Gold nano pumpkin array, SERS (Surface Enhanced Raman Scattering) sensor for detecting gastric precancerous lesion biomarkers as well as preparation method and application of SERS sensor

Abstract

The invention belongs to the technical field of biological detection, particularly relates to a gold nano pumpkin array, and relates to an SERS (Surface Enhanced Raman Scattering) sensor of a gastric precancerous lesion biomarker as well as a preparation method and application thereof. The preparation method of the gold nano pumpkin arrays (Au NPAs) comprises the steps that a PS single-layer template is obtained through a gas-liquid interface self-assembly technology, the PS single-layer template is sequentially subjected to plasma etching technology treatment, annealing treatment and ion sputtering treatment, and the gold nano pumpkin arrays (Au NPAs) are obtained. The SERS sensor and the SERS microarray chip constructed on the basis of the gold nano pumpkin array show excellent chemical stability, high molecular specificity and excellent reproducibility, ultra-trace detection of gastric cancer precancerous lesion biomarkers miR-196b and miR-221 is realized, and technical support is provided for early discovery, timely intervention and treatment of GC (Gas Chromatography).

Description

Technical Field

[0001] The present invention relates to the field of biological detection technologies, and particularly to gold nano-pumpkin arrays, SERS sensors for detecting biomarkers of precancerous gastric lesions, and preparation methods and applications thereof. Background Art

[0002] Gastric cancer (GC) is one of the most common malignant tumors in the field of the digestive system, and its incidence and mortality rates continue to rank among the top of malignant tumors. The occurrence and development of GC follow a complex biological carcinogenesis process driven by multiple stages and multiple factors. Existing screening methods such as the urea breath test (UBT) and imaging (CT) examinations are often used as auxiliary means due to their deficiencies in sensitivity and specificity, and need to be comprehensively evaluated in combination with clinical aspects. Although gastroscopy + pathological biopsy, as the gold standard for diagnosis, is accurate, it is difficult to popularize screening due to disadvantages such as strong invasiveness and poor patient compliance. In view of this, developing non-invasive detection technologies with both high sensitivity and reliability has become a key requirement for early screening and blocking of precancerous gastric lesions.

[0003] Nanozymes are a new type of catalyst that combines the catalytic characteristics of biological enzymes and the advantages of artificial catalysis. A new generation of catalysts represented by noble metal nanozymes such as gold (Au), platinum (Pt), and palladium (Pd) exhibit revolutionary advantages by precisely regulating the kinetics of enzymatic reactions. Compared with natural enzymes, these materials not only have a catalytic activity increased by several orders of magnitude, but also have characteristics such as high stability, low cost, and resistance to extreme environments, breaking through the technical bottleneck of insufficient sensitivity in traditional colorimetry and electrochemical detection. Surface-enhanced Raman spectroscopy (SERS), as an innovative technology in the field of molecular vibration spectroscopy, realizes an exponential amplification of Raman scattering signals by up to 10 10 -10 12 times through the local electromagnetic field enhancement effect of a rough metal surface at the nanoscale. This technology relies on the strong near-field enhancement effect generated by local surface plasmon resonance (LSPR), greatly improving the detection sensitivity of molecular "fingerprint" information. Its unique molecular vibration "fingerprint" recognition ability provides a non-invasive, fast, and highly sensitive solution for biomarker detection.

[0004] CHA is an enzyme-free amplification strategy based on hairpin DNA, which achieves exponential amplification of signals through strand displacement reactions. Compared with other signal amplification strategies such as polymerase chain reaction (PCR) and hybridization chain reaction (HCR), CHA has a unique cyclic amplification mechanism and does not require temperature cycling and participation, and can rapidly enhance the SERS signal. By integrating the CHA amplification strategy with SERS technology, a novel molecular diagnostic system was constructed. This platform innovatively combines the electromagnetic field enhancement effect of SERS with the intelligent signal amplification function of CHA: the SERS substrate (such as gold-platinum nanoparticles) not only provides a Raman signal enhancement interface, but its nanozyme properties can also catalyze color reactions; while the CHA system converts the target recognition event into the aggregation of reporter molecules, and realizes the detection of trace markers through the cascade amplification of "recognition-amplification-transduction". This dual-functional synergistic strategy opens up a new way for the ultrasensitive detection of gastric cancer biomarkers. Summary of the Invention

[0005] Based on this, the present invention provides a gold nanopyramid array (Au NPAs), a SERS sensor for detecting precancerous biomarkers of gastric cancer, and its preparation method and application.

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

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] On the one hand, the present invention provides a gold nanopyramid array (AuNPAs), and its preparation method includes: obtaining a PS monolayer template through gas-liquid interface self-assembly technology, and sequentially performing plasma etching technology treatment, annealing treatment and ion sputtering treatment on the PS sphere monolayer template to obtain a gold nanopyramid array (Au NPAs).

[0009] Preferably, the preparation method of the above gold nanopyramid array (Au NPAs) includes:

[0010] 1) Mix a PS microsphere suspension with a size of 120 nm and ethanol at a volume ratio of 1:1, and obtain a PS monolayer template through gas-liquid interface self-assembly technology;

[0011] 2) Using plasma etching technology (RIE), with SF6, O2, and Ar gases flowing at a rate of 20 sccm / 5 sccm / 10 sccm to obtain reaction gases, under the condition that the etching power is set to 100 W, the sample is etched for 3 minutes to form an ordered array of a PS microsphere / nanocone silicon hybrid structure; then, an annealing treatment is carried out to obtain a silicon cone array with a nano-platform on the surface; finally, an ion sputtering technology (IBS) treatment is used to obtain a gold nanopyramid array.

[0012] More preferably, in the method for preparing the above-mentioned gold nano-pumpkin arrays (AuNPAs), in step 2), the temperature of the annealing treatment is 350 °C and the time is 30 min; the ion sputtering technology treatment sputters for 1 min with a current of 20 mA, and then sputters for 4 min with a large current of 30 mA.

[0013] On the other hand, the present invention provides a method for preparing a SERS sensor for detecting biomarkers of precancerous gastric lesions, and the preparation method includes:

[0014] 1) Synthesize platinum-coated gold nanorods (Au@PtNRs) by the seed growth method;

[0015] 2) Modify the AuNPAs array described in any one of claims 1 to 3 with hairpin DNA-HP2 (HP2-1 and HP2-2) to obtain two functionalized AuNPAs arrays, Au NPAs@HP2-1 and Au NPAs@HP2-2; embed the AuNPAs array into the PDMS layer to obtain a SERS microarray chip;

[0016] 3) Modify the Au@PtNRs synthesized in step 1) with hairpin DNA-HP1 (HP1-1 and HP1-2) to obtain two SERS nanoprobes, Au@PtNRs@HP1-1 and Au@PtNRs@HP1-2;

[0017] 4) Drop the target and the nanoprobe obtained in step 3) onto the detection area of the SERS microarray chip. The target triggers the catalytic hairpin self-assembly (CHA) amplification strategy, and the probe assembles on the surface of AuNPAs due to the complementary pairing of the HP1 strand and the HP2 strand, constructing a "bifunctional" SERS sensing system with high peroxidase (POD) activity and excellent SERS enhancement ability;

[0018] Among them, the sequence of HP1-1 is shown in SEQ ID NO.1, the sequence of HP1-2 is shown in SEQ ID NO.2, the sequence of HP2-1 is shown in SEQ ID NO.3, and the sequence of HP2-2 is shown in SEQ ID NO.4.

