SERS (Surface Enhanced Raman Scattering) analysis platform for detecting early gastric cancer protein biomarkers as well as preparation method and application of SERS analysis platform

Abstract

The invention discloses a surface enhanced Raman scattering (SERS) analysis platform for detecting early gastric cancer protein biomarkers as well as a preparation method and application thereof, and belongs to the technical field of detection. The preparation method comprises the following steps: (1) modifying the surface of Au (at) Pt nano particle (Au (at) PtNPs) nano enzyme with DNA1 (hpDNA1) of a hairpin structure to form two SERS probes; (2) modifying hpDNA2 on the surface of the special-shaped nanorod array (Au / SiNPA) to serve as a capture substrate; (3) assembling a 4 * 4 microarray chip as an SERS (Surface Enhanced Raman Scattering) analysis platform; the SERS analysis platform has the advantages of simplicity in operation, high throughput, low price, high sensitivity and specificity and the like; moreover, the repeatability is good, and large-scale preparation can be realized.

Description

Technical Field

[0001] The present invention relates to the field of detection technologies, and in particular, to a SERS analysis platform for detecting early gastric cancer protein biomarkers, a preparation method thereof, and an application thereof. Background Art

[0002] Gastric cancer (GC) is a common malignant tumor that poses a major threat to human health. Actively carrying out the "secondary prevention" of GC is the key to effectively improving the prognosis of GC and reducing the mortality rate. It is worth noting that traditional serological tumor markers, such as carcinoembryonic antigen (CEA), carbohydrate antigen 72-4 (CA72-4), and carbohydrate antigen 19-9 (CA19-9), are not sufficient to detect GC due to their poor sensitivity and specificity. Although gastroscopy with pathological analysis is considered the "gold standard" for diagnosing GC, invasive examinations can cause obvious discomfort to patients and are therefore not suitable for large-scale clinical screening. Therefore, it is necessary to explore potential biomarkers related to GC to provide new ideas for the early diagnosis of GC. A large number of studies have shown that the protein Ephrin-A1 (EFNA1) encoded by the EFNA1 gene is overexpressed in GC tissues, and its upregulation is closely related to tumor malignancy, metastatic potential, and patient prognosis. Matrix metalloproteinase 13 (MMP13) is specifically activated in GC and is an important regulator of tumor growth, which can promote the invasiveness of primary tumors. There is literature indicating that compared with single biomarkers, the area under the curve (AUC) for the combined detection of serum EFNA1 and MMP13 in early GC is improved (0.794). Currently, there are various methods for detecting EFNA1 and MMP13, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and electrochemical sensors. However, most of the above analysis methods have problems such as high cost, time-consuming for multiple detections, low sensitivity, and poor anti-interference ability. Therefore, developing a low-cost, simple, rapid, sensitive, and specific EFNA1 / MMP13 detection method has become the key to breaking the routine.

[0003] Nanozymes are nanomaterials with catalytic activities similar to those of natural enzymes. Compared with natural enzymes, nanozymes have the advantages of high stability, tunable catalytic activity, low cost, etc., and can avoid the problem of easy inactivation of biological enzymes. Therefore, they show great potential in biosensing, therapy, energy conversion, etc. Generally, the research on nanozymes in the analysis field mainly focuses on the visualization of the color change of enzyme substrates after signal catalytic reactions. However, due to low sensitivity and color interference caused by the material itself and the sample matrix, their practical applications are limited. To improve the detection sensitivity and accuracy, researchers have adopted techniques such as electrochemical and fluorescence detection in the study of nanozyme-mediated catalytic platforms. They can provide rapid and sensitive detection, but also have obvious disadvantages such as unstable electrodes, susceptibility to fluorescence quenching, and complex operations. Surface-enhanced Raman scattering (SERS) is a powerful inelastic scattering spectroscopic technique that has developed rapidly in the field of biosensing due to its fast, non-destructive, and water-intolerant characteristics. SERS technology can easily obtain the "fingerprint information" of each molecule, including the structure, molecular composition, and conformation of the substance, making it possible to distinguish biomarkers from complex samples. However, due to the relatively low Raman scattering cross-section of small molecule proteins and their relatively weak interaction with substrates, the sensitivity of label-free SERS detection strategies is insufficient.

[0004] Due to the extremely low concentration of biomarkers in serum during early GC, traditional detection methods cannot meet the demanding sensitivity requirements. Therefore, it is of great significance to develop a SERS analysis platform with high sensitivity, strong specificity, simple operation, fast detection speed, and high throughput. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a preparation method, product, and application of a SERS analysis platform for detecting early gastric cancer protein biomarkers. This SERS analysis platform is based on nanozyme-catalyzed signal amplification and introduces a catalytic hairpin assembly (CHA) nucleic acid amplification strategy to achieve the simultaneous quantitative detection of EFNA1 and MMP13, with excellent detection specificity and sensitivity.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] A preparation method of a SERS analysis platform for detecting early gastric cancer protein biomarkers, the preparation method comprising the following steps:

[0008] S1: Modify hpDNA1 on the surface of Au@PtNPs nanozymes to form two SERS probes;

[0009] S2: Modify hpDNA2 on the surface of the shaped nano-column array Au / SiNPA as a capture substrate;

[0010] S3: Assemble the capture substrate prepared in step S2 into a microarray chip as the SERS analysis platform;

[0011] The hpDNA1 includes hpDNA1-1 and hpDNA1-2. The sequence of hpDNA1-1 is shown in SEQ ID NO:3, and the sequence of hpDNA1-2 is shown in SEQ ID NO:5. The hpDNA2 includes hpDNA2-2 and hpDNA2-2. The sequence of hpDNA2-2 is shown in SEQ ID NO:4, and the sequence of hpDNA2-2 is shown in SEQ ID NO:6.

