SERS sensor for detecting serum biomarkers related to colorectal cancer and preparation method thereof

Abstract

The present invention relates to the field of biomedical technology, and in particular to a SERS sensor for detecting serum biomarkers related to colorectal cancer and a preparation method thereof. The SERS sensor of the present invention is composed of a double-stranded nucleic acid structure formed by a hairpin structure DNA specifically binding to a relevant serum biomarker and a single-stranded DNA partially complementary to the hairpin structure DNA, which is modified on the surface of gold-coated ferroferric oxide magnetic nanoparticles with Raman signal molecules, and a single-stranded DNA partially complementary to the hairpin structure DNA. ‑1 and 1346cm ‑1 By measuring the position and intensity of the characteristic peaks, rapid, highly sensitive and synchronous detection of miR-21 and miR-122 in serum can be achieved; the SERS sensor of the present invention has good specificity, stability and reproducibility. When detecting clinical serum samples, the operation is simple and the detection of miR-21 and miR-122 can be completed in a short time.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a SERS sensor for detecting serum biomarkers related to colorectal cancer and a preparation method thereof. Background Art

[0002] Colorectal cancer (CRC) is the third most common cancer in the world, with global morbidity and mortality increasing year by year. Studies have shown that the 5-year survival rate of early CRC is >90%, while the 5-year survival rate of advanced cancer is <10%. Therefore, early diagnosis and timely treatment play an important role in improving the survival rate of CRC patients and improving their physical health. At present, the conventional screening methods for CRC include fecal occult blood test (FOBT), colonoscopy and histopathological examination. However, these methods all have certain shortcomings. FOBT has low sensitivity and specificity and is prone to false positives; although colonoscopy has high sensitivity, it has the disadvantages of being expensive and invasive; as the current "gold standard" for diagnosing malignant tumors, histopathological examination is time-consuming, traumatic, and requires high professional talents, which limits its development. Therefore, there is an urgent need to develop a fast, convenient, non-invasive, and highly sensitive screening technology to diagnose and monitor the development of CRC.

[0003] In recent years, nucleic acid tumor markers have become a research hotspot, among which microRNA (miRNA) has attracted the most attention. miRNAs are a class of endogenous non-coding single-stranded nucleic acid molecules composed of 19 to 23 nucleotides. They play an important regulatory role in various physiological or pathological processes such as cell proliferation, metabolism, differentiation, gene expression, and immune response, and are closely related to the occurrence and development of the disease. miRNAs can be used as a non-invasive biomarker to play an active role in the early diagnosis and prognosis monitoring of CRC. Yanlei Ma et al. analyzed the expression of miRNAs in CRC patients and normal human mucosa and found that the expression of miR-30, miR-139, miR-145, miR-125, and miR-133 in tumor tissues was significantly lower than that in normal tissues; Peacock et al. found that the levels of miR-21 and miR-31 were upregulated in the serum of CRC patients; Pardini et al. used a machine learning model to discover 25 differentially expressed miRNAs in CRC patients. However, some inherent characteristics of miRNAs, such as short sequence, easy degradation, low abundance, and high homology, pose challenges to the rapid and accurate detection of miRNAs. Existing conventional detection methods such as polymerase chain reaction (PCR), strand displacement amplification (SDA), colorimetry, and electrochemical methods have shortcomings such as complex operation and low sensitivity, which are difficult to meet the detection needs of actual samples. Therefore, the development of a rapid, simple, and highly sensitive miRNAs detection method is crucial for the early diagnosis of CRC.

[0004] Surface enhanced Raman spectroscopy (SERS) is a powerful analytical technique that is widely used in chemical and biological sensing applications. With the help of the "hot spots" on the surface of noble metal nanostructures, a significantly enhanced local electric field is formed, and the molecules around it are gathered to produce a strong SERS signal. In recent years, the application of SERS technology in biomedicine has attracted much attention. It has the unique advantages of strong anti-interference ability, high sensitivity, fast analysis speed and non-invasiveness in the detection of biological samples. With the deepening of SERS research spectrum, due to the synergistic effect of the electromagnetic field enhancement effect of noble metal nanoparticles and the charge transfer mechanism of metal oxides, a variety of gold or silver modified metal oxide composite nanomaterial structures have been confirmed to be excellent SERS substrates. In addition to acting as a SERS enhancement substrate, Fe3O4@AuMNPs can also be enriched and separated under the action of micro-magnets, so that the SERS signal is double-amplified, which is feasible in improving the sensitivity of detection, thereby solving the problem of low sensitivity of miRNAs detection. In addition, the detection of miRNAs mainly relies on the "sandwich" strategy based on aptamers. Although this strategy is specific, it requires two incubations, the steps are cumbersome, and it is easily affected by human operation factors. Summary of the invention

[0005] In view of the above-mentioned deficiencies of the prior art, the present invention aims to provide a serum biomarker, a SERS sensor and a preparation method for detecting colorectal cancer-related biomarkers, so as to solve the problems of cumbersome process and high operation requirements of the existing methods for detecting miRNAs for colorectal cancer.

