Preparation method of magnetic SERS (Surface Enhanced Raman Scattering) biosensor and application of magnetic SERS biosensor in ctDNA mutant gene detection

Abstract

The invention discloses a preparation method of a magnetic SERS (Surface Enhanced Raman Scattering) biosensor and application of the magnetic SERS biosensor in ctDNA mutant gene detection, and relates to the technical field of Raman spectrum detection. According to the technical scheme, the method comprises the following steps: (1) synthesizing gold-coated gold nanoparticles which have gap structures and are modified with Raman signal molecules in the gap structures by using a DNA single chain as a skeleton; (2) preparing gold-coated ferroferric oxide nanoparticles marked by Raman signal molecules by a hydrothermal synthesis method; and (3) designing a corresponding hairpin DNA structure according to the mutation sequence of the ctDNA, and respectively modifying the hairpin DNA structure on the surfaces of the gold-coated gold nanoparticles in the step (1) and the gold-coated ferroferric oxide nanoparticles in the step (2) to construct a cyclic amplification system so as to realize the detection of the ctDNA mutant gene. The magnetic SERS biosensor provided by the invention has the advantages of high sensitivity, strong specificity, good stability, no need of enzyme participation and the like, and ctDNA mutant genes can be rapidly detected in a complex reaction system.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and particularly relates to a preparation method of a magnetic SERS biosensor and its application in the detection of ctDNA mutant genes. Background Art

[0002] Circulating tumor DNA (ctDNA) is a DNA fragment with the genetic characteristics of tumor cells, which is generally released from cancer cells into body fluids such as blood. The detection of ctDNA has unique advantages, such as high timeliness, low false positive rate, and high specificity. However, problems such as high fragmentation, rarity, interference from wild-type genes, and scarcity make the detection of ctDNA in body fluids challenging. Currently, common methods for ctDNA detection include NGS, Amplification Refractory Mutation System (ARMS), digital PCR, etc., but these methods have disadvantages such as complex operation, long time, and high cost, and cannot meet the needs of patients and doctors simultaneously. Therefore, there is an urgent need for a new method for ctDNA genotyping detection that is convenient, fast, and highly sensitive.

[0003] In recent years, surface-enhanced Raman spectroscopy technology has become one of the most promising tools in biomedical analysis due to its excellent optical properties. However, due to the low abundance of mutant ctDNA in blood, a single SERS technology has been unable to achieve the required ultrasensitive detection level. To improve the ability to detect low-abundance ctDNA, a large number of studies have combined various signal amplification strategies with SERS technology, such as rolling circle amplification (RCA), CRISPR / Cas system, etc. However, most of these methods require enzymes as catalysts and are easily interfered by complex biological environments. Catalytic hairpin assembly (CHA) is a typical isothermal enzyme-free signal amplification strategy that can achieve hundreds of times of catalytic amplification, overcoming the limitations of specific reaction conditions and complex operations required for enzyme amplification. In addition, the CHA reaction strictly follows the base complementary pairing principle, ensuring high specificity for ctDNA detection. Therefore, in order to achieve rapid, simple, highly accurate, and highly sensitive analysis of ctDNA mutant types in body fluids, the present invention designs a kit for multiplex detection of ctDNA mutant genes based on a magnetic SERS biosensor. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a preparation method of a magnetic SERS biosensor and its application in the detection of ctDNA mutant genes, to determine the mutant types of ctDNA in actual samples isothermally, enzyme-free, rapidly, highly sensitively, and highly accurately.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A preparation method of a magnetic SERS biosensor, comprising the following steps: (1) Using single-stranded DNA as the backbone, prepare gold-coated gold nanoparticles Au@Gap-Raman reporter@Au NPs with a gap structure and Raman signal molecules modified in the gap structure. (2) Prepare Raman signal molecule-labeled gold-coated iron oxide nanoparticles Fe3O4@Raman reporter@Au NPs by hydrothermal synthesis. (3) Design corresponding hairpin DNA structures according to the mutant sequences of ctDNA, and modify them on the surfaces of the gold-coated gold nanoparticles in step (1) and the gold-coated iron oxide nanoparticles in step (2) respectively to construct a cyclic amplification system.