[0012] The present invention also aims to provide an SERS analysis platform for detecting early gastric cancer protein biomarkers prepared by the above preparation method.

[0013] The present invention also aims to provide the application of the above-mentioned SERS analysis platform in simultaneously detecting early gastric cancer protein biomarkers EFNA1 and MMP13 for non-disease diagnosis and treatment purposes.

[0014] As Figure 1 shown, a novel SERS analysis platform constructed by the present invention combines nanozyme and SERS technologies well. On the one hand, the nanozyme has peroxidase (POD)-like activity and can catalyze probe molecules into SERS-active reporter molecules, thus generating strong SERS signals. On the other hand, due to its large surface area and numerous active sites, the nanozyme effectively increases the number of interacting signal molecules, thereby improving the detection sensitivity.

[0015] The bimetallic Au@Pt nanoparticles (Au@PtNPs) selected in the present invention have excellent POD-like activity and SERS enhancement effect. The enhanced catalytic activity of Au@PtNPs is mainly attributed to its porous structure and the formation of an electron-rich Pt shell, which changes the catalytic pathway from the generation of hydroxyl radicals to an electron transfer process. The selected SERS substrate is a heteromorphic nano-column array (Au / SiNPA) prepared by colloidal sphere template-assisted reactive ion etching (RIE), which has excellent uniformity and sensitivity.

[0016] The present invention also uses catalytic hairpin assembly (CHA), which is a versatile isothermal enzyme-free amplification technique. Compared with other amplification strategies, such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and rolling circle amplification (RCA), CHA has significant advantages of simple reaction conditions, high hybridization efficiency, low cost, low background signal, and good stability. An antibody-DNA conjugate formed by binding a specific antibody to a carefully designed oligonucleotide chain can not only specifically bind to the target biomarker but also trigger CHA to amplify the signal; that is, applying the CHA technique to the SERS detection of protein biomarkers can further improve the detection sensitivity.

[0017] The beneficial effects of the present invention at least include:

[0018] The core-shell structure of the Au@Pt nanoparticles (Au@PtNPs) nanozyme prepared by the present invention combines good peroxidase (POD)-like activity and SERS enhancement activity.

[0019] The shaped nanopillar array (Au / SiNPA) prepared by the present invention has the advantages of stable structure, regular arrangement, dense hot spots, and uniform distribution, and can be prepared on a large scale.

[0020] The SERS analysis platform prepared by the present invention has the advantages of high sensitivity, strong specificity, simple operation, fast detection speed, and high throughput; that is, it can successfully detect EFNA1 and MMP13, and the limit of detection (LOD) is as low as pg / mL.

[0021] The SERS analysis platform prepared by the present invention has excellent stability, outstanding specificity, and good reproducibility, providing strong technical support for the early detection, timely intervention, and treatment of GC. Brief Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the preparation process of the SERS analysis platform in the embodiment of the present invention and the detection of early gastric cancer protein biomarkers EFNA1 and MMP13.

[0023] Figure 2 It is the SEM photograph (a) of Au@PtNPs in the embodiment of the present invention; (low magnification) high-resolution TEM image (b); (high magnification) high-resolution TEM image (c); SAED diffraction pattern photograph (d); HAADF-STEM image and corresponding elemental mapping (e); EDX spectrum (f); UV-vis-NIR spectrum (g); steady-state kinetic analysis of Au@PtNPs and its corresponding double-reciprocal Lineweaver-Burk plot (h); pure ox-TMB (1×10 -1 M) and ox-TMB (1×10 -6SERS spectra of M)-labeled Au@Pt NPs (i).

[0024] Figure 3 SEM images of monolayer PS colloidal spheres (a); SEM images of SiNPA (b); SEM images of Au / SiNPA and its local magnification (c); SERS spectra of 40 randomly selected points on Au / SiNPA (d), corresponding peak intensities at 1080 cm -1 and corresponding peak intensities at 1592 cm -1 (e); SERS spectra of 4-MBA-labeled Au / SiNPA (g); pure 4-MBA (1×10 -1 M) and 4-MBA (1×10 -8 M)-labeled Au / SiNPA SERS spectra (h); corresponding SERS intensity line graphs of 4-MBA-labeled Au / SiNPA at 1592 cm -1 after storage in N2 and air for 0 - 30 d (i).

[0025] Figure 4 Agarose gel electrophoresis images of the intermediate complex of the CHA reaction in the examples of the present invention (a); agarose gel electrophoresis images verifying the CHA amplification reaction (b).

[0026] Figure 5 Optimization of the SERS nanolabel volume (a); optimization of the incubation time (b); optimization of the incubation temperature (c); optimization of the pH value of the reaction system (d) in the examples of the present invention.