[0006] In order to solve the above problems, the present invention adopts the following technical solutions:

[0007] The signal "Off-On" strategy is a new miRNAs detection strategy. Through the binding of ssDNA to the target miRNAs and their release from the substrate surface, the hpDNA is restored to a hairpin structure, causing the Raman signal molecules at the end of the hpDNA to approach the substrate surface, and the SERS signal changes from "Off" to "On", and the SERS signal is enhanced. The intensity of the SERS signal can be used to quickly and quantitatively detect the target miRNAs in the sample. This strategy is different from the secondary hybridization of the sandwich structure. As long as a simple one-step pairing hybridization is performed, miRNA detection can be achieved. Based on this, SERS, Fe3O4@Au MNPs enrichment and separation, and the signal "Off-On" strategy are combined to detect miR-21 and miR-122 in the serum of CRC patients.

[0008] In a first aspect, the present invention provides a method for detecting serum biomarkers associated with colorectal cancer, wherein the serum biomarkers associated with colorectal cancer include miR-21 and miR-122.

[0009] Furthermore, the nucleotide sequences of miR-21 and miR-122 are shown in SEQ ID No.1 and SEQ ID No.2.

[0010] In a second aspect, the present invention provides a SERS sensor for detecting serum biomarkers related to colorectal cancer, which is composed of a double-stranded nucleic acid structure formed by a hairpin structure DNA that specifically binds to the relevant serum biomarker and a single-stranded DNA that is partially complementary to the hairpin structure DNA, which is modified on the surface of gold-coated ferroferric oxide magnetic nanoparticles with a Raman signal molecule marker.

[0011] Furthermore, the nucleotide sequences of the hairpin structure DNA specifically binding to the relevant serum biomarkers labeled by Raman signal molecules are shown as SEQ ID No.3 and SEQ ID No.4; the nucleotide sequences of the single-stranded DNA partially complementary to the hairpin structure DNA labeled by Raman signal molecules are shown as SEQ ID No.5 and SEQ ID No.6.

[0012] In a third aspect, the present invention provides a method for preparing a SERS sensor for detecting serum biomarkers related to colorectal cancer, comprising:

[0013] S10, using electrostatic adsorption to modify negatively charged gold nanoparticles onto the surface of positively charged polyethyleneimine-modified ferroferric oxide nanoparticles to prepare gold-coated ferroferric oxide magnetic nanoparticles;

[0014] S20. A double-stranded nucleic acid structure formed by a hairpin structure DNA that specifically binds to relevant serum biomarkers and a single-stranded DNA that is partially complementary to the hairpin structure DNA is modified on the surface of gold-coated ferroferric oxide magnetic nanoparticles with Raman signal molecules to obtain a SERS sensor for detecting serum biomarkers related to colorectal cancer.

[0015] Further, the S10 includes:

[0016] S11, 400 mL of 0.25 mM chloroauric acid solution and 4 mL of 30.57 mM sodium citrate solution were mixed, and then 6 mL of 200 mM NaBH4 solution was added, and the mixture was stirred at room temperature until the solution turned into wine red to obtain negatively charged gold nanoparticles;

[0017] S12, adding 1 mL of 10 mg / mL Fe3O4 solution to 100 mL of ultrapure water, ultrasonicating for 10 min, then adding 10 mL of 0.5 mg / mL PEI solution, ultrasonicating for 30 min, using a micro magnet to enrich and separate the product, and removing excess PEI, resuspending in 10 mL of ultrapure water to obtain positively charged polyethyleneimine-modified ferrosoferric oxide nanoparticles;

[0018] S13, mixing the negatively charged gold nanoparticles with the positively charged polyethyleneimine-modified ferroferric oxide nanoparticles, ultrasonicating for 30 min, enriching and separating the products using a micro magnet, and resuspending them in 5 mL of ultrapure water to obtain gold-coated ferroferric oxide magnetic nanoparticles;

[0019] The volume ratio of the negatively charged gold nanoparticles to the positively charged polyethyleneimine-modified ferroferric oxide nanoparticles is 40-60:1; AuNPs with a diameter of 3 nm are uniformly covered on the surface of Fe3O4 with a diameter of 100 nm, and the lattice spacing of Au MNPs is 0.24 nm.

