steps of exosome detection, reducing the dependence on expensive instruments, and also improving the reliability of the detection results; through the specific recognition of nucleic acid aptamers and exosomes, the SERS biosensor has a high specificity and can effectively identify and distinguish exosomes from different cell sources or different subtypes of exosomes; the method of indirect qualitative and quantitative analysis of exosomes by Raman signal molecules solves the problem of direct SERS detection of exosomes with multiple spectral peaks and difficulty in analysis. At the same time, Raman signal molecules have clearly visible characteristic fingerprint peaks, which can improve the sensitivity and accuracy of the detection results; this detection method is universal and can be used for the simultaneous detection of multiple exosomes or the simultaneous detection of other exosome subtypes.
[0079] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention.
Claims
1. A method for preparing a SERS biosensor, characterized in that: The following steps are involved: S1. In situ growth of star-shaped gold nanoparticles on the surface of molybdenum disulfide nanosheets to prepare MoS2-AuNSs nanocomposite materials; magnetic nanoparticles are assembled on the surface of the MoS2-AuNSs nanocomposite materials to form MoS2-Fe3O4-AuNSs composite materials, which serve as the active substrate of the SERS biosensor; S2, thiol-modified probe DNA was assembled on the surface of MoS2-Fe3O4-AuNSs composite material through Au-S bond to obtain SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform; S3. The signal probe is fixed on the surface of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform to identify and capture exosomes. The signal probe is a nucleic acid aptamer modified with a Raman signal molecule.
2. The method for preparing a SERS biosensor according to claim 1, wherein: S1 includes the following steps: S11, stirring and mixing the molybdenum disulfide nanosheet solution and the polyvinylpyrrolidone aqueous solution, and then adding a chloroauric acid precursor to prepare a MoS2-AuNPs nanocomposite material solution; S12, adding chloroauric acid aqueous solution to the hexadecyltrimethylammonium bromide aqueous solution, then adjusting the pH value of the reaction solution; adding the MoS2-AuNPs nanocomposite material solution to react; finally, adding ascorbic acid and silver nitrate solution simultaneously to obtain MoS 2- AuNSs nanocomposite solution; S13, adding magnetic nanoparticles to the MoS2-AuNSs nanocomposite solution, stirring and mixing, centrifuging, washing with anhydrous ethanol and ultrapure water, and dispersing the washed product in ultrapure water to assemble the magnetic nanoparticles into the MoS 2- The surface of the AuNSs nanocomposite material was prepared to obtain a MoS2-Fe3O4-AuNSs nanocomposite material solution.
3. The method for preparing a SERS biosensor according to claim 1, wherein: S2 specifically involves adding thiol-modified probe DNA activated with TCEP to the MoS2-Fe3O4-AuNSs nanomaterial solution and mixing them evenly. The solution is then subjected to magnetic separation and rinsed with phosphate buffer, and the rinsed product is dissolved in phosphate buffer to obtain the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform.
4. The method for preparing a SERS biosensor according to claim 1, wherein: Specifically, S3 is as follows: the signal probe is bound to the surface of the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform through base complementarity, the free signal probe in the solution is removed by magnetic separation and washed with phosphate buffer, and the washed product is dissolved in phosphate buffer to obtain the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with the signal probe.
5. A SERS biosensor, characterized in that: The SERS biosensor is made by the preparation method of the SERS biosensor according to any one of claims 1 to 4.
6. Use of the SERS biosensor according to claim 5 in detecting exosomes.
7. Use of the SERS biosensor according to claim 6 in detecting exosomes, characterized in that: The following steps are involved: Exosomes were added to the SH-DNA / MoS2-Fe3O4-AuNSs Raman detection platform with a signal probe. The exosomes bound to the signal probe with high specificity to form a signal probe-exosome complex, which then separated from the Raman detection platform and was then removed. The SERS signal intensity in the substrate after removal of the signal probe-exosome complex was analyzed using a Raman microscopy system.
8. Use of the SERS biosensor according to claim 7 in detecting exosomes, characterized in that: Density gradient centrifugation is performed on the exosomes to obtain highly pure exosomes.
9. Use of the SERS biosensor according to claim 7 in detecting exosomes, characterized in that: The product from which the signal probe-exosome complex was removed was dropped onto the surface of a silicon wafer, and the SERS signal of the product was detected using a Raman microscope system.