[0006] Further, step (1) is specifically as follows: Add the single-stranded DNA 5’-NH2-C6-aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-HS-3’ to the Au NPs solution, incubate at 25 °C, and sequentially add phosphate buffer solution, sodium chloride solution, and sodium chloride solution every 20 minutes during the incubation. After the addition is complete, incubate overnight at 25 °C, and then centrifuge and concentrate to obtain the Au@DNA NPs solution; Add the Raman signal molecule solution to the Au@DNA NPs solution, incubate at 25 °C for 2 h, then centrifuge, collect the precipitate and resuspend it with ultrapure water to obtain the Au@DNA-Raman reporter NPs solution; Add the Au@DNA-Raman reporter NPs solution to reaction solution A, react at 25 °C for 5 min, then immediately add the HAuCl4 solution, continue to react at 25 °C for 30 min, then add the HS-PEG compound solution, incubate at 25 °C for 0.5 h, then centrifuge, collect the precipitate and resuspend it with ultrapure water to obtain the Au@Gap-Raman reporter@Au NPs solution.

[0007] Further, step (2) is specifically as follows: Add the polyethyleneimine solution to the Fe3O4 solution, sonicate at room temperature for 15 min, magnetically separate the solid product, wash it with ultrapure water and resuspend it to obtain the Fe3O4@PEI NPs solution; Add the Fe3O4@PEI NPs solution to the Au NPs solution, sonicate at room temperature for 30 min, magnetically separate the solid product, wash it with ultrapure water and resuspend it to obtain the Fe3O4@Au-seed NPs solution; Add the Raman signal molecule solution to the Fe3O4@Au-seed NPs solution, incubate at room temperature for 2 h, magnetically separate the solid product, wash it with ultrapure water and add it to reaction solution B, sonicate at room temperature for 30 min to obtain the Fe3O4@Raman reporter@Au NPs solution.

[0008] Further, step (3) above is specifically as follows: Design hairpin DNA probes HP1 and HP2 according to the mutant sequences of ctDNA; Modify the hairpin DNA probe HP1 on Fe3O4@Raman reporter@Au NPs to obtain the Fe3O4@Raman reporter@Au@HP1 NPs probe, and modify the hairpin DNA probe HP2 on Au@Gap-Raman reporter@Au NPs to obtain the Au@Gap-Raman reporter@Au@HP2 NPs probe; Mix the Fe3O4@Raman reporter@Au@HP1 NPs probe solution with the Au@Gap-Raman reporter@Au@HP2 NPs probe solution and the ctDNA target strand to be detected, incubate at 37 °C for 0.5 - 3 h, magnetically separate the solid product, and wash the solid product with phosphate buffer solution containing Tween and ultrapure water to obtain the magnetic SERS biosensor.

[0009] Further, the above Raman signal molecules include but are not limited to 4-mercaptobenzoic acid, mercaptobenzonitrile, 2-mercapto-4-methyl-5-thiazoleacetic acid, 4-mercaptobenzeneboronic acid, 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid.

[0010] Further, the above reaction solution A is composed of cetyltrimethylammonium chloride solution and ascorbic acid solution, wherein the molar ratio of cetyltrimethylammonium chloride to ascorbic acid is 1000:12.

[0011] Further, the above reaction solution B is composed of polyvinylpyrrolidone solution, hydroxylamine hydrochloride solution and HAuCl4 solution, wherein the mass ratio of polyvinylpyrrolidone, hydroxylamine hydrochloride and HAuCl4 is 8:3:0.024.

[0012] A magnetic SERS biosensor is prepared by the above preparation method.