[0020] Further, the S20 includes:

[0021] S21, adding 10 μL of a mixed solution of 5-FAM-labeled hairpin structure DNA1 and Cy5-labeled hairpin structure DNA2, and 10 μL of a mixed solution of single-stranded DNA1 and single-stranded DNA2 into PBS buffer, and incubating at 37°C for 12 h to generate a double-stranded nucleic acid structure;

[0022] S22. Add 40 μL of double-stranded nucleic acid structure and 0.5 mL of 3.5 mM magnesium chloride solution into 5 mL of gold-coated ferroferric oxide magnetic nanoparticle solution. The hairpin structure DNA is attached to the surface of the gold-coated ferroferric oxide magnetic nanoparticles through Au-S bonds. After stirring, the product is centrifuged for 10 minutes and resuspended in Tris-HCl buffer to obtain a SERS sensor for detecting colorectal cancer-related serum biomarkers.

[0023] Furthermore, the concentration of the 5-FAM labeled hairpin structure DNA1 and the Cy5 labeled hairpin structure DNA2 is not less than 10 μM.

[0024] In a fourth aspect, the present invention provides a system for detecting the colorectal cancer-related serum biomarkers, characterized in that it comprises a collection module, a detection module and a calculation module;

[0025] The collection module is used to collect serum test samples;

[0026] The detection module is used to perform SERS detection on the collected serum test sample using the SERS sensor for detecting colorectal cancer-related serum biomarkers;

[0027] The calculation module is used to calculate the results of SERS detection at 1170 cm -1 and 1346cm -1 The SERS intensity of the characteristic peak at was substituted into the working curve to calculate the concentration of miR-21 and miR-122 in the serum test sample.

[0028] Further, the working curve is:

[0029] y1=2342.10x1+43127.65, where y1 is 1170cm -1 is the SERS intensity, x1 is the concentration of miR-21 in the serum test sample;

[0030] y2=2374.92x2+42999.05, where y2 is 1346cm -1 SERS intensity at SERS intensity, x2 is the concentration of miR-122 in the serum test sample;

[0031] In the SERS detection of the collected serum test sample, the hybridization reaction time of the SERS sensor and the serum test sample is not less than 10 minutes.

[0032] The beneficial effects of the present invention are as follows: the present invention is based on colorectal cancer-related serum biomarkers, combined with the designed SERS sensor, ssDNA as a "placeholder" chain hybridizes with the hpDNA part, causing the hpDNA terminal Raman signal molecule to be away from the Fe3O4@Au MNPs surface, showing a lower SERS signal. When the target miRNAs are present, ssDNA binds to it and is released from the Fe3O4@Au MNPs surface, hpDNA recovers to a hairpin structure, and the hpDNA terminal Raman signal molecule is close to the Fe3O4@AuMNPs surface, and the SERS signal is enhanced (primary amplification). Finally, the Fe3O4@Au MNPs are enriched and separated by micro-magnets, and a large number of "hot spots" are generated between the nanoparticles, which can greatly enhance the SERS signal and improve the sensitivity of detection (secondary amplification). According to 5-FAM and Cy5 at 1170cm -1 and 1346cm -1 By analyzing the position and intensity of the characteristic peaks, rapid, highly sensitive and synchronous detection of miR-21 and miR-122 in serum can be achieved.

[0033] The SERS sensor can quantitatively detect miR-21 and miR-122 in a wide linear range, with a detection limit (LOD) as low as aM. At the same time, the SERS sensor exhibits good specificity, stability and reproducibility. When testing clinical serum samples, the operation is simple, and the detection of miR-21 and miR-122 can be completed in a short time. The required amount of samples to be tested is also small, only a few microliters are needed. The accuracy of the SERS results was verified using qRT-PCR as the gold standard. The results show that the SERS sensor of the present invention achieves rapid, quantitative and highly sensitive detection of low-abundance miRNA markers in clinical samples, providing technical support for expanding the wide application of SERS in early detection of CRC. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the preparation of gold-coated ferroferric oxide magnetic nanoparticles in Example 1 of the present invention.

[0035] Figure 2 This is a transmission electron microscope (TEM) image of a single Fe3O4@Au MNPs prepared in Example 1 of the present invention.

[0036] Figure 3 TEM images of multiple Fe3O4@Au MNPs prepared in Example 1 of the present invention.

[0037] Figure 4 The lattice fringes and the magnified image of Fe3O4@Au MNPs prepared in Example 1 of the present invention.