[0027] Figure 6 Selectivity evaluation of the SERS analysis platform for the detection of EFNA1 and MMP13 in the examples of the present invention (concentrations of CEA, AFP, CA19-9, CA72-4, MMP13, and EFNA1 are 1 mg / mL) (a); effects of potential interfering substances such as small molecules glucose, uric acid, GSH, urea, AA, lactic acid, and cholesterol, and protein markers CEA, AFP, CA19-9, CA72-4, and MMP13 on the detection of EFNA1 (1 μg / mL) (b); SERS spectra obtained from different batches of the SERS analysis platform (c), and SERS intensities of the corresponding characteristic peaks at 1602 cm -1 (d).

[0028] Figure 7SERS spectra of EFNA1 at different concentrations (blank, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 mg / mL) in serum (a); SERS spectra of MMP13 at different concentrations (blank, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 mg / mL) in serum (b); linear curve of the logarithm of EFNA1 concentration versus the intensity of the characteristic peak at 1602 cm -1 ; linear curve of the logarithm of MMP13 concentration versus the intensity of the characteristic peak at 1602 cm -1 .

[0029] Figure 8 Top view and side view of the SERS microarray chip in the embodiment of the present invention (a); average SERS spectra of EFNA1 and MMP13 in the sera of 20 healthy individuals and 20 early GC patients (b), and the intensity of the characteristic peak at 1602 cm -1 . Detailed implementation manners

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0032] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement them. When the combination of technical solutions conflicts with each other or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.

[0033] The embodiment of the present invention provides a surface-enhanced Raman scattering (SERS) analysis platform for detecting early gastric cancer protein biomarkers, and its preparation method includes: (1) Modifying the surface of Au@Pt nanoparticles (Au@Pt NPs) nanoenzymes with hairpin-structured DNA1 (hpDNA1) to form two SERS probes. (2) Modifying hpDNA2 on the surface of the shaped nano-column array (Au / SiNPA) as a capture substrate; (3) Assembling a 4×4 microarray chip as the SERS analysis platform. Among them, hpDNA1 includes hpDNA1-1 and hpDNA1-2, and their sequences are shown in Table 1; hpDNA2 includes hpDNA2-1 and hpDNA2-2, and their sequences are shown in Table 1.

[0034] During the detection process, the present invention triggers the CHA amplification by designing antibody-DNA conjugates and generating antigen@antibody-DNA conjugates that specifically bind to target proteins, enabling more Au@PtNPs nanozymes to be linked to the Au / SiNPA surface, which means a higher catalytic efficiency for the catalytic conversion of 3,3’,5,5’-tetramethylbenzidine (TMB) to SERS-active oxidized TMB (ox-TMB). The close proximity of the SERS nanotags to the capture substrate significantly amplifies the plasmon coupling effect, generating more "hot spots" to further amplify the ox-TMB signal. Using this strategy, EFNA1 and MMP13 can be successfully detected with a detection limit (LOD) as low as pg / mL. This SERS analysis platform has good specificity, anti-interference ability, and stability, and has been successfully applied to the serum detection of early gastric cancer, with the detection results consistent with those of ELISA. This study not only provides a reference for constructing a SERS analysis platform based on bifunctional nanozymes, but also offers a new solution for introducing the CHA strategy to achieve highly sensitive SERS detection of protein biomarkers.

[0035] In some specific embodiments, the preparation method of the above Au@PtNPs nanozyme may include: taking gold sol, adding deionized water, AA, and trisodium citrate to form solution A. Adding HAuCl4 solution to solution A and continuing the reaction for more than 30 minutes to obtain solution B. Adding H2PtCl6 solution to solution B and mixing well while maintaining the temperature in the reaction flask not lower than 80°C. Then, adding AA and trisodium citrate dropwise to the mixed solution until the reaction is complete, thus obtaining the Au@PtNPs nanozyme.

[0036] In some preferred embodiments, the preparation method of the above Au@PtNPs nanozyme may include: taking gold sol, adding it to deionized water, AA solution (1.0 - 1.5 mL, 28 mM), and trisodium citrate solution (1.0 - 1.5 mL, 10 mM) to form solution A. Adding HAuCl4 solution (0.20 - 0.35 mL, 24 mM) to solution A and reacting for more than 30 minutes while maintaining 90 - 100°C to obtain solution B. Adding H2PtCl6 solution (4 - 4.5 mL, 0.096 mM) to solution B and mixing well while maintaining the temperature in the reaction flask not lower than 80°C. Then, adding AA solution (3.5 - 4 mL, 4 mM) and trisodium citrate solution (0.5 - 0.7 mL, 10 mM) dropwise to the mixed solution and reacting for more than 30 min until the reaction is complete, thus obtaining the Au@PtNPs nanozyme.

[0037] In some specific embodiments, the above gold sol has a gold seed diameter of 16 - 18 nm.

[0038] In some specific embodiments, the formation of two SERS probes by surface modification of Au@PtNPs nanozyme with hpDNA1 may include: after activating hpDNA1, co-incubating it with Au@PtNPs nanozyme for more than 12 h, and dispersing the obtained mixture in bovine serum albumin (BSA) solution. Centrifuging and purifying to collect two SERS nanolabels: Au@PtNPs@hp1-1 and Au@PtNPs@hp1-2.