[0013] The application of the above magnetic SERS biosensor in the preparation of a ctDNA mutant gene detection kit.

[0014] A ctDNA mutant gene detection kit contains the above magnetic SERS biosensor.

[0015] A method for detecting ctDNA mutant genes in an actual sample based on the above magnetic SERS biosensor includes the following steps: S1: Mix the Fe3O4@Raman reporter@Au@HP1 NPs solution with the Au@Gap-Raman reporter@Au@HP2NPs solution and standard solutions of different concentrations (0-10 nM), incubate at 37°C for 0.5-3 h, wash alternately with phosphate buffer containing Tween and ultrapure water, and collect spectral information with the help of a portable Raman spectrometer; construct the working curve of the standard with the Raman intensity ratio of the Raman Reporter characteristic peaks on Fe3O4@Raman reporter@Au@HP1 NPs and Au@Gap-Raman reporter@Au@HP2 NPs; S2: The actual sample needs to be unwound first to obtain ssDNA: first incubate at 95°C for 10 min, then incubate at 0°C for 10 min; then proceed according to step S1; collect the Raman intensity ratio of the actual sample, and calculate the corresponding concentration according to the standard working curve.

[0016] The working principle of the magnetic SERS biosensor of the present invention for detecting ctDNA mutant genes is as follows: Figure 1 ): When the target mutant gene sequence chain (hereinafter referred to as the target chain) is present, the target chain combines with the stem part of HP1 through the principle of base complementary pairing, thereby opening the hairpin DNA probe to form a double strand. Since HP2 has more complementary bases with HP1, it replaces the target chain by competitive binding, and the released target chain re-enters the system to trigger the next cycle, namely, the enzyme-free isothermal cyclic amplification reaction (CHA). Among them, with the stable double strand formed by HP1 and HP2 as the connecting medium, the Fe3O4@Ramanreporter@Au@HP1 NPs probe and the Au@Gap-Raman reporter@Au@HP2 NPs probe form a core-satellite structure, so the characteristic peak intensity ratio of the Reporter in the two probes is positively correlated with the target chain concentration. On the contrary, if there is no target chain, HP1 cannot be opened, and the CHA reaction cannot be triggered, that is, the Fe3O4@Raman reporter@Au@HP1 NPs probe and the Au@Gap-Raman reporter@Au@HP2 NPs probe cannot form a core-satellite structure.

[0017] It can be seen from the above technical solutions that the beneficial effects of the present invention are: 1) The CHA reaction has the characteristics of being enzyme-free and isothermal, overcoming the limitations of specific reaction conditions and complex operations required for enzyme amplification. In addition, based on the principle of complementary base pairing, the CHA reaction has high specificity for the detection of ctDNA.

[0018] 2) The Au@Gap-Raman reporter@Au NPs, by virtue of the gap structure, on the one hand, greatly enhance the characteristic peak intensity of the embedded labeled signal molecules, and on the other hand, avoid the loss of signal molecules during reactions and other processes.

[0019] 3) The core-satellite structure formed by the Fe3O4@Raman reporter@Au@HP1 NPs probe and the Au@Gap-Raman reporter@Au@HP2 NPs provides a large number of "hot spots", combined with the CHA reaction, to achieve double amplification of the detection signal.

[0020] 4) Using the ratio of the characteristic peak intensities in the core-satellite structure as the output signal can correct the signal fluctuations caused by physical factors such as the external environment and instruments, greatly improving the detection accuracy. Description of the Drawings

[0021] Figure 1 : The working principle of the magnetic SERS biosensor for detecting ctDNA mutant genes.

[0022] Figure 2 : The TEM image of Au@Gap-TFMBA@Au NPs.

[0023] Figure 3 : The TEM image of Fe3O4@MBN@Au NPs.

[0024] Figure 4 : A, The Raman spectral curve of the magnetic SERS biosensor for detecting the EGFR Exon 19deletion standard; B, The standard working curve of the magnetic SERS biosensor for detecting the EGFR Exon 19deletion standard.