[0019] Preferably, the above preparation method satisfies one or more combinations of the following conditions:

[0020] (i) Step 1) includes:

[0021] 1.1) Add HAuCl4 to the CTAB solution, and then quickly inject the NaBH4 solution into the above solution under vigorous stirring to obtain a gold seed solution (Au NRs);

[0022] 1.2) Dissolve CTAC and NaOL sufficiently in an aqueous solution to obtain a mixed solution; add the AgNO3 solution and the HAuCl4 solution to the above-mentioned mixed solution, and stir at 30 °C for 150 min; subsequently, add the HCl solution and stir for another 15 min; finally, add the AA solution and stir vigorously for 30 min to prepare a growth solution; then quickly add 0.4 - 0.6 mL of the gold seed solution to the above growth solution, stir for 1 min, and let it stand overnight at 30 °C for growth to obtain AuNRs.

[0023] 1.3) Sequentially add the Au NRs seed solution, the sodium chloroplatinate (Na2PtCl4) solution, and the AA solution to the cetylpyridinium chloride monohydrate (CPC) solution, stir vigorously, and then let it stand at room temperature. After centrifugal purification, Au@PtNRs are prepared.

[0024] (ii) Step 2) includes:

[0025] 2.1) Dissolve the hairpin DNA (HP1 and HP2) in the PBS buffer solution, and subject it to denaturation treatment at a high temperature of 95 °C for 5 min; incubate with the TCEP solution for 2 h to activate the hairpin structure; modify the AuNPAs array with the activated hairpin DNA - HP2 (HP2 - 1 and HP2 - 2) to obtain two functionalized Au NPAs arrays, Au NPAs@HP2 - 1 and AuNPAs@HP2 - 2; embed the above - mentioned functionalized arrays into the PDMS layer to obtain the SERS microarray chip.

[0026] 2.2) Based on the AutoCAD software, design the size and structure of the microarray chip. The microarray chip consists of two parts: detection area 1 integrating 8 circular areas with a corresponding diameter of 4 mm and detection area 2 configured with 8 square areas with a corresponding side length of 4 mm; first, use soft lithography technology to prepare a mold on the silicon wafer that matches the chip design; then, evenly pour the vacuum - treated PDMS prepolymer and cross - linker mixture (mass ratio 10:1) onto the surface of the mold, and place it in an oven for curing treatment (80 °C, 2 h); after curing, carefully peel the PDMS layer from the master mold, cut, punch holes, and clean it according to the design drawing; subsequently, use a plasma machine to perform ultrasonic treatment on the PDMS layer for 1 min. Finally, through plasma bonding technology, seal the hydrophilized PDMS layer with the glass slide and embed the functionalized AuNPAs prepared in step 2.1) to obtain the SERS microarray chip.

[0027] (iii) Step 3) includes:

[0028] Add the activated hairpin DNA-HP1 (HP1-1 and HP1-2) solution to the Au@PtNRs solution prepared in step 1) and react. Then disperse the obtained mixed solution in the BSA solution and incubate to obtain functionalized nanoprobes, Au@Pt NRs@HP1-1 and Au@PtNRs@HP1-2;

[0029] (iv) Step 4) includes:

[0030] 4.1) Add miR-196b and miR-221 to fetal bovine serum respectively to prepare miRNAs solutions with different concentrations, and obtain two targets with different concentrations;

[0031] 4.2) Drop the two targets with different concentrations prepared in step 4.1) and the nanoprobes prepared in step 3) onto the detection area of the SERS microarray chip prepared in step 3) for incubation. Au@Pt NRs are assembled on the surface of AuNPAs through the CHA technique. After incubation, rinse off the unbound Au@PtNRs with PBS buffer to prepare a SERS sensing system.

[0032] More preferably, in the above preparation method,

[0033] In step 1.3), set the reaction temperature condition to 65 °C, stir vigorously for 2 min, and let stand for 30 min; and / or

[0034] In step 2.1), incubate HP2 with Au NPAs at 25 °C for 12 h; and / or

[0035] In step 3), the volume ratio of the Au@Pt NRs solution to the BSA solution is 50:1, the reaction temperature is 25 °C, the reaction time is 12 h, and the incubation time is 1 h; and / or

[0036] In step 4.2), the incubation time is 20 min.

[0037] On the other hand, the present invention provides a SERS sensor for detecting biomarkers of precancerous gastric lesions prepared by the preparation method in the present invention.

[0038] On the other hand, the present invention provides a method for detecting miR-196b and miR-221 in a sample to be detected by the SERS sensor for detecting biomarkers of precancerous gastric lesions in the present invention. The method includes:

[0039] 1) Add TMB solution and H2O2 solution to the SERS sensing system in the present invention. The solvent for both is PBS buffer (pH = 4.0). React for 15 min at 30 °C, and the reaction solution will show an obvious blue color. Subsequently, perform Raman spectroscopy detection on the detection area, capture the characteristic signal of oxTMB, and construct a concentration logarithm-signal intensity working curve based on the wavenumber at 1606 cm -1 to build a concentration logarithm-signal intensity working curve.

[0040] 2) Collect 30 serum samples from healthy people, patients with precancerous gastric lesions, and GC patients. Drop the samples to be detected and the nanoprobes onto the detection area of the SERS microarray chip, and place them in an incubator at 30 °C for the CHA reaction. After reacting for a certain time, wash several times with PBS buffer to obtain a bifunctional SERS sensor. Add TMB solution and H2O2 solution to the bifunctional SERS sensing system for reaction. The solvent of the solution is PBS buffer (pH = 4.0). When the nanozyme catalyzes the reaction of TMB and H2O2, blue oxTMB can be generated. Subsequently, perform Raman spectroscopy detection on the detection area, capture the characteristic signal of oxTMB, and substitute the intensity of the characteristic peak of oxTMB obtained above at 1606 cm -1 into the working curve determined in step 1) to determine the concentrations of miR-196b and miR-221 in the samples to be detected.

[0041] 3) Substitute the intensity of the characteristic peak of the samples to be detected obtained in step 2) at 1606 cm -1 into the working curve determined in step 1) to determine the concentrations of miR-196b and miR-221 in the samples to be detected.