[0039] In some preferred embodiments, the formation of two SERS probes by surface modification of Au@PtNPs nanozyme with hpDNA1 includes: after activating hpDNA1, co-incubating it with Au@PtNPs nanozyme for more than 12 h, and dispersing the obtained mixture in bovine serum albumin (BSA) solution (160 - 180 L, 1 wt%). Centrifuging and purifying to collect two SERS nanolabels: Au@PtNPs@hp1-1 and Au@Pt NPs@hp1-2.

[0040] In some specific embodiments, the above-mentioned centrifugation speed is 10000 - 12000 rpm, and the centrifugation time is 10 - 12 min.

[0041] In some specific embodiments, the preparation method of the above-mentioned Au / SiNPA may include: obtaining a monolayer of densely arranged PS microsphere arrays by the gas-liquid interface self-assembly method, and performing ion etching on its surface using three etching gases SF6, O2, and Ar. Then, heat-treating the obtained intermediate product to remove the residual PS microspheres, i.e., obtaining SiNCA. Finally, depositing gold nanoparticles on the surface of SiNCA by ion sputtering technology to obtain Au / SiNCA.

[0042] In some preferred embodiments, the preparation method of the above-mentioned Au / SiNPA may include:

[0043] (1) Using a PS microsphere suspension (500 nm) as a precursor, it is mixed with ethanol at a volume ratio of 1:1, and an ordered monolayer PS microsphere array is self-assembled at the gas-liquid interface. (2) Then, reactive ion etching is employed. First, SF6 and O2 are used as etching gases to perform ion etching on the obtained monolayer PS colloidal sphere template for 6 - 8 min (gas flow rate 20 - 25 sccm / 10 - 15 sccm, etching power 100 W), and then Ar gas is used to physically etch its ionized surface (gas flow rate 10 - 15 sccm, etching power 100 W) to obtain an ordered array of PS microsphere / silicon pillar hybrid structures. (3) After superstructure annealing (≥350 °C, 30 min), a silicon pillar array with a nano-platform on the surface (SiNCA) is obtained. Finally, using Au as a target, gold nanoparticles are deposited on the surface of SiNCA by ion sputtering technology, and Au / SiNPA is successfully prepared. Its structural unit consists of deformed nano-columns. Then, hpDNA2 (hpDNA2-1 and hpDNA2-2) activated by TECP buffer is modified on the surface of Au / SiNCA to prepare capture substrates: Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2.

[0044] In some embodiments of the present invention, after obtaining the above monolayer PS microsphere array, it further includes the step of transferring the PS microsphere array using a hydrophilized silicon wafer. The present invention does not make special limitations on the method of hydrophilizing the silicon wafer, and a technical solution well-known to those skilled in the art can be selected.

[0045] In some specific embodiments, the sputtering time of the above ion sputtering technology is 5 - 8 min, the current is 30 - 35 mA, and the deposition rate is 0.5 - 0.7 nm / s.

[0046] Preferably, the temperature for activating hpDNA2 using TECP buffer is 80 - 95 °C, and the time is not less than 30 min.

[0047] In some specific embodiments, the above assembly of a 4×4 microarray chip as a SERS analysis platform includes: an ITO glass substrate embedded with Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2 and a 4×4 PDMS microhole array interlayer are aligned and bonded by oxygen plasma technology, and a PDMS cover slip is covered. The chip size is 40 mm × 40 mm, and it contains 16 microholes (8 square and 8 circular).

[0048] In some preferred embodiments, the above assembly of a 4×4 microarray chip as a SERS analysis platform may include:

[0049] (1) First, the PDMS elastomer material prepared by mixing the polydimethylsiloxane (PDMS) prepolymer and the curing agent in a ratio of 10:1 was poured into a mold and cured at 80 - 90 °C for 2 - 2.5 h. (2) Next, the PDMS elastomer material was cut and perforated to prepare a PDMS microporous array sandwich and a PDMS cover slip respectively, and ultrasonically cleaned and dried with deionized water and ethanol. (3) Subsequently, the prepared Au / SiNPA was cut into 16 parts of corresponding sizes and embedded in the ITO glass substrate by laser etching. (4) Then, the ITO glass substrate and the PDMS microporous array sandwich were aligned and bonded by oxygen plasma technology to ensure a tight connection between the two. (5) Finally, the PDMS cover slip was covered to successfully construct a microarray chip. (6) The chip consists of three parts: the ITO glass substrate embedded with Au / SiNPA@hp2 - 1 and Au / SiNPA@hp2 - 2, the PDMS microporous array sandwich, and the PDMS cover slip. The chip size is 40 mm × 40 mm, containing 16 micropores (8 squares and 8 circles), with a diameter / side length of 4 mm, a spacing of 6 mm, and a height of 6 mm.