[0038] Figure 5 This is a selected area electron diffraction (SAED) image of the crystal material of Fe3O4@Au MNPs prepared in Example 1 of the present invention.

[0039] Figure 6 This is a high-resolution transmission electron microscopy (HRTEM) image of Fe3O4@Au MNPs prepared in Example 1 of the present invention.

[0040] Figure 7 This is the element mapping image of the gold element in the Fe3O4@Au MNPs prepared in Example 1 of the present invention.

[0041] Figure 8 This is an element mapping image of the iron element in the Fe3O4@Au MNPs prepared in Example 1 of the present invention.

[0042] Fig. 9 This is the element mapping image of oxygen in Fe3O4@Au MNPs prepared in Example 1 of the present invention.

[0043] Fig.10 This is the energy dispersive X-ray (EDX) spectrum of Fe3O4@Au MNPs prepared in Example 1 of the present invention.

[0044] Fig.11 This is the ultraviolet-visible (UV-vis) absorption spectra of Fe3O4, Fe3O4@PEI, and Fe3O4@Au MNPs in Example 1 of the present invention.

[0045] Fig.12 is the DSNB (1×10 -1 M) and DSNB (1×10 -8M) Raman spectrum of labeled Fe3O4@Au MNPs.

[0046] Fig.13 1170cm in Example 2 of the present invention -1 and 1346cm -1 Figure 3. The relationship between the intensity of the characteristic peak and the hpDNA concentration.

[0047] Fig.14 This is an optimization diagram of the hybridization time of the SERS sensor prepared in Example 2 of the present invention.

[0048] Fig.15 The SERS spectra of different targets detected by the SERS sensor in Example 3 of the present invention are shown.

[0049] Fig.16 The SERS spectra of the SERS sensor in Example 3 of the present invention for detecting different targets are at 1170 cm -1 and 1346cm -1 Histogram of the intensity at .

[0050] Fig.17 These are SERS spectra of the SERS sensor in Example 3 of the present invention after storage for 0 days, 7 days, 14 days and 21 days respectively.

[0051] Fig.18 The SERS spectra at 1170 cm after storage for 0 d, 7 d, 14 d and 21 d in Example 3 of the present invention are shown in FIG. -1 and 1346cm -1 Line graph of SERS intensity at

[0052] Fig.19 This is a SERS spectrum diagram of the same sample detected by three SERS sensors in Example 3 of the present invention.

[0053] Fig. 20 The SERS spectra of the same sample detected by the three SERS sensors in Example 3 of the present invention are at 1170 cm -1 and 1346cm -1 Histogram of SERS intensity at .

[0054] Fig.21 The SERS spectra of the SERS sensor in Example 4 of the present invention detecting mixed solutions of miR-21 and miR-122 of different concentrations.

[0055] Fig. 22 is the logarithm of the concentration of miR-21 in Example 4 of the present invention and 1170 cm -1 Linear fitting diagram of SERS intensity at .

[0056] Fig.23 is the logarithm of the concentration of miR-122 in Example 4 of the present invention and 1346 cm -1 Linear fitting diagram of SERS intensity at .

[0057] Fig.24 This is the average SERS spectrum of the serum of healthy subjects and CRC patients in Example 4 of the present invention.

[0058] Fig.25 The average SERS spectrum of the serum of healthy people and CRC patients in Example 4 of the present invention at 1170 cm -1 and 1346cm -1 Histogram of SERS signal intensity at . DETAILED DESCRIPTION

[0059] The present invention is further described in detail below in conjunction with specific embodiments.

[0060] It should be noted that these embodiments are only used to illustrate the present invention rather than to limit the present invention. Simple improvements to the method based on the concept of the present invention all fall within the scope of protection claimed by the present invention.

[0061] The instruments and test conditions used in the present invention are as follows:

[0062] TEM images, SAED images, elemental mapping images and EDX spectra were obtained using a Tecnai G2F30S-TWIN field emission transmission electron microscope produced by Hitachi, Japan.

[0063] Raman and SERS spectra were measured by Invia Reflex laser microscope Raman spectrometer produced by Renishaw, UK. The test conditions were laser wavelength of 785nm, exposure time of 10s, laser intensity of 50mW, and 50× objective lens.

[0064] The UV-vis absorption spectrum was measured by a Cary UV-5000 ultraviolet-visible absorption spectrometer produced by Agilent Corporation of the United States.