[0042] Preferably, in the above method, the CHA reaction in step 2) is 20 min; the reaction temperature for adding TMB solution and H2O2 solution to the bifunctional SERS sensing system is 30 °C, and the reaction time is 15 min.

[0043] Compared with the existing miRNA detection methods, the present invention has the following advantages:

[0044] The surface of the Au NPAs array presents rough near-spherical particles, and high-density "hot spots" can be generated in the existing nanogaps, which can significantly enhance the Raman signal intensity and greatly enhance the sensitivity of SERS detection. The CHA technology further amplifies the SERS signal. When the target is present, Au@Pt NRs are assembled on the surface of the Au NPAs array due to the complementary pairing of HP1 and HP2 chains, and a target-triggered SERS sensing system is successfully constructed. This system has "dual functions" of high-efficiency peroxidase-like (POD) activity and excellent SERS enhancement ability, catalyzes the generation of oxTMB from TMB, and CHA further amplifies the SERS signal. According to the oxTMB at 1606 cm-1 The SERS signal intensity at [specific location] was used to obtain the concentrations of miR-196b and miR-221. This SERS sensing system can quantitatively detect miR-196b and miR-221 within a wide linear range, with an LOD as low as the aM level. It has the advantages of high sensitivity, strong specificity, simple operation, and high throughput, achieving ultra-trace detection of the gastric precancerous biomarker miR-196b and miR-221, providing technical support for the early detection, timely intervention, and treatment of GC. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a schematic diagram of the preparation process of the SERS microarray chip of the present invention and the detection of gastric precancerous biomarkers miR-196b and miR-221.

[0046] Figure 2 It is a characterization diagram of the Au NPAs array of the present invention; among them, A and B are SEM images of the PS monolayer template at different magnifications; C and D are SEM images of the SiNCAs with nano-platforms at different magnifications; E and F are SEM images of the Au NPAs at different magnifications; G is the FDTD simulation image of the Au NPAs; H is the SERS spectrum of pure 4-MBA (10 -1 M) and 4-MBA (10 -8 M) modified Au NPAs; I is the SERS mapping image of the Au NPAs; J is the full spectrum of 30 random points on the surface of the Au NPAs; K is the scatter plot of the SERS intensity at 1075 cm -1 ; L is the line graph of the SERS intensity of the AuNPAs stored under nitrogen conditions at room temperature for 30 days.

[0047] Figure 3 It is a characterization diagram of the Au@PtNRs; among them, A, B, C, D, and E are the SEM image, TEM image, HRTEM image, SAED image, and EDX image and elemental mapping image of the Au@PtNRs respectively; F is the ultraviolet-visible absorption spectrum and solution color change during the catalytic process of the Au@PtNRs; G is the Michaelis-Menten equation curve and double-reciprocal curve of the Au@PtNRs nanozyme; H is the SERS spectrum of pure DTNB (10 - 1 M) and DTNB (10 -8 M) modified Au@PtNRs.

[0048] Figure 4 It is the physical image of the microarray chip at different angles. Among them, A and C are the top views of the physical image of the microarray chip; B and D are the side views.

[0049] Figure 5 PAGE verification based on the CHA sensing strategy; Lane 1: Marker; Lane 2: miR-196b; Lane 3: HP1-1; Lane 4: HP1-1 + HP2-1; Lane 5: HP1-1 + miR-196b; Lane 6: HP1-1 + HP2-1 + miR-196b.

[0050] Figure 6 Optimization of experimental parameters; where A is the CHA reaction time, B is the TMB concentration, C is the H2O2 concentration, D is the pH, E is the temperature; F is the incubation time.

[0051] Figure 7 Evaluation of the reproducibility and specificity of the SERS sensing system; where A and B are the reproducibility evaluations; C is the bar chart of the SERS signal intensity at 1606 cm -1 ; D and E are the specificity evaluations and F is the bar chart of the SERS signal intensity at 1606 cm -1 .

[0052] Figure 8 Sensitivity test of the SERS sensing system; where A is the SERS spectra of different miR-196b concentrations (10 aM - 10 pM) in serum, and B is the linear regression equation of the logarithm of the concentration at 1606 cm -1 versus the SERS signal intensity; C is the SERS spectra of different miR-221 concentrations (10 aM - 10 pM) in serum; D is the linear regression equation of the logarithm of the concentration at 1606 cm -1 versus the SERS signal intensity.

[0053] Figure 9 Characterization of clinical samples; where A, B, and C are the gastroscopy images, CT scan images, and pathological examination images of healthy people respectively; D, E, and F are the gastroscopy images, CT scan images, and pathological examination images of patients with precancerous gastric lesions respectively; G, H, and I are the gastroscopy images, CT scan images, and pathological examination images of GC patients respectively.

[0054] Figure 10 Expression levels of targets in the serum of clinical samples in different disease states; where A and B are the concentration half-box plots of miR-196b and miR-221 in the serum of actual samples detected by the SERS sensing system respectively; C and D are the correlation studies of the expression levels of miR-196b and miR-221 in the serum of actual samples detected by the SERS sensing system and qRT-PCR respectively; E is the ROC curve to evaluate the accuracy of dual-target combined detection in diagnosing healthy people and patients with precancerous gastric lesions; F is the ROC curve to evaluate the accuracy of dual-target combined detection in diagnosing patients with precancerous gastric lesions and gastric cancer patients.

[0055] Figure 11 The average SERS spectra of the targets (miR-196b and miR-221) in the serum of clinical samples under different disease states were measured; among them, A and B are the average SERS spectra of the serum of clinical samples related to miR-196b and miR-221 under different disease states; C and D are the histograms of the characteristic peak intensities of miR-196b and miR-221 at 1606 cm -1 . Detailed implementation manners

[0056] To deepen the understanding of the present invention, the present invention will be further described in detail in conjunction with embodiments and drawings.

[0057] The schematic diagram of the preparation process of the SERS microarray chip of the present invention and the detection of gastric precancerous lesion biomarkers miR-196b and miR-221 is as Figure 1 shown.

[0058] The instruments and equipment used in the present invention and the test conditions are as follows: The optical absorption spectrum of the nanoparticles was measured using a Cary 60 UV-Vis spectrophotometer produced by Agilent Technologies, USA. The morphology of the nanomaterials was characterized using an S-4800 field emission scanning electron microscope produced by Hitachi, Japan. The size and structure of the nanomaterials were characterized using a Tecnai G2 F30 field emission transmission electron microscope produced by FEI Company, USA. The Raman spectrum of the sample was measured using an Invia Reflex laser micro-Raman spectrometer produced by Renishaw, UK. The test conditions were a laser wavelength of 785 nm, an integration time of 10 s, a laser intensity of 50 mW, an objective lens of 50×, and a detection range of 600-1800 cm -1 .