[0025] Figure 5 : The Raman spectrogram of the magnetic SERS biosensor for detecting ctDNA mutant genes in HCC827 cells and A549 cells.

[0026] Figure 6 : The Raman spectrogram of the anti-interference experiment of the magnetic SERS biosensor for detecting ctDNA mutant genes. Detailed Embodiments

[0027] In order to enable those skilled in the art to more clearly understand the technical solutions of this application, the technical solutions of this application will be described in detail below with specific embodiments. Obviously, the embodiments described below are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0028] In the embodiments of the present invention, the hairpin DNA probes used are all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0029] In the embodiments of the present invention, the pH value of the phosphate buffer solution used is 7.4 and the concentration is 0.01 M.

[0030] In the embodiments of the present invention, the reaction solution A used is formed by mixing a 0.1 M cetyltrimethylammonium chloride aqueous solution and a 0.1 M ascorbic acid aqueous solution in a volume ratio of 1000:12.

[0031] The formula of the reaction solution B used in the embodiments of the present invention is: polyvinylpyrrolidone (molecular weight 50000) 3 mg / mL, hydroxylamine hydrochloride 0.5 mg / mL, HAuCl4 0.004 mg / mL, and the rest is ultrapure water.

[0032] Example 1: (1) Preparation of Au@Gap-Raman reporter@Au NPs TFMBA was selected as the Raman signal molecule (Raman reporter) to prepare Au@TFMBA(gap)@Au NPs; the diameter of the AuNPs used was 10 nm. The specific steps are as follows: S1: Add the DNA single strand 5’-NH2-C6-aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-HS-3’ to 10 mL of a 100 nM Au NPs solution, control the molar ratio of the DNA single strand to Au NPs to be 75:1, incubate at 25 °C for 20 min, then add 100 μL of 0.01 M phosphate buffer solution, continue to incubate at 25 °C for 20 min, then add 600 μL of 0.02 M sodium chloride solution, continue to incubate at 25 °C for 20 min, then add 500 μL of 0.1 M sodium chloride solution, incubate overnight at 25 °C, and then centrifuge (11000 rpm, 15 min) to concentrate to 1 / 2 of the original volume to obtain the Au@DNANPs solution.

[0033] S2: Add 10 μL of 1 mM TFMBA solution to the Au@DNANPs solution in S1, incubate at 25 °C for 2 h, then centrifuge (9000 rpm, 12 min), collect the precipitate and resuspend it with ultrapure water to the original volume to obtain the Au@DNA-TFMBANPs solution.

[0034] S3: Add 1 mL of the Au@DNA-TFMBANPs solution from S2 to 10 mL of reaction solution A, react at 25 °C for 5 min, then immediately add 75 μL of 0.1 mM HAuCl4 solution, continue to react at 25 °C for 30 min, and then add 100 μL of 10 μM HS-PEG (where the PEG part M W 2000) solution, incubate at 25 °C for 0.5 h to block excess sites and reduce non-specific adsorption, then centrifuge (7000 rpm, 8 min), collect the precipitate and resuspend it with ultrapure water to the original volume to obtain the Au@Gap-TFMBA@Au NPs solution.

[0035] Characterize the morphology of Au@Gap-TFMBA@Au NPs using TEM. As Figure 2 can be seen, Au@Gap-TFMBA@Au NPs have a gap structure, and there is a gap of approximately 1.5 nm between the Au core and the Au shell.

[0036] (2) Preparation of Fe3O4@MBN@Au NPs Select MBN as the Raman signal molecule (Raman reporter) to prepare Fe3O4@Raman reporter@Au NPs; the diameter of the Au NPs used is 3 nm. The specific steps are as follows: S1: Add 1 mL of 1 mg / mL polyethyleneimine (molecular weight 25000) solution to 5 mL of 1 mg / mL Fe3O4 solution, sonicate at room temperature with a power of 30 W for 15 min, magnetically separate the solid product, wash it with ultrapure water and resuspend it to a concentration of 1 mg / mL to obtain the Fe3O4@PEI NPs solution.