[0065] Example 1

[0066] Preparation and characterization of Fe3O4@Au MNPs

[0067] See Figure 1 , using electrostatic adsorption, negatively charged gold nanoparticles AuNPs were modified onto the surface of positively charged polyethyleneimine-modified ferroferric oxide nanoparticles Fe3O4@PEI to obtain gold-coated ferroferric oxide magnetic nanoparticles Fe3O4@Au MNPs:

[0068] 1) After 400 mL of 0.25 mM HAuCl4 solution and 4 mL of 30.57 mM sodium citrate solution were fully mixed, 6 mL of 200 mM NaBH4 solution was added. The mixed solution was vigorously stirred at room temperature for 4 h until the solution turned into wine red, indicating that the reaction was complete and negatively charged AuNPs were obtained.

[0069] 2) Add 1 mL of Fe3O4 solution (10 mg / mL) to 100 mL of ultrapure water and perform intense ultrasound for 10 minutes to disperse the Fe3O4 evenly. Then add 10 mL of PEI solution (0.5 mg / mL) and perform ultrasound to rapidly modify the polyamino cationic polymer PEI on the surface of the Fe3O4 particles. After 30 minutes of reaction, use a micromagnet to enrich and separate the product (Fe3O4@PEI), and wash it several times with ultrapure water to remove excess PEI. Resuspend the Fe3O4@PEI in 10 mL of ultrapure water to obtain positively charged Fe3O4@PEI.

[0070] 3) 40 mL of the negatively charged AuNPs synthesized in step 1) was mixed with 1 mL of the positively charged Fe3O4@PEI synthesized in step 2) and ultrasonicated for 30 min to firmly bind the AuNPs to the surface of Fe3O4@PEI. The separated product was enriched using a micro magnet and resuspended in 5 mL of ultrapure water to form Fe3O4@Au MNPs.

[0071] Characterization of morphology and SERS effect of Fe3O4@Au MNPs.

[0072] The morphology, SAED images, elemental mapping images and EDX spectra of Fe3O4@Au MNPs were detected by TEM. -8 M DSNB solution was stored at 4°C. 10 μL of 1×10 -8 M of DSNB solution was added into 0.1 mL of the prepared Fe3O4@Au MNPs solution, mixed evenly, and the SERS spectrum was detected after standing for 0.5 h.

[0073] See also Figure 2 , AuNPs with a diameter of 3nm are uniformly covered on the 100nm Fe3O4 surface, and the nanogaps between Au particles can generate a large number of “hot spots” to enhance the SERS effect.

[0074] See also Figure 3 , showing that the prepared Fe3O4@Au MNPs have uniform morphology and size and good dispersibility.

[0075] See also Figure 4, the lattice spacing of Au MNPs is 0.24 nm, and the lattice fringes correspond to the {111} crystal planes of Au.

[0076] See also Figure 5 , the diffraction rings represent the {111}, {200}, {220} and {311} crystal planes, respectively, illustrating the polycrystalline structure of Fe3O4@Au MNPs.

[0077] See also Figure 6 , showing the TEM image structure of Fe3O4@Au MNPs.

[0078] See also Figure 7-9 The EDS imaging in the figure shows the element distribution of Fe3O4@Au MNPs, with Fe concentrated in the central area and Au concentrated in the outside, suggesting a core-shell structure of Fe3O4@Au MNPs.

[0079] After further analysis by EDX, see Fig.10 , obvious Fe, O and Au peaks can be seen, and the peak of Fe is higher than the peak of Au, indicating that the proportion of Fe element is higher than that of Au element, and the peaks of Cu and C are attributed to the copper mesh.

[0080] Fig.11 The UV-vis absorption spectra of Fe3O4, Fe3O4@PEI and Fe3O4@Au MNPs show that Fe3O4@Au MNPs has an obvious absorption peak at 558nm.

[0081] Fig.12 DSNB (1×10 -1 M) and DSNB (1×10 -8 From the SERS spectrum of Fe3O4@Au MNPs labeled with DSNB, it can be clearly observed that the Raman signal of high concentration DSNB is weak, while the SERS signal of Fe3O4@Au MNPs labeled with DSNB is significantly enhanced. This shows that Fe3O4@Au MNPs have excellent SERS enhancement effect. The enhancement factor (EF) of Fe3O4@Au MNPs is calculated by the following formula: EF=(I SERS / C SERS ) / (I RS / C RS ), where I and C represent the Raman signal intensity and concentration of the analyte, respectively, with DSNB at 1341 cm -1 The characteristic peak at is used to calculate EF. SERS =1×10 -8 M,C RS =1×10 -1 When M, the EF value of Fe3O4@Au MNPs was calculated to be 3.11×108 This is because the coupling between AuNPs and Fe3O4 particles produces a large number of “hot spots”, resulting in a strong SERS signal.