[0050] It should also be noted that the application of any of the above - mentioned SERS analysis platforms for detecting early gastric cancer protein biomarkers in detecting early gastric cancer protein biomarkers EFNA1 and MMP13 while not for the purpose of disease diagnosis and treatment includes the following steps:

[0051] (1) First, demonstrate the EFNA1 detection process. Dilute 5.0 - 5.5 mL of serum sample and 3.0 - 3.5 mL of antibody - DNA conjugate (Ab1 - 1@DNA1 - 1 and Ab2 - 1@DNA2 - 1) were incubated with shaking at 37 °C for 2 - 3 h to form an antigen@antibody - DNA conjugate through antigen - antibody reaction. Then, the conjugate solution was dropped into the microarray chip (square sample inlet), and at the same time, the SERS nanoprobe (Au@Pt NPs@hp1 - 1) was also added to the microarray chip.

[0052] (2) The conjugate and the SERS nanoprobe trigger the CHA reaction in the reaction chamber, allowing more and more Au@Pt NPs nanoenzymes to attach to the SERS capture substrate. After the reaction is terminated, the excess SERS nanoprobe is removed by washing three times with PBS buffer and deionized water.

[0053] (3) Finally, add 15 - 20 mL of freshly prepared solution, an acetic acid / sodium acetate buffer (0.5 M) of TMB (1 mM) and H2O2. After drying, proceed to the next step of SERS detection. The MMP13 detection process is the same as above, and the detection area is the circular sample addition port. Use a Raman microscope with a 50× objective lens and a 785 nm laser with a power of 5 mW to obtain SERS spectra on the reaction chamber. Each spectrum is obtained within 10 seconds. Scan each sample at five random positions, with a spot size of 20 mm2 in the range of 400 - 1800 cm-1 to generate an average SERS spectrum, and the error bars indicate the standard deviation of five measurements.

[0054] Preferably, the volume of the SERS probe Au@PtNPs@hp1 - 1 added is 3.0 - 3.5 μL, and the volume of the SERS probe Au@Pt NPs@hp2 - 1 added is 4.0 - 4.5 μL.

[0055] Preferably, the temperature of the reaction system is 36 - 38 °C, and the pH value is 4.2 - 4.3.

[0056] To better understand the present invention, the following further clarifies the content of the present invention with specific examples, but the content of the present invention is not limited to the following examples. The preparation process of the SERS analysis platform of the present invention and the schematic diagram of detecting early gastric cancer biomarkers EFNA1 and MMP13 are as Figure 1 shown.

[0057] The raw materials and reagents used in the examples can be obtained through commercial channels without special instructions.

[0058] The instruments and equipment as well as the test conditions used in the experiments of the present invention are as follows:

[0059] The transmission electron microscope (TEM) images were measured by a TECNAI12 transmission electron microscope produced by Philips Company of the Netherlands; the high - resolution transmission electron microscope (HRTEM) images and selected area electron diffraction analysis (SAED) were measured by a FEI field - emission transmission electron microscope combined with an X - ray energy spectrometer (EDS); the scanning electron microscope (SEM) images were measured by an S - 4800 field - emission scanning electron microscope produced by Hitachi Company of Japan; the Raman spectra were measured by an Invia Reflex type laser micro - Raman spectrometer produced by Renishaw Company of the UK. The laser wavelength is 785 nm, the exposure time is 10 s, the laser intensity is 50 mW, and the objective lens is 50×; the SERS imaging was measured by a DXRxi micro - Raman imaging spectrometer produced by Thermo Fisher Scientific of the United States.

[0060] The following specifically illustrates the solutions proposed by the present invention through specific examples:

[0061] Example 1 Synthesis and Characterization of Au@PtNPs Nanozyme

[0062] 1. Synthesis of Au@PtNPs Nanozyme

[0063] (1) Take gold sol (16 - 18 nm), and add it to deionized water, AA solution (1.0 - 1.5 mL, 28 mM) and trisodium citrate solution (1.0 - 1.5 mL, 10 mM) to form solution A.

[0064] (2) Add HAuCl4 solution (0.20 - 0.35 mL, 24 mM) to solution A, and react in an environment of 90 - 100 °C for more than 30 minutes to obtain solution B.

[0065] (3) Add H2PtCl6 solution (4 - 4.5 mL, 0.096 mM) to solution B and mix well, while keeping the temperature in the reaction flask not lower than 80 °C. Then, add AA solution (3.5 - 4 mL, 4 mM) and trisodium citrate solution (0.5 - 0.7 mL, 10 mM) dropwise to the mixed solution, and react for more than 30 minutes until the reaction is complete, that is, Au@PtNPs nanozyme is obtained.

[0066] 2. Morphology, POD-like Activity and SERS Performance of Au@PtNPs Nanozyme

[0067] As shown in Figure 2 a, the TEM image shows that Au@PtNPs are regular spheres with a height of 52 nm and high uniformity. It can be seen that many smaller granular platinum atom clusters (about 4 nm) are evenly distributed on the surface of the Au core, assembling them into a porous outer layer, as shown in the HRTEM image ( Figure 2 b). The lattice fringes of typical particles with a crystal spacing of 0.226 nm ( Figure 2 c) correspond well to the Pt(111) surface. In the selected area electron diffraction (SAED) pattern ( Figure 2 d), four unique diffraction rings match the (111), (200), (220) and (311) crystal planes, reflecting its polycrystalline nature. In addition, the elemental composition of Au@PtNPs was confirmed by energy dispersive X-ray (EDX) spectroscopy, in which Au and Pt elements are dominant ( Figure 2 f). To further clarify the elemental distribution of Au@PtNPs, high-angle annular dark-field scanning TEM (HAADF-STEM) images and corresponding elemental maps ( Figure 2 e) show that Pt is distributed around the Au core and forms a metal shell, indicating the successful synthesis of core-shell structured Au@PtNP.