[0059] Example 1 Preparation and characterization of Au NPAs arrays

[0060] 1) A PS microsphere suspension with a size of 120 nm was mixed with ethanol at a volume ratio of 1:1, and a PS monolayer template was obtained by the gas-liquid interface self-assembly technique.

[0061] 2) Using plasma etching technology (RIE), reaction gases were obtained with SF6, O2, and Ar gases flowing at a rate of 20 sccm / 5 sccm / 10 sccm. Under the condition that the etching power was set to 100 W, the sample was etched for 3 min to form an ordered array of a hybrid structure of PS microspheres / nanocone silicon. Then, an annealing treatment was carried out at 350 °C for 30 min to obtain a silicon cone array with a nano-platform on the surface. Finally, using ion beam sputtering technology (IBS), gold was selected as the target material. First, it was sputtered with a current of 20 mA for 1 min, and then sputtered with a large current of 30 mA for 4 min to successfully prepare a gold nanopumpkin array.

[0062] 3) Evaluation of the morphological structure and performance of the Au NPAs array. The morphology of the gold nanopumpkin array was detected by SEM to evaluate the uniformity of the SERS substrate; the EF value was calculated with 4-MBA as the signal molecule to evaluate the SERS activity of the Au NPAs; the surface of the Au NPAs was detected in the form of a large-area and high-precision surface scan to evaluate the signal uniformity of the Au NPAs; the Raman spectra of random points on the surface of the Au NPAs prepared in different batches were detected to evaluate the signal reproducibility of the Au NPAs; the gold nanopumpkin array modified with 4-MBA was stored in air and nitrogen for different times to verify the storage stability of the AuNPAs array.

[0063] Figure 2 A and 2B characterized the morphological changes during the preparation of the Au NPAs array, which were SEM images of the PS monolayer template at different magnifications. The monodisperse PS microsphere solution formed a tight and orderly arranged PS monolayer template after self-assembly at the gas-liquid interface. After the PS monolayer template was treated by plasma etching technology and annealed for 30 min, the remaining PS spheres were burned to obtain a silicon cone array (Si NCAs) with a nano-platform on the surface. Figure 2 C and 2D are SEM images of the intermediate product SiNCAs at different magnifications. The upper layer shows a clear nano-platform, and the lower layer shows a nano-cone structure. Figure 2 E and 2F are SEM images of the Au NPAs at different magnifications. Using Au as the target material for ion beam sputtering, a rough gold film was deposited on the surface of the Si NCAs with a nano-platform to form a gold nanopumpkin array (Au NPAs). The surface of the Au NPAs presents rough near-spherical particles, and high-density "hot spots" can be generated in the existing nano-gaps. Figure 2 G is the FDTD simulation image of the Au NPAs. To understand the "hot spot" distribution of the Au NPAs, a three-dimensional model was constructed using the finite-difference time-domain (FDTD) and the electromagnetic field distribution of this structure was simulated. Figure 2 H is for pure 4-MBA (10 -1M) and 4-MBA (10 -8 SERS spectra of Au NPAs modified with M), using 4-MBA as the signal molecule. According to the formula: EF = (I SERS / C SERS ) / (I Raman / C Raman ), the calculated EF value is 3.48×10 8 , indicating that Au NPAs have excellent SERS activity. Figure 2 I is the SERS mapping image of Au NPAs. The surface of Au NPAs is detected with large area and high precision in the form of area scanning. The obtained SERS mapping image shows large area green, indicating good signal uniformity of Au NPAs. Figure 2 J is the complete spectra of 30 random points on the surface of Au NPAs. Figure 2 K is the scatter plot of the SERS intensity at 1075 cm -1 . The relative standard deviation (RSD) of the Raman intensity at 1075 cm -1 is calculated to be 7.20% through statistics, indicating good signal reproducibility of surface Au NPAs. Figure 2 L is the line chart of the SERS intensity of Au NPAs stored under nitrogen conditions at room temperature for 30 days. The prepared Au NPAs are stored under nitrogen conditions at room temperature for 1 month, and the detection interval is 6 days. The Raman intensity at 1075 cm -1 decreases over time, which can be explained by the degradation of the Au-S bond on the surface of 4-MBA modified Au NPAs. Compared with the initial state, the Raman intensity at 1075 cm- 1 only decreases by 14.01% on the 30th day, and AuNHAs shows good storage stability.

[0064] Example 2 Preparation and Characterization of Au@Pt NRs

[0065] 1) First, Au NRs were synthesized by the seed growth method. HAuCl4 (10 mM, 0.25 mL) was added to the CTAB (200 mM, 5 mL) solution, and then the freshly prepared NaBH4 solution (10 mM, 0.6 mL) was rapidly injected into the above solution under vigorous stirring. The color of the obtained solution changed to a brown-yellow solution, and the gold seed solution (Au NRs) was obtained. The gold seed solution was aged at 30 °C for 30 min before use. 3.08 g of CTAC and 0.77 g of NaOL were fully dissolved in an aqueous solution and made up to 250 mL. The AgNO3 solution (4 mM, 6 mL) and the HAuCl4 solution (10 mM, 250 mL) were added to the above mixed solution, and the mixture was stirred at 30 °C for 150 min. Subsequently, HCl solution (37 wt%, 2 mL) was added, and the mixture was stirred for another 15 min. Finally, AA solution (64 mM, 0.62 mL) was added and stirred vigorously for 30 min to prepare the growth solution. Then, 0.4 - 0.6 mL of the gold seed solution was quickly added to the above growth solution, stirred for 1 min, and left to stand overnight at 30 °C for growth to obtain AuNRs.

[0066] 2) Au@Pt NRs were synthesized by the seed growth method. The temperature was set at 65 °C. To the cetylpyridinium chloride monohydrate (CPC) solution (10 mM, 20 mL), the Au NRs seed solution (2 mL), sodium chloroplatinate (Na2PtCl4) solution (10 mM, 200 μL), and freshly prepared AA solution (100 mM, 400 μL) were added in sequence. After vigorous stirring for 2 min, the mixture was left to stand for 30 min. After centrifugal purification (8000 rpm / min, 5 min), Au@Pt NRs were prepared.

[0067] 3) Characterization of the morphology, structure, and SERS effect of Au@Pt NRs. The morphology and structure of Au@Pt NRs were detected by SEM, TEM, HRTEM, and SAED imaging diffraction patterns. The UV-Vis-NIR absorption spectrum of Au@Pt NRs was detected by an ultraviolet absorption spectrometer. At room temperature, DTNB solutions with concentrations of 10 -1 M and 10 -8 M were prepared, and SERS detection of Au@PtNRs labeled with DTNB (10 -1 M and 10 -8 M) was carried out by a laser micro-Raman spectrometer.