[0037] S2: Add the Fe3O4@PEI NPs solution from S1 to 100 mL of 100 nM Au NPs solution, sonicate at room temperature with a power of 30 W for 30 min, magnetically separate the solid product, wash it with ultrapure water and resuspend it to a concentration of 1 mg / mL to obtain the Fe3O4@Au-seed NPs solution.

[0038] S3: Add 100 μL of 1 mM MBN solution to the Fe3O4@Au-seed NPs solution from S2, incubate at room temperature for 2 h, magnetically separate the solid product, wash it with ultrapure water and add it to 60 mL of reaction solution B, sonicate at room temperature with a power of 30 W for 30 min to obtain the Fe3O4@MBN@Au NPs solution.

[0039] Characterize the morphology of Fe3O4@MBN@Au NPs using TEM. The results are as Figure 3as shown

[0040] Example 2: Establish a standard working curve for EGFR Exon 19deletion Select the EGFR Exon 19deletion standard as the target strand, with the sequence 5’-CGTCGCTATCAAAACATCTCCG-3’; the hairpin DNA probe HP1 sequence is 5’-SH-(CH2)6-TTCCGATAGCTGATTGGACCGTCGCTATCAAAACAGACGGTCCAATCA-3’, and the hairpin DNA probe HP2 sequence is 5’-SH-(CH2)6-CGGAGATGTTTTGATAGCGACGGTCCAATCAGCTATCAAAACCCGGACT-3’. Both hairpin DNA probes HP1 and HP2 need to be annealed at 95 °C for 5 min before use, and then naturally cooled to room temperature.

[0041] (1) Preparation of Fe3O4@MBA@Au@HP1 NPs probe Add 10 μL of 10 μM hairpin DNA probe HP1 solution to 1 mL of Fe3O4@MBN@Au NPs solution, incubate at 25 °C for 20 min, then add 10 μL of phosphate buffer solution, continue to incubate at 25 °C for 20 min, then add 10 μL of 0.02 M sodium chloride solution, continue to incubate at 25 °C for 20 min, then add 10 μL of 0.1 M sodium chloride solution, and incubate overnight at 25 °C to obtain the Fe3O4@MBA@Au@HP1 NPs probe solution.

[0042] (2) Preparation of Au@Gap-TFMBA@Au@HP2 NPs probe Add 10 μL of 10 μM hairpin DNA probe HP2 solution to 1 mL of Au@Gap-TFMBA@Au NPs solution, incubate at 25 °C for 20 min, then add 10 μL of phosphate buffer solution, continue to incubate at 25 °C for 20 min, then add 10 μL of 0.02 M sodium chloride solution, continue to incubate at 25 °C for 20 min, then add 10 μL of 0.1 M sodium chloride solution, and incubate overnight at 25 °C to obtain the Au@Gap-TFMBA@Au@HP2 NPs probe solution.

[0043] (3) Assembly of magnetic SERS biosensor Mix 50 μL of Fe3O4@MBN@Au@HP1 NPs probe solution with 50 μL of Au@Gap-TFMBA@Au@HP2 NPs probe solution and 10 μL of target strand solution with different concentrations (0 - 10 nM), incubate at 37 °C for 1.5 h, magnetically separate the solid product, and after washing the solid product alternately with phosphate buffer solution containing 0.1 vol% Tween 80 and ultrapure water twice, collect spectral information with a portable Raman spectrometer. Using the Raman intensity ratio of the characteristic peaks on Fe3O4@MBN@Au@HP1 NPs and Au@Gap-TFMBA@Au@HP2 NPs (i.e., I 1377 / I 2230 ), construct the working curve of the target strand.