[0068] Peroxidase (POD)-like activity is one of the important properties of Au@PtNPs, which was verified by the classical colorimetric method ( Figure 2 g). Using TMB as the colorimetric substrate, the experimental results showed that the color quickly turned blue only when TMB, Au@PtNPs, and H2O2 were simultaneously present in the solution, clearly indicating that Au@PtNPs exhibited good POD-like activity. In addition, the steady-state kinetics of Au@PtNPs was analyzed. As Figure 2 shown in h, the Michaelis-Menten curve and its corresponding double-reciprocal plot were obtained. The V max and K m of Au@PtNPs catalyzing TMB were 0.853 mM·s -1 and 0.570 mM, respectively, which were superior to those of the natural enzyme HRP, and this could be attributed to its porous structure that could enhance the adsorption of TMB. The enhancement factor (EF) is an important indicator to measure the SERS enhancement ability. As Figure 2 shown in i, Au@PtNPs significantly enhanced the Raman signal of ox-TMB, and the EF was calculated to be 4.05×10 SERS / C SERS ) / (I Raman / C Raman ) as 4.05×10 6 , where ISERS and I Raman were the intensities at the 1602 cm -6 peak obtained from Au@PtNPs labeled with ox-TMB (1×10 -1 M) and pure ox-TMB (1×10 -1 M), respectively. In summary, in this example, bifunctional Au@PtNPs nanozymes with strong POD-like activity and SERS performance were successfully prepared.

[0069] Example 2 Preparation and Characterization of Au / SiNPA

[0070] 1. Preparation of Au / SiNPA

[0071] (1) Using a PS microsphere suspension (500 nm) as the precursor, it was mixed with ethanol at a volume ratio of 1:1, and an ordered monolayer PS microsphere array was self-assembled at the gas-liquid interface.

[0072] (2) Then, reactive ion etching was used. First, SF6 and O2 were used as etching gases to perform ion etching on the obtained monolayer PS colloidal sphere template for 6 - 8 min (gas flow rate 20 - 25 sccm / 10 - 15 sccm, etching power 100 W), and then Ar gas was used to physically etch its ionized surface (gas flow rate 10 - 15 sccm, etching power 100 W) to obtain an ordered array of PS microsphere / silicon column hybrid structure.

[0073] (3) After superstructure annealing (≥350 °C, 30 min), a silicon pillar array with a nano-platform on the surface (SiNCA) was obtained. Finally, using Au as the target, by means of ion sputtering technology, sputtering was carried out at a current of 30 - 35 mA for 5 - 8 min, and Au / SiNPA (deposition rate: 0.5 - 0.7 nm / s) was successfully prepared, and its structural unit consists of deformed nano-columns.

[0074] 2. Morphology and SERS effect characterization of Au / SiNPA

[0075] As Figure 3 shown in a, in this example, by means of the gas - liquid interface self - assembly method and overall transfer, a layer of uniformly and densely organized PS colloidal spheres (diameter 500 nm) was successfully formed on the surface of the hydrophilized silicon wafer. As Figure 3 shown in b, by optimizing the etching process and annealing operation, a silicon pillar array with a nano - platform on the surface (SiNPA) was prepared, where it was observed that the silicon pillars showed a high degree of regularity and a smooth surface. Finally, highly ordered Au / SiNPA was obtained by ion - sputtering gold ( Figure 3 c), which consists of deformed nano - columns with a height of 290 nm, a period of 500 nm, and uniformly arranged in an ordered pattern. Figure 3 The lower right corner of c is a local enlarged view of Au / SiNPA, revealing that the rough array surface is characterized by many protruding near - spherical particles with abundant nano - gaps between them, which can generate a large number of SERS "hot spots".

[0076] Subsequently, SERS detection was carried out on 40 randomly selected points on 4 - MBA - labeled Au / SiNPA to evaluate the uniformity of the array ( Figure 3 d). The results show that the overall spectral shape shows a consistent trend, with only slight intensity differences. Through statistical calculation, the relative standard deviation (RSD) values of the characteristic peak intensities of 4 - MBA at 1080 and 1592 cm -1 are only 5.84% and 5.48% respectively, where the red horizontal line represents the average intensity, and the area between the two blue lines represents the deviation range ( Figure 3 e and f). The results show that Au / SiNPA shows satisfactory uniformity and signal reproducibility. In addition, Figure 3 g shows the SERS map of 4 - MBA - labeled Au / SiNPA in a 50×50 mm area, which shows an overall uniform green (the color represents the SERS signal intensity), further demonstrating the significant uniformity of the array. Figure 3 h shows pure 4 - MBA (1×10 -1 M) and 4 - MBA (1×10 -8SERS spectra of the M)-labeled Au / SiNPA, with the calculated EF value of 1.09×10 9 . The results indicate that Au / SiNPA has significant SERS enhancement ability, which is consistent with the previous hypothesis.