[0068] Figure 3 A is the SEM image of Au@PtNRs. The Au@Pt NRs prepared in this study have good uniformity and can be prepared in batches. Figure 3B is the TEM image of Au@Pt NRs. Au@Pt NRs are rough nanorods with an average length of 92 nm and a width of 40 nm, and the aspect ratio is about 2.3:1. Figure 3 C is the HRTEM image of Au@Pt NRs. Au@Pt NRs exhibit an obvious core-shell structure. The lattice fringes with a spacing of 0.238 nm correspond to the {111} plane of face-centered cubic (FCC) Au, and the lattice fringes with a spacing of 0.225 nm correspond to the FCC Pt {111} plane. Figure 3 D is the SAED pattern of single-crystalline Au@Pt NR. Figure 3 E is the energy-dispersive X-ray spectroscopy (EDX) combined with elemental mapping images. Au@Pt NRs consist of a nanorod core composed of Au element and a Pt nanoparticle shell tightly wrapped on the outer layer. In addition, the detected Cu element comes from the copper mesh used in the experiment. Figure 3 F is the ultraviolet-visible absorption spectrum. The ultraviolet absorption peak of Au@Pt NRs is located at 728 nm, and the solution shows an obvious grayish-black color. Only when Au@Pt NRs + TMB + H2O2 coexist, an obvious absorption peak of oxTMB will appear at 652 nm, and the solution changes from grayish-black to blue. Figure 3 G is to test the peroxidase-like reaction kinetics of Au@Pt NRs as nanozymes, and the Michaelis-Menten equation curve and double-reciprocal curve of this nanozyme are obtained by fitting. The maximum reaction rate (Vmax) of the Au@Pt NRs nanozyme is 28.905 μM s -1 , and the Michaelis constant (Km) is 0.352 mM. Au@Pt NRs have excellent peroxidase-like catalytic activity. Figure 3 H is the SERS spectra of pure DTNB (10 -1 M) and Au@Pt NRs modified with DTNB (10 -8 M). Using DTNB as the signal molecule, the calculated EF value is 1.59×10 6 , indicating that Au@Pt NRs have excellent SERS activity. The comprehensive experimental results prove that Au@Pt NRs have dual functions of nanozyme and SERS activity.

[0069] Example 3 Preparation of SERS Sensing System

[0070] 1) Prepare AuNPAs arrays as in Example 1.

[0071] 2) Prepare Au@Pt NRs as in Example 2.

[0072] 3) Dissolve the hairpin DNA (HP1 and HP2) in PBS buffer solution with a concentration of 20 mM (containing Mg with a concentration of 10 mM) 2+and placed in a buffer solution with pH = 7.4), and denatured at a high temperature of 95 °C for 5 min. Incubate with TCEP solution (1 mM) for 2 h to activate the hairpin structure. The sequence of HP1-1 is shown in SEQ ID NO.1, and the sequence of HP1-2 is shown in SEQ ID NO.2; the sequence of HP2-1 is shown in SEQ ID NO.3, and the sequence of HP2-2 is shown in SEQ ID NO.4.

[0073] 4) Fix the activated hairpin DNA2 (HP2-1 and HP2-1) on the Au NPAs substrate prepared in Example 1 through the binding of Au-S bonds, and incubate at 25 °C for 12 h to obtain functionalized Au NPAs (Au NPAs@HP2-1 and Au NPAs@HP2-2). According to experimental requirements, design the size and structure of the microarray chip based on AutoCAD software. Figure 4 are physical images of SERS microarray chips at different angles. As Figure 4 shown, the microarray chip consists of two parts: detection area 1 integrating 8 circular areas with a corresponding diameter of 4 mm and detection area 2 configured with 8 square areas with a corresponding side length of 4 mm to detect different tumor markers. Embed the two functionalized Au NPAs (Au NPAs@HP2-1 and Au NPAs@HP2-2) substrates into the PDMS chip to obtain the SERS microarray chip.

[0074] 5) Add the activated hairpin DNA1 (HP1-1 and HP1-2) solution to the Au@Pt NRs solution (5 mL) prepared in Example 2, react at 25 °C for 12 h, and then disperse the obtained mixed solution in a BSA (1 wt%, 100 μL) solution and incubate for 1 h. HP1 binds to Au@Pt NRs through Au-S bonds to obtain functionalized nanoprobes Au@Pt NRs (Au@Pt NRs@HP1-1 and Au@Pt NRs@HP1-2). The sequence of HP1-1 is shown in SEQ ID NO.1, and the sequence of HP1-2 is shown in SEQ ID NO.2.

[0075] 6) Add miR-196b and miR-221 to fetal bovine serum respectively to prepare miRNA solutions with different concentrations, and obtain two targets with different concentrations (10 aM - 10 pM). Drop the two targets with different concentrations prepared and the nanoprobes prepared in 5) onto the detection areas of the SERS microarray chip prepared in 4). The targets trigger the CHA reaction, enabling the SERS nanoprobes to be anchored on the Au NPAs array. After 20 min of the CHA reaction, wash away the unbound Au@Pt NRs with PBS buffer to prepare the SERS sensing system.

[0076] Feasibility Analysis of CHA Amplification in Example 4

[0077] To verify the feasibility of CHA amplification, polyacrylamide gel electrophoresis (PAGE) was carried out. After diluting and annealing the synthesized single-stranded DNA, the synthesized single-stranded DNA sample was appropriately diluted and annealed, and then electrophoretic analysis was completed on a 20% polyacrylamide gel system (containing 0.5×TBE buffer and 5% glycerol). First, pre-electrophoresis was carried out under the conditions of 100V / 30min, and then electrophoretic separation was carried out under the conditions of 120V / 100min. After electrophoresis, the gel was placed in an acetate buffer solution (pH = 4.6) and stained with SYBR Gold nucleic acid dye for 30 min. Finally, a gel imaging system was used to take pictures and record the experimental results. The results are as Figure 5 shown: Lane 1: Marker; Lane 2: miR-196b; Lane 3: HP1-1; Lane 4: HP1-1 + HP2-1; Lane 5: HP1-1 + miR-196b; Lane 6: HP1-1 + HP2-1 + miR-196b. The results show that, as shown in Lanes 2, 3, and 4, the target nucleic acid, hairpin probes HP1 and HP2 do not hybridize in the absence of the target miRNA, indicating that the amplification strategy is specific in principle. After adding the target miRNA in Lane 5, HP1 was activated and opened, forming a new nucleic acid complex. In Lane 6, after further adding HP2, a larger nucleic acid complex was formed (Lane 6), demonstrating the feasibility of the CHA signal amplification strategy.