[0082] Example 2

[0083] Preparation of SERS sensor

[0084] A double-stranded nucleic acid structure formed by a hairpin DNA (hpDNA) specifically binding to a relevant serum biomarker and a single-stranded DNA (ssDNA) partially complementary to the hairpin DNA was modified on the surface of gold-coated ferroferric oxide magnetic nanoparticles Fe3O4@Au MNPs to obtain a SERS sensor for detecting serum biomarkers related to colorectal cancer:

[0085] 1) Example 1 Preparation of Fe3O4@Au MNPs.

[0086] 2) 10 μL of 5-FAM labeled hpDNA1 and molecular Cy5 labeled hpDNA2 mixed solution of different concentrations (10nM to 100 μM), and 10 μL of ssDNA1 and ssDNA2 mixed solution were added to PBS buffer and incubated at 37°C for 12 hours to generate double-stranded nucleic acid structures. Next, 40 μL of double-stranded nucleic acid structure and 0.5 mL of 3.5 mM magnesium chloride solution were added to 5 mL of the Fe3O4@Au MNPs solution prepared above, and hpDNA was attached to the Fe3O4@Au MNPs surface through Au-S bonds. After stirring for 12 hours, the product was centrifuged for 10 minutes and resuspended in Tris-HCl buffer, enriched and separated using a micro magnet, and washed several times with PBS buffer. SERS detection was performed at room temperature to screen the optimal concentration of hpDNA.

[0087] 3) The mixed solution of miR-21 and miR-122 to be tested was incubated with the SERS sensor constructed with the optimal concentration of hpDNA and placed in a 37°C incubator for hybridization reaction. Every 5 minutes, a micro magnet was used to enrich and separate, and the samples were washed several times with PBS buffer. SERS detection was performed at room temperature to screen the optimal hybridization time. Three locations were randomly selected in the test area for SERS detection, and the average SERS spectrum was obtained.

[0088] The concentration of hpDNA determines the number of double-stranded nucleic acid structures assembled on the surface of Fe3O4@Au MNPs. Fig.13 As shown in the figure, as the concentration of hpDNA gradually increased, the 1170cm -1 and 1346cm -1The SERS intensity at the position first increases and then gradually stabilizes. This is because the hpDNA modified on the Fe3O4@Au MNPs has reached saturation. Therefore, hpDNA with a concentration of 10 μM or above is selected to prepare SERS sensors.

[0089] The hybridization time between SERS sensors and target miRNAs is another key parameter to achieve good performance of SERS sensors. Fig.14 As shown, 1170cm -1 and 1346cm -1 The SERS intensity at the position increases with time, and when the time reaches 10 min, the growth trend almost disappears. Therefore, the optimal hybridization time is 10 min and above.

[0090] Example 3

[0091] Specificity, stability and reproducibility of SERS sensors

[0092] 1) Example 2: Construction of SERS sensor.

[0093] 2) 10 μL of target miRNAs (miR-21 and miR-122), random sequence miRNA, single-base mismatch miRNA and triple-base mismatch miRNA (see Table 1) were incubated with the SERS sensor respectively, and the optimal hybridization time in Example 2 was hybridized in a 37°C incubator. Use a micromagnet to enrich and separate, and wash several times with PBS buffer. Perform SERS detection at room temperature to evaluate the specificity of the SERS sensor. The sensor was placed at room temperature for 0d, 7d, 14d and 21d, and the mixed solution of miR-21 and miR-122 was subjected to SERS detection to evaluate the stability of the SERS sensor. Prepare 3 different SERS sensors and use them to detect the same sample to evaluate the reproducibility of the SERS sensor.

[0094] Table 1 Nucleotide sequences used in the specificity experiment of SERS sensor

[0095]

[0096] like Fig.15 and 16 As shown, the target miRNAs group is at 1170 cm -1 and 1346cm -1 The characteristic peak intensity at the 400 nm position was significantly higher than that of the blank group and the interference group. Therefore, the SERS sensor can effectively and specifically identify miR-21 and miR-122.

[0097] Fig.17 and 18It shows that after 21 days of storage, the sensor is 1170cm -1 and 1346cm -1 The SERS intensity at the two locations only decreased by 7.33% and 5.86%, respectively, showing good stability.

[0098] like Fig.19 and 20 As shown, there is no obvious difference in the waveform and intensity of the SERS spectra of the same sample detected by three different SERS sensors.