[0077] Finally, the prepared 4-MBA-labeled Au / SiNPA was placed in air and N2 for different days to evaluate the stability of the array. The intensity of the characteristic peak at 1592 cm -1 remained at a high value in both N2 and air ( Figure 3 i). After 30 days, the SERS intensity decreased by only 4.5% and 9.5% respectively, indicating that Au / SiNPA has significant stability. Therefore, after evaluating all the properties of Au / SiNPA, it was confirmed that it can meet the subsequent practical applications.

[0078] Example 3 Feasibility Evaluation of the CHA Nucleic Acid Amplification Strategy

[0079] Before applying it to quantify EFNA1 and MMP13, the feasibility of the CHA nucleic acid amplification strategy must be evaluated. Therefore, agarose gel electrophoresis ( Figure 4 ) was used to analyze the amplification process. Here, in this example, refDNA (see Table 1) was innovatively used to simulate the complex structure of Ab1-1@EFNA1@Ab2-1, which allows the coupling between DNA1-1 and DNA2-1 to form a cooperative complex (DNA1-1-refDNA-DNA2-1) that triggers the CHA reaction. In Figure 4 a, lanes 2 to 4 clearly show the bands of individual DNA1-1, DNA2-1, and refDNA respectively, demonstrating that the experimental materials in this example are complete and effective. When DNA1-1 and DNA2-1 were mixed in lane 5, no significant hybridization reaction bands were observed. However, when refDNA was incorporated into the mixture of DNA1-1 and DNA2-1, a new band appeared in lane 6, proving the generation of DNA1-1-refDNA-DNA2-1.

[0080] In Figure 4In lane 2 and 3 of b, bright bands of single hpDNA1-1 and hpDNA2-1 are clearly shown respectively, confirming the integrity of the hairpin structure. In lane 4, a mixture of DNA1-1, DNA2-1, hpDNA1-1 and hpDNA2-1 is included, but no new bands are observed, indicating that the CHA nucleic acid amplification is not spontaneous without a trigger. After adding the DNA1-1+refDNA+DNA2-1 co-complex as a trigger in lane 5, new bands appear, marking the successful start of the CHA nucleic acid amplification. Meanwhile, the DNA1-1-refDNA-DNA2-1 band is clearly visible in lane 5, further confirming that the target-triggered CHA nucleic acid amplification proceeds effectively.

[0081] Table 1 Nucleotide sequences used in the experiment

[0082]

[0083] Example 4 Assembly and parameter optimization of the SERS analysis platform

[0084] 1. Assembly of the SERS analysis platform

[0085] (1) First, the PDMS elastomer material prepared by mixing the PDMS prepolymer and the curing agent in a ratio of 10:1 is poured into a mold and cured at 80-90 °C for 2-2.5 h.

[0086] (2) Next, the PDMS elastomer material is cut and perforated to prepare a PDMS microporous array sandwich and a PDMS cover slip respectively, and ultrasonically cleaned with deionized water and ethanol and dried.

[0087] (3) Subsequently, the prepared Au / SiNPA is cut into 16 corresponding-sized parts and embedded in the ITO glass substrate by laser etching.

[0088] (4) Then, the ITO glass substrate and the PDMS microporous array sandwich are aligned and bonded by oxygen plasma technology to ensure a tight connection between the two.

[0089] (5) Finally, the PDMS cover slip is covered to successfully construct the microarray chip.

[0090] (6) The chip consists of three parts: the ITO glass substrate embedded with Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2, the PDMS microporous array sandwich and the PDMS cover slip. The chip size is 40 mm × 40 mm, containing 16 micropores (8 squares, 8 circles), with a diameter / side length of 4 mm, a spacing of 6 mm, and a height of 6 mm.

[0091] 2. Parameter optimization of the SERS analysis platform

[0092] The experimental parameters such as the volume of SERS nanolabels, incubation time, temperature and pH value of the reaction system were optimized. First, the sensing ability of the SERS analysis platform at different volumes of SERS nanolabels was evaluated. Figure 5 a shows that when the volume of SERS nanolabels for EFNA1 increased from 2 mL to 3 mL, the SERS signal increased, but then decreased with the increase in the volume of nanolabels. This is because the increase in the molecular collision frequency in the solution limits the assembly efficiency of SERS nanolabels. Similarly, the optimal volume of SERS nanolabels for MMP13 is 4 mL. This volume difference may be related to the steric site blocking effect caused by the length of hpDNA, affecting the assembly affinity and hybridization efficiency. Next, as Figure 5 shown in b, the SERS signal intensity increased during the 3 - 12 min incubation period and stabilized after 12 min. Therefore, for the following tests, the incubation time was set to 12 min. The temperature and pH value of the reaction system greatly affect the POD-like activity of SERS nanolabels, which in turn affects the catalytic efficiency of TMB. As Figure 5 shown in c and Figure 5 d, the intensity of the SERS signal first increased and then decreased with the increase in temperature and pH value. Therefore, the optimal temperature is 37 °C and the pH value is 4.25.