[0078] Example 5 Optimization of Experimental Conditions

[0079] 1) Screening of CHA Reaction Time

[0080] To systematically screen out the optimal reaction time of CHA, the present invention adopted a strategy combining multi-gradient time-point sampling and quantitative characterization, setting time gradients of 5, 10, 15, 20, 25, and 30 min, and respectively detecting the SERS spectra of the sensing system. As Figure 6 shown in A, with the increase of the CHA reaction time, more Au@PtNRs are assembled on the surface of Au NPAs; the SERS sensing system has peroxidase-like activity, catalyzing the reaction of H2O2 with TMB to generate blue oxTMB, which gradually tends to saturation; after the time reaches 20 min, the SERS signal intensity at 1606 cm -1 does not increase significantly, and the optimal CHA reaction time is 20 min.

[0081] 2) Optimization of Peroxidase-like Activity

[0082] The present invention optimizes the peroxidase - like (POD) activity of the SERS sensing system. The POD - like activity is the main factor affecting the detection performance. The present invention respectively examines the effects of TMB concentration, H2O2 concentration, pH value and temperature on the performance of the sensing system. To systematically screen out the appropriate TMB concentration, the present invention adopts a strategy combining TMB concentration gradient sampling and quantitative characterization, setting concentration gradients of 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM, 1.4 mM and 1.6 mM, and respectively detecting the SERS spectra of the sensing system. The results are as Figure 6 shown in B. As the TMB concentration increases, the SERS signal intensity at 1606 cm -1 increases and reaches the peak at 0.8 mM and basically remains stable. To systematically screen out the appropriate H2O2 concentration, the present invention sets concentration gradients of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M and 0.7 M, and respectively detects the SERS spectra of the sensing system. The results are as Figure 6 shown in C. As the H2O2 concentration increases, the SERS signal intensity at 1606 cm -1 increases and reaches the peak at 0.5 M and basically remains stable. To systematically screen out the appropriate pH value, the present invention sets pH gradients of 2, 3, 4, 5 and 6, and respectively detects the SERS spectra of the sensing system. As Figure 6 shown in D, as the pH value increases, the SERS signal intensity at 1606 cm -1 increases and reaches the peak at pH = 4 and basically remains stable. The incubation temperature is the main factor affecting the detection performance. To systematically screen out the incubation temperature, the present invention sets temperature gradients of 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, 30 °C, 32 °C, 34 °C and 36 °C, and respectively detects the SERS spectra of the sensing system. As the temperature increases, the SERS signal intensity at 1606 cm -1 increases and reaches the peak at 30 °C and basically remains stable. After careful analysis, it is found that as these factors change, the SERS signal intensity at 1606 cm -1 shows a trend of first increasing and then decreasing. When the TMB concentration is 0.8 mM, the H2O2 concentration is 0.5 M, the pH value is 4, and the temperature is 30 °C, the POD - like activity of the SERS sensing system reaches the optimum and shows the best detection performance.

[0083] 3) Drop TMB (0.8 mM, 8 μL) and H2O2 (0.5 M, 8 μL) onto the reaction area of the sensing system and incubate at a constant temperature of 30 °C. Take it out every 5 minutes and immediately perform SERS detection to determine the optimal incubation time. As Figure 6 shown in F, as the incubation time increases, the SERS signal intensity at 1606 cm-1 The SERS signal intensity at -1 gradually increases. When the incubation time reaches 15 min, the SERS intensity gradually stabilizes and does not increase significantly. Therefore, the optimal incubation time is 15 min.

[0084] Example 6 Specificity and reproducibility of the SERS sensing system

[0085] 1) Prepare the SERS sensing system as in Example 3.

[0086] 2) To improve the reliability of the detection results, the performance of the reproducibility and specificity of the SERS sensing system was evaluated in this study. As shown in Figure 7 Figures 7A and 7B, five different batches of SERS sensing systems were prepared respectively, and the SERS spectra of detection area 1 (miR-196b) and detection area 2 (miR-221) were recorded. As shown in Figure 7 Figure 7C, the SERS signal intensities of the two targets (miR-196b and miR-221) at -1 show slight fluctuations, and the relative standard deviations (RSDs) are 7.31% and 6.45% respectively. The SERS sensing system exhibits good signal reproducibility.

[0087] 3) Specificity is an important indicator for identifying and distinguishing target molecules from other non-target molecules. In this study, miR-196b and miR-221 were used as targets, and single-mismatch sequences (MT1-1 and MT1-2), three-base mismatch sequences (MT3-1 and MT3-2), random sequences, and blank buffer were used as control interferents. The nucleotide sequences used in the experiment are shown in Table 1, and the measured SERS spectra are shown in Figure 7 Figures 7D and 7E. It was observed that the targets miR-196b and miR-221 showed significant SERS signals. In contrast, all interference samples did not show distinguishable SERS characteristic peaks. As shown in Figure 7 Figure 7F, its spectral curve highly coincides with that of the blank control group. The results indicate that the SERS sensing system constructed in this study exhibits excellent specificity and signal reproducibility, ensuring the accuracy and reliability of the data, and providing a reliable technical guarantee for the precise detection of trace miRNAs in clinical serum samples.

[0088] Table 1 Nucleotide sequences used in the experiment

[0089]

[0090]

[0091] Example 7 Quantitative detection of the SERS sensing system

[0092] 1) Prepare the SERS sensing system as in Example 3.

[0093] 2) Add TMB solution (0.8 mM, 8 μL) and H2O2 solution (0.5 M, 8 μL) to the SERS sensing system. The solvents are both PBS buffer (pH = 4.0). React for 15 min at 30 °C, and the reaction solution will show an obvious blue color. Subsequently, perform Raman spectroscopy detection on the detection area, capture the characteristic signal of oxTMB, and construct a concentration logarithm-signal intensity working curve based on the wavenumber of 1606 cm -1 to build a concentration logarithm-signal intensity working curve.

[0094] 3) To evaluate the sensitivity of the SERS sensing system, use the optimized SERS sensing system to simultaneously detect two targets (10 aM - 10 pM), and record the SERS spectra of detection area 1 (miR-196b) and detection area 2 (miR-221). As Figure 8 shown in A and C, as the target concentration increases, more Au@Pt NRs are induced to assemble on the surface of Au NPAs through the CHA amplification strategy, and the SERS sensing system is prepared. This system has high peroxidase-like activity and strong SERS activity, catalyzes the reaction of H2O2 and TMB to generate a blue oxTMB solution. The SERS signal is further amplified by the CHA amplification strategy, and the SERS signal intensity at 1606 cm -1 significantly increases. As Figure 8 shown in B and 8D, the SERS signal intensity at 1606 cm -1 has a significant linear relationship with the logarithm of the target (miR-196b and miR-221) concentration. The linear regression equations are y = 1649.04x + 245.15 (R 2 = 0.9845) and y = 2015.25x + 434.73 (R 2 = 0.9861) respectively. The detection limits of miR-196b and miR-221 are calculated to be 2.21 aM and 3.09 aM respectively.