[0044] As Figure 4 shown in A, the Raman signal of Au@Gap-TFMBA@Au@HP2 NPs is normalized with the intensity of Fe3O4@MBA@Au@HP1 NPs at 2230 cm -1 . When the concentration of the EGFR Exon 19deletion standard solution decreases, the intensity of the characteristic peak of the Raman signal of Au@Gap-TFMBA@Au@HP2 NPs decreases accordingly. As Figure 4 shown in B, there is a good linear relationship between the concentration of the EGFR Exon19deletion standard solution and the Raman intensity ratio (i.e., I 1377 / I 2230 ), and the linear equation is y = 0.4873x + 0.8025, R 2 = 0.9925. According to the three-fold signal-to-noise ratio formula, the detection limit is calculated to be 0.27×10 -13 M.

[0045] Example 3: On the basis of Example 2, change the analyte to genomic DNA extracted from HCC827 cells or A549 cells; there is an EGFR Exon 19deletion mutation in HCC827 cells, and there is no EGFR mutation in A549 cells. Mix 10 μL of genomic DNA with 50 μL of Au@Gap-TFMBA@Au@HP2 NPs solution and 50 μL of Fe3O4@MBN@Au@HP1 NPs solution, incubate at 37 °C for 1.5 h, magnetically separate the solid product, and after washing the solid product alternately with phosphate buffer solution containing 0.1 vol% Tween 80 and ultrapure water twice, collect spectral information with a portable Raman spectrometer.

[0046] The results are as Figure 5 shown, and only the signal response of the TFMBA characteristic peak appears on the Raman curve of the HCC827 cell group.

[0047] Example 4: Specific Detection of Magnetic SERS Biosensor On the basis of Example 2, the analytes were changed to DNA sequences (M 1-1 , M 1-2 , M 2-1 , M 2-2 ) of wild type, L858R mutation, and single-base mutation based on EGFR Exon 19deletion of EGFR Exon 19deletion, EGFRExon19deletion standard or their mixture; among them, the concentration of the target strand EGFR Exon 19deletion standard was 10 -12 M, and the concentration of the non-target strand was 10 -10 M. The corresponding sequences are shown in the following table. Mix 10 μL of the analyte with 50 μL of Au@Gap-TFMBA@Au@HP2 NPs solution and 50 μL of Fe3O4@MBN@Au@HP1 NPs solution, incubate at 37 °C for 1.5 h, magnetically separate the solid product, and after washing the solid product alternately with phosphate buffer solution containing 0.1 vol% Tween 80 and ultrapure water twice, collect spectral information with a portable Raman spectrometer.

[0048] The results are as Figure 6 shown. When only the target strand EGFR Exon 19deletion standard was present, a characteristic peak signal response of TFMBA appeared on the Raman curve; for the non-target strand, even in the case of single-base mutation, the detected signal value had no significant difference from the blank control (ultrapure water). It shows that the magnetic SERS biosensor has high specificity for the target strand EGFR Exon 19deletion.

[0049] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent transformation made by using the specification and drawings of the present invention, or directly or indirectly applied in the related technical field, shall be included in the patent protection scope of the present invention by the same token.

Claims

1. A preparation method of a magnetic SERS biosensor, characterized in that: It includes the following steps: (1) Using a single-stranded DNA as a backbone, prepare gold-coated gold nanoparticles Au@Gap-Raman reporter@Au NPs with a gap structure and Raman signal molecules modified in the gap structure; (2) Prepare Raman signal molecule-labeled gold-coated iron oxide nanoparticles Fe3O4@Raman reporter@Au NPs by hydrothermal synthesis; (3) Design corresponding hairpin DNA structures according to the mutant sequences of ctDNA, and modify them on the surfaces of the gold-coated gold nanoparticles in step (1) and the gold-coated iron oxide nanoparticles in step (2) respectively to construct a cyclic amplification system.