[0099] 1170cm -1 and 1346cm -1 The SERS signal intensity at Fig. 20 As shown, the relative standard deviations (RSDs) are 5.03% and 5.78%, respectively, indicating that the SERS sensor has good reproducibility.

[0100] Example 4

[0101] SERS sensor detects miR-21 and miR-122 in the sample to be tested

[0102] 1) Example 2 Preparation of SERS sensor.

[0103] 2) Add miR-21 and miR-122 to serum to prepare mixed solutions of miRNAs with different concentrations (C miR-21 :C miR-122 =1:1). The mixed solution of miR-21 and miR-122 with different concentrations was added to the centrifuge tube containing the SERS sensor for detecting CRC-related serum biomarkers miR-21 and miR-122, and placed in a 37°C incubator for hybridization reaction. The cells were enriched and separated using a micro magnet, and washed several times with PBS buffer. Finally, SERS detection was performed at room temperature. According to the 5-FAM and Cy5 at 1170 cm -1 and 1346cm -1 The working curves between the logarithm of miR-21 and miR-122 concentrations and SERS signal intensity were drawn for the characteristic peaks.

[0104] 3) Collect 30 samples of serum from healthy people and 30 samples of serum from CRC patients. Add the samples to be tested into a centrifuge tube equipped with a SERS sensor for detecting CRC-related serum biomarkers miR-21 and miR-122, and place it in a 37°C incubator for the optimal hybridization time in Example 3 of the hybridization reaction. Use a micro magnet to enrich and separate, and wash several times with PBS buffer. Perform SERS detection at room temperature.

[0105] Fig.21The SERS spectra obtained by reacting the SERS sensor with different concentrations of miRNAs mixed solutions (10aM, 100aM, 1fM, 10fM, 100fM, 1pM, 10pM). The SERS signal gradually becomes stronger with the increase of concentration.

[0106] like Fig. 22 As shown in the figure, in the concentration range of 10aM to 10pM, the logarithm of miR-21 concentration and 1170cm -1 The SERS signal intensity at the position is linearly related, and the linear regression equation is y=2342.10x+43127.65, and the correlation coefficient (R 2 )=0.982.

[0107] like Fig.23 As shown, the logarithm of miR-122 concentration is related to 1346 cm -1 The SERS signal intensity at the position has a good linear relationship, and the linear regression equation is y = 2374.92x + 42999.05, R 2 =0.986.

[0108] The LODs of SERS sensors for miR-21 and miR-122 were calculated to be 6.39aM and 6.02aM, respectively. The results showed that the constructed SERS sensor had highly sensitive sensing performance for trace miR-21 and miR-122 in serum.

[0109] Furthermore, a detection system was designed based on the detection process of the above-mentioned CRC-related serum biomarkers miR-21 and miR-122, including an acquisition module, a detection module, and a calculation module;

[0110] The collection module is used to collect serum test samples;

[0111] The detection module is used to perform SERS detection on the collected serum test sample using the SERS sensor for detecting colorectal cancer-related serum biomarkers as described in claim 3 or 4;

[0112] The calculation module is used to convert the SERS detection results at 1170 cm -1 and 1346cm -1 The SERS intensity of the characteristic peak at was substituted into the working curve to calculate the concentration of miR-21 and miR-122 in the serum test sample.

[0113] Serum samples of 30 healthy subjects and 30 CRC patients were collected according to imaging examination, colonoscopy and pathological sections.

[0114] Fig.24The average SERS spectra of miR-21 and miR-122 in serum samples of healthy subjects and CRC patients are shown in Figure 2. The results show that the SERS signal intensity of CRC patients is much higher than that of healthy subjects.

[0115] Fig.25 1170cm -1 and 1346cm -1 The histogram of SERS signal intensity was plotted, and the SERS signal intensity was substituted into the corresponding working curve to calculate the concentration of miR-21 and miR-122. At the same time, the SERS results were compared with the qRT-PCR detection results. Table 2 shows that the detection results of the two methods are consistent, and the RSD is less than 10%. Therefore, the SERS sensor has good accuracy and clinical application potential for early diagnosis of CRC.