[0095] 4) The comparison results of the SERS sensing system with other methods in biomarker detection are shown in Table 2. Compared with traditional detection methods such as electrochemical method, fluorescence method and colorimetric method, the SERS sensing system constructed in this study shows significant performance advantages, such as a wider detection range and lower sensitivity, etc., providing a new detection scheme with both high sensitivity and wide linear range for early cancer screening.

[0096] Table 2 Comparison of SERS microarray chip with other methods in biomarker detection

[0097]

[0098] Example 8 Characterization of clinical samples

[0099] 1) The clinical samples were characterized by gastroscopy, CT, and pathological examinations, as Figure 9 shown. A, B, and C are the gastroscopy image, CT scan image, and pathological examination image of a healthy person, respectively; A is the gastroscopy image of a healthy person. For the healthy population, the gastroscopy image shows the smoothness and mucosal integrity of the gastric cavity; B is the CT scan image of a healthy person, and the CT scan clearly shows the normal structure of the stomach; C is the pathological examination image of a healthy person, and the HE pathological section further confirms the microscopic normal state of the gastric tissue. At the pre-gastric cancer lesion stage, D is the gastroscopy image of a patient with pre-gastric cancer, and the gastroscopy image captures possible minor morphological changes in the gastric wall; E is the CT scan image of a patient with pre-gastric cancer, and the CT scan reveals the thickening of local gastric tissue; F is the pathological examination image of a patient with pre-gastric cancer, and the HE pathological section clearly indicates the increased cellular atypia, all of which are important signs of pre-cancerous lesions. At the gastric cancer stage, H is the CT scan image of a GC patient, and the gastroscopy image visually shows the malignant lesions of the gastric wall, such as ulcers or masses; G is the gastroscopy image of a GC patient, and the CT scan further depicts the infiltration range and depth of the tumor; I is the pathological examination image of a GC patient, and the HE pathological section details the malignant features such as the infiltration of tumor cells and the increase in mitotic figures, providing indispensable information for the timely diagnosis of GC and the formulation of treatment plans.

[0100] Example 9 Detection of miR-196b and miR-221 in a sample to be tested by the SERS sensing system

[0101] 1) Thirty serum samples from healthy people, patients with pre-gastric cancer, and GC patients were collected. The sample to be tested and the nanosensor prepared in Example 2 were dropped onto the detection area of the SERS microarray chip prepared in Example 3 and placed in an incubator at 30 °C for 20 min for the CHA reaction. After washing several times with PBS buffer, the SERS sensing system was obtained. At a temperature of 30 °C, TMB (0.8 mM, 8 μL) and H2O2 (0.5 M, 8 μL) were dropped into the reaction area of the SERS sensing system. The sensing system catalyzed the generation of oxTMB from TMB. After incubation for 15 min, Raman spectroscopy was performed on the detection area.

[0102] 2) To verify the clinical applicability of the SERS sensing system, the expression levels of the targets (miR-196b and miR-221) in the sera of 90 clinical samples were quantitatively analyzed using this system. The obtained SERS signal intensities were substituted into the corresponding regression equations in Example 7 to obtain the expression levels of the targets in the sera of clinical samples in different disease states. The results are as Figure 10 shown in A and B. The average SERS spectra of the targets (miR-196b and miR-221) in the sera of clinical samples in different disease states are as Figure 11As shown, as the individual develops from a healthy state to precancerous gastric lesions and then to GC, the expression levels of target biomarkers increase continuously. As Figure 10 shown in Figures C and 10D, the correlation coefficients of miR-196b and miR-221 are 0.9950 and 0.9988, respectively. The receiver operating characteristic (ROC) curve was introduced to evaluate the diagnostic efficacy of the SERS sensing system for detecting precancerous gastric lesions. The results are as Figure 10 shown in Figures E and 10F. According to the analysis of the area under the ROC curve (AUC) results, the combined detection of dual biomarkers shows significantly better diagnostic performance than single-index detection. The results indicate that the SERS sensing system developed in this study exhibits high accuracy and excellent diagnostic efficacy in clinical detection, demonstrating broad application potential in the fields of liquid biopsy and point-of-care testing (POCT).

[0103] 3) The SERS detection results were compared with those of a real-time quantitative polynucleotide chain reaction (qRT-PCR) kit (purchased from Jidan Biotechnology Co., Ltd.). Compared with healthy individuals, the expression levels of miR-196b and miR-221 in the sera of patients with precancerous GC lesions and GC patients gradually increased. The detection results of the SERS sensing system were highly consistent with those of qRT-PCR (Table 3).

[0104] Table 3 Comparison of SERS sensors and qRT-PCR for detecting clinical specimens

[0105]

Claims

1. Gold nanopyramid arrays (Au NPAs), characterized in that, The preparation method includes: obtaining a PS monolayer template through the gas-liquid interface self-assembly technique, and successively subjecting the PS sphere monolayer template to plasma etching technique treatment, annealing treatment, and ion sputtering treatment to obtain a gold nanometer pumpkin array (Au NPAs).

2. The gold nanosphere pumpkin arrays (AuNPAs) according to claim 1, wherein The preparation method includes: 1) Mix a PS microsphere suspension with a size of 120 nm and ethanol at a volume ratio of 1:1, and obtain a PS monolayer template through the gas-liquid interface self-assembly technique; 2) Using the plasma etching technique (RIE), with SF6, O2, and Ar gases flowing at a rate of 20 sccm / 5 sccm / 10 sccm to obtain reaction gases, under the condition that the etching power is set to 100 W, perform an etching treatment on the sample for 3 minutes to form an ordered array of a hybrid structure of PS microspheres / nanocone silicon; then, obtain a silicon cone array with a nanoscale platform on the surface through annealing treatment; finally, perform ion sputtering technique (IBS) treatment to obtain a gold nanometer pumpkin array.

3. The gold nanosphere pumpkin arrays (Au NPAs) according to claim 2, characterized in that, In step 2), the temperature of the annealing treatment is 350 °C and the time is 30 minutes; the ion sputtering technique treatment sputters at a current of 20 mA for 1 minute, and then sputters at a large current of 30 mA for 4 minutes.

4. Preparation method of SERS sensor for detecting biomarkers of precancerous gastric lesions, characterized in that, Including: 1) Synthesize platinum-coated gold nanorods (Au@PtNRs) using the seed growth method; 2) Modify the AuNPAs array described in any one of claims 1 to 3 with hairpin DNA-HP2 (HP2-1 and HP2-2) to obtain two functionalized AuNPAs arrays, Au NPAs@HP2-1 and Au NPAs@HP2-2; embed the AuNPAs array into the PDMS layer to obtain a SERS microarray chip; 3) Modify the Au@PtNRs synthesized in step 1) with hairpin DNA-HP1 (HP1-1 and HP1-2) to obtain two SERS nanoprobes, Au@PtNRs@HP1-1 and Au@PtNRs@HP1-2; 4) Drop the target and the nanoprobe obtained in step 3) onto the detection area of the SERS microarray chip, and the target triggers the catalytic hairpin self-assembly (CHA) amplification strategy. The probe is assembled on the surface of AuNPAs due to the complementary pairing of the HP1 strand and the HP2 strand, constructing a "dual-functional" SERS sensing system with high peroxidase (POD) activity and excellent SERS enhancement ability; Among them, the sequence of HP1-1 is as shown in SEQ ID NO.1, the sequence of HP1-2 is as shown in SEQ ID NO.2, the sequence of HP2-1 is as shown in SEQ ID NO.3, and the sequence of HP2-2 is as shown in SEQ ID NO.