2. The preparation method according to claim 1, characterized in that: Step (1) is specifically as follows: Add the single-stranded DNA 5’-NH2-C6-aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-HS-3’ to the Au NPs solution, incubate at 25 °C, and sequentially add phosphate buffer solution, sodium chloride solution, and sodium chloride solution every 20 min during the incubation. After the addition is complete, incubate overnight at 25 °C, and then centrifuge and concentrate to obtain the Au@DNA NPs solution; Add the Raman signal molecule solution to the Au@DNA NPs solution, incubate at 25 °C for 2 h, then centrifuge, collect the precipitate and resuspend it with ultrapure water to obtain the Au@DNA-Raman reporter NPs solution; Add the Au@DNA-Raman reporter NPs solution to reaction solution A, react at 25 °C for 5 min, then immediately add the HAuCl4 solution, continue to react at 25 °C for 30 min, then add the HS-PEG compound solution, incubate at 25 °C for 0.5 h, then centrifuge, collect the precipitate and resuspend it with ultrapure water to obtain the Au@Gap-Raman reporter@Au NPs solution.

3. The preparation method according to claim 1, characterized in that: Step (2) is specifically as follows: Add the polyethyleneimine solution to the Fe3O4 solution, sonicate at room temperature for 15 min, magnetically separate the solid product, wash it with ultrapure water and resuspend it to obtain the Fe3O4@PEI NPs solution; Add the Fe3O4@PEI NPs solution to the Au NPs solution, sonicate at room temperature for 30 min, magnetically separate the solid product, wash it with ultrapure water and resuspend it to obtain the Fe3O4@Au-seed NPs solution; Add the Raman signal molecule solution to the Fe3O4@Au-seed NPs solution, incubate at room temperature for 2 h, magnetically separate the solid product, wash it with ultrapure water and add it to reaction solution B, sonicate at room temperature for 30 min to obtain the Fe3O4@Raman reporter@Au NPs solution.

4. The preparation method according to claim 1, wherein: Step (3) specifically includes: designing hairpin DNA probes HP1 and HP2 according to the mutant sequence of ctDNA; modifying the hairpin DNA probe HP1 on Fe3O4@Raman reporter@Au NPs to obtain the Fe3O4@Raman reporter@Au@HP1 NPs probe, and modifying the hairpin DNA probe HP2 on Au@Gap-Raman reporter@Au NPs to obtain the Au@Gap-Raman reporter@Au@HP2 NPs probe; mixing the Fe3O4@Raman reporter@Au@HP1 NPs probe solution with the Au@Gap-Raman reporter@Au@HP2 NPs probe solution and the ctDNA target strand to be detected, incubating at 37 °C for 0.5 - 3 h, magnetically separating the solid product, and washing the solid product with phosphate buffer solution containing Tween and ultrapure water to obtain the magnetic SERS biosensor.

5. The preparation method according to claim 1, characterized in that: The Raman signal molecules include but are not limited to 4-mercaptobenzoic acid, mercaptobenzonitrile, 2-mercapto-4-methyl-5-thiazoleacetic acid, 4-mercaptobenzeneboronic acid, 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid.

6. The preparation method according to claim 2, wherein: The reaction solution A is composed of cetyltrimethylammonium chloride solution and ascorbic acid solution, wherein the molar ratio of cetyltrimethylammonium chloride to ascorbic acid is 1000:

12.

7. The preparation method according to claim 3, characterized in that: The reaction solution B is composed of polyvinylpyrrolidone solution, hydroxylamine hydrochloride solution and HAuCl4 solution, wherein the mass ratio of polyvinylpyrrolidone, hydroxylamine hydrochloride and HAuCl4 is 18:3:0.

024.

8. A magnetic SERS biosensor, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the magnetic SERS biosensor according to claim 8 in the preparation of a ctDNA mutant gene detection kit.

10. A ctDNA mutation gene detection kit, characterized in that: Comprising the magnetic SERS biosensor according to claim 8.

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