[0116] Table 2 Expression levels of miR-21 and miR-122 in serum samples detected by SERS and qRT-PCR

[0117]

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A SERS sensor for detecting serum biomarkers related to colorectal cancer, characterized in that: The double-stranded nucleic acid structure is formed by modifying the surface of gold-coated ferroferric oxide magnetic nanoparticles with Raman signal molecules, a hairpin structure DNA specifically binding to relevant serum biomarkers, and a single-stranded DNA partially complementary to the hairpin structure DNA; The nucleotide sequences of the hairpin structure DNA specifically binding to the relevant serum biomarker and labeled by Raman signal molecules are shown in SEQ ID No.3 and SEQ ID No.4; the nucleotide sequences of the single-stranded DNA partially complementary to the hairpin structure DNA and labeled by Raman signal molecules are shown in SEQ ID No.5 and SEQ ID No.6; The colorectal cancer-related serum biomarkers include miR-21 and miR-122; The method for preparing the SERS sensor for detecting colorectal cancer-related serum biomarkers comprises: S10, using electrostatic adsorption to modify negatively charged gold nanoparticles onto the surface of positively charged polyethyleneimine-modified ferroferric oxide nanoparticles to prepare gold-coated ferroferric oxide magnetic nanoparticles; S20, modifying the surface of gold-coated ferroferric oxide magnetic nanoparticles with a double-stranded nucleic acid structure formed by a hairpin structure DNA specifically binding to a relevant serum biomarker and a single-stranded DNA partially complementary to the hairpin structure DNA, to obtain a SERS sensor for detecting serum biomarkers related to colorectal cancer; The S10 includes: S11, 400 mL of 0.25 mM chloroauric acid solution and 4 mL of 30.57 mM sodium citrate solution were mixed, and then 6 mL of 200 mM NaBH4 solution was added, and the mixture was stirred at room temperature until the solution turned into wine red to obtain negatively charged gold nanoparticles; S12, adding 1 mL of 10 mg / mL Fe3O4 solution to 100 mL of ultrapure water, ultrasonicating for 10 min, then adding 10 mL of 0.5 mg / mL PEI solution, ultrasonicating for 30 min, using a micro magnet to enrich and separate the product, and removing excess PEI, resuspending in 10 mL of ultrapure water to obtain positively charged polyethyleneimine-modified ferrosoferric oxide nanoparticles; S13, mixing the negatively charged gold nanoparticles with the positively charged polyethyleneimine-modified ferroferric oxide nanoparticles, ultrasonicating for 30 min, enriching and separating the products using a micro magnet, and resuspending them in 5 mL of ultrapure water to obtain gold-coated ferroferric oxide magnetic nanoparticles; Wherein, the volume ratio of the negatively charged gold nanoparticles to the positively charged polyethyleneimine-modified ferroferric oxide nanoparticles is 40-60:1; The S20 includes: S21, adding 10 μL of a mixed solution of 5-FAM-labeled hairpin structure DNA1 and Cy5-labeled hairpin structure DNA2, and 10 μL of a mixed solution of single-stranded DNA1 and single-stranded DNA2 into PBS buffer, and incubating at 37°C for 12 h to generate a double-stranded nucleic acid structure; S22, adding 40 μL of double-stranded nucleic acid structure and 0.5 mL of 3.5 mM magnesium chloride solution into 5 mL of gold-coated ferroferric oxide magnetic nanoparticle solution, the hairpin structure DNA is attached to the surface of the gold-coated ferroferric oxide magnetic nanoparticles through Au-S bonds, and after stirring, the product is centrifuged for 10 min and resuspended in Tris-HCl buffer to obtain a SERS sensor for detecting serum biomarkers related to colorectal cancer; The concentrations of the 5-FAM-labeled hairpin structure DNA1 and the Cy5-labeled hairpin structure DNA2 are not less than 10 μM.

2. The SERS sensor for detecting colorectal cancer-related serum biomarkers according to claim 1, characterized in that: The nucleotide sequences of miR-21 and miR-122 are shown in SEQ ID No.1 and SEQ ID No.

2.

3. A system for detecting serum biomarkers related to colorectal cancer, characterized in that: It includes an acquisition module, a detection module and a calculation module; The collection module is used to collect serum test samples; The detection module is used to perform SERS detection on the collected serum test sample using the SERS sensor for detecting colorectal cancer-related serum biomarkers according to claim 1 or 2; The calculation module is used to calculate the results of SERS detection at 1170 cm -1 and 1346cm -1 The SERS intensity of the characteristic peak at was substituted into the working curve to calculate the concentration of miR-21 and miR-122 in the serum test sample; The working curve is: y1=2342.10x1+43127.65, where y1 is 1170cm -1 is the SERS intensity, x1 is the concentration of miR-21 in the serum test sample; y2=2374.92x2+42999.05, where y2 is 1346cm -1 is the SERS intensity, x2 is the concentration of miR-122 in the serum test sample; In the SERS detection of the collected serum test sample, the hybridization reaction time between the SERS sensor and the serum test sample is not less than 10 minutes.

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