4.

5. The preparation method according to claim 4, wherein The preparation method satisfies one or more combinations of the following conditions: (i) Step 1) includes: 1.1) Add HAuCl4 to the CTAB solution, and then quickly inject the NaBH4 solution into the above solution under vigorous stirring to obtain a gold seed solution (Au NRs); 1.2) CTAC and NaOL were fully dissolved in an aqueous solution to obtain a mixed solution; an AgNO3 solution and an HAuCl4 solution were added to the above-mentioned mixed solution, and stirred at 30 °C for 150 min; subsequently, an HCl solution was added, and stirred for another 15 min; finally, an AA solution was added and stirred vigorously for 30 min to prepare a growth solution; then, 0.4 - 0.6 mL of the gold seed solution was quickly added to the above growth solution, stirred for 1 min, and left to grow overnight at 30 °C to obtain AuNRs. 1.3) To a cetylpyridinium chloride monohydrate (CPC) solution, an Au NRs seed solution, a sodium chloroplatinate (Na2PtCl4) solution and an AA solution were added in sequence. After vigorous stirring, it was left to stand at room temperature, and after centrifugal purification, Au@Pt NRs were prepared. (ii) Step 2) includes: 2.1) The hairpin DNAs (HP1 and HP2) were dissolved in PBS buffer solution and denatured at a high temperature of 95 °C for 5 min; incubated with a TCEP solution for 2 h to activate the hairpin structure; the activated hairpin DNA - HP2 (HP2 - 1 and HP2 - 2) was used to modify the AuNPAs array to obtain two functionalized Au NPAs arrays, Au NPAs@HP2 - 1 and AuNPAs@HP2 - 2; the above functionalized arrays were embedded in the PDMS layer to obtain a SERS microarray chip. 2.2) Based on AutoCAD software, the size and structure of the microarray chip were designed. The microarray chip consisted of two parts: detection area 1 integrated with 8 circular areas with a corresponding diameter of 4 mm and detection area 2 configured with 8 square areas with a corresponding side length of 4 mm; first, a mold matching the chip design was prepared on a silicon wafer by soft lithography technology; then, the vacuum - treated PDMS prepolymer and cross - linker mixture were evenly poured onto the surface of the mold and placed in an oven for curing; after curing, the PDMS layer was carefully peeled off from the master mold, cut, punched and cleaned according to the design drawing; subsequently, the PDMS layer was ultrasonically treated for 1 min by a plasma machine; finally, the hydrophilized PDMS layer was sealed with a glass slide by plasma bonding technology and the functionalized Au NPAs prepared in step 2.1) were embedded to obtain a SERS microarray chip. (iii) Step 3) includes: The activated hairpin DNA - HP1 (HP1 - 1 and HP1 - 2) solution was added to the Au@PtNRs solution prepared in step 1) for reaction, and then the obtained mixed solution was dispersed in a BSA solution for incubation to obtain functionalized nanoprobes, Au@Pt NRs@HP1 - 1 and Au@PtNRs@HP1 - 2. (iv) Step 4) includes: 4.1) miR - 196b and miR - 221 were respectively added to fetal bovine serum to prepare miRNAs solutions with different concentrations, obtaining two targets with different concentrations. 4.2) Drop the two kinds of targets with different concentrations configured in step 4.1) and the nanoprobes prepared in step 3) onto the detection area of the SERS microarray chip prepared in step 3) for incubation. Au@Pt NRs are assembled on the surface of AuNPAs through the CHA technology. After incubation, unbound Au@PtNRs are washed off with PBS buffer to prepare a SERS sensing system.

6. The preparation method according to claim 5, wherein in step 1.3), the reaction temperature condition is set to 65 °C, with vigorous stirring for 2 min and standing for 30 min; and / or in step 2.1), HP2 and Au NPAs are incubated at 25 °C for 12 h; and / or in step 3), the volume ratio of the Au@Pt NRs solution to the BSA solution is 50:1, the reaction temperature is 25 °C, the reaction time is 12 h, and the incubation time is 1 h; and / or in step 4.2), the incubation time is 20 min.

7. A SERS sensor for detecting biomarkers of precancerous gastric lesions prepared by the preparation method according to any one of claims 1 to 6.

8. A method for detecting miR-196b and miR-221 in a sample to be detected using the SERS sensor for detecting biomarkers of gastric precancerous lesions according to claim 7, characterized in that the method Comprising: 1) Add TMB solution and H2O2 solution to the SERS sensing system described in claim 7. The solvent is PBS buffer (pH = 4.0). React for 15 min at 30 °C. The reaction solution will show an obvious blue color. Subsequently, perform Raman spectroscopy detection on the detection area, capture the characteristic signal of oxTMB, and construct a concentration logarithm-signal intensity working curve based on the wavenumber of 1606 cm -1 -1 2) Collect 30 serum samples from healthy individuals, patients with gastric precancerous lesions, and GC patients. Drop the samples to be detected and the nanoprobes onto the detection area of the SERS microarray chip, and place it in an incubator at 30 °C for the CHA reaction. After reacting for a certain period of time, wash it several times with PBS buffer to obtain a bifunctional SERS sensor. Add TMB solution and H2O2 solution to the bifunctional SERS sensing system for reaction. The solvent of the solution is PBS buffer (pH = 4.0). When the nanozyme catalyzes the reaction of TMB and H2O2, blue oxTMB can be generated. Subsequently, perform Raman spectroscopy detection on the detection area to capture the characteristic signal of oxTMB. Substitute the intensity of the characteristic peak of oxTMB at 1606 cm -1 obtained from the above detection into the working curve determined in step 1) to determine the concentrations of miR-196b and miR-221 in the samples to be detected. 3) Substitute the characteristic peak intensity at 1606 cm of the sample to be detected obtained in step 2) into the working curve determined in step 1) to measure the concentrations of miR-196b and miR-221 in the sample to be detected. -1 ​ 9. The method for detecting gastric precancerous biomarker miR-196b and miR-221 in a sample to be detected according to claim 8, wherein in step 2), the CHA reaction is 20 min; adding TMB solution and H2O2 solution to the bifunctional SERS sensing system, the reaction temperature is 30 °C, and the reaction time is 15 min.

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