[0093] Example 5 Specificity, anti-interference ability and reproducibility of the SERS analysis platform

[0094] 1. Assemble the SERS analysis platform as in Example 4.

[0095] 2. Expose the established SERS analysis platform to various clinical tumor markers and serum interferents at the same concentration (1 mg / mL) to investigate its selectivity and anti-interference ability. The results of the selectivity experiment are shown in Figure 6 a, where significant SERS signals were observed in the presence of EFNA1 and MMP13, while the SERS signals of other non-target proteins (CEA, AFP, CA19-9 and CA72-4) seemed weaker and similar to the blank spectrum. In addition, as Figure 6 shown in b, the present invention measured the mixed solution of key interfering substances and target proteins. It was found that even when the concentration of these interfering substances reached 10 times that of the target protein, they hardly affected the SERS intensity of the target protein. The above research results indicate that the platform has strong anti-interference ability and specificity, and is completely suitable for the accurate identification of biomarkers in complex samples.

[0096] In addition, as Figure 6As shown in Figures 6c and 6d, the present invention also records the SERS spectra of five SERS analysis platforms prepared in different batches for detecting EFNA1. The results show that there are no obvious differences in the Raman spectra and the intensities of the main characteristic peaks of different platforms, indicating that the platform has good reproducibility.

[0097] Example 6 Quantitative analysis of two target protein biomarkers (EFNA1 and MMP13)

[0098] 1. Assemble the SERS analysis platform as in Example 4.

[0099] 2. Under the optimized experimental conditions, the sensitivity of different concentrations of target proteins (EFNA1 and MMP13) in serum was detected and analyzed using this SERS analysis platform. Figure 7 Figures 7a and 7c record the Sers spectra, and it can be clearly found that the intensity of the SERS spectra gradually increases with the increase in the concentrations of EFNA1 and MMP13. It should be noted that the SERS signal intensity of ox-TMB indirectly reflects the concentration of the target protein.

[0100] Taking the characteristic peak intensity of ox-TMB at 1602 cm -1 and plotting a linear curve with the logarithm of the target protein concentration, the corresponding linear regression equations are obtained ( Figure 7 Figures 7b and 7d). The regression equation for FENA1 is y = 4059.28x + 51059.74, and the determination coefficient (R 2 ) is 0.984, with a linear range of 1 pg / mL - 1 mg / mL. The regression equation for MMP13 is y = 3702.48x + 46785.73, and R 2 is 0.982, with a linear range of 1 pg / mL - 1 mg / mL. Therefore, the nanozyme-mediated catalytic signal amplification combined with the CHA nucleic acid amplification strategy adopted in this SERS analysis platform can effectively quantify EFNA1 and MMP13. In addition, the detection limits (LOD) of EFNA1 and MMP13 are calculated to be 0.75 and 0.84 pg / mL (signal-to-noise ratio S / N = 3), respectively. Compared with other tests (see Table 2), this scheme can quickly and accurately quantify and detect EFNA1 and MMP13 in a complex serum environment.

[0101] Table 2 Comparison of the detection method in this experiment with other reported methods

[0102]

[0103] Example 7 Analysis of clinical samples by the SERS analysis platform

[0104] 1. Assemble the SERS analysis platform as in Example 4.

[0105] 2. The SERS analysis platform was used to test 20 serum samples from healthy individuals and 20 serum samples from early GC patients. Figure 8 Figure a shows the top view and side view of the microarray chip with a three - layer structure, which ensures that SERS detection can be carried out under contamination - free conditions. The chip contains 16 detection units, and the detection areas of EFNA1 and MMP13 are distinguished by the fine layout of square and circular micro - pores, enabling high - throughput detection of target proteins. As Figure 8 shown in Figure b, when EFNA1 and MMP13 are detected, the total intensity of the average SERS spectrum of early GC patients is higher than that of the average SERS spectrum of healthy individuals. The significance of the characteristic peak intensity at 1602 cm -1 was evaluated by Student’t test ( Figure 8 Figure c), which reveals a significant difference in the expression levels of EFNA1 and MMP13 in the sera of early GC patients and healthy individuals.

[0106] In addition, by substituting the SERS intensity at 1602 cm -1 into the linear regression equation obtained in human serum, the concentrations (ng / mL) of EFNA1 and MMP13 were calculated. Subsequently, the ELISA method was established as the gold standard for detecting the expression levels of EFNA1 and MMP13 in clinical samples and compared with the results of the SERS analysis platform (Table 3 - 4). The relative deviations (RD) of the detection results of the two methods were within the acceptable range. The linear fitting results show a high correlation between the two detection methods for EFNA1 and MMP13, with R 2 values of 0.973 and 0.981 respectively, indicating good consistency between them. Although the combination of gastroscopy and biopsy can diagnose early GC, due to its invasiveness and complications, it is not suitable as a large - scale screening tool. Therefore, the SERS analysis platform constructed in the present invention is expected to better meet the needs of clinical early GC screening, providing an efficient and low - risk solution.

[0107] Table 3 SERS and ELISA detection results of EFNA1 and MMP13 concentrations in the sera of healthy individuals

[0108]

[0109] Table 4 SERS and ELISA detection results of EFNA1 and MMP13 concentrations in the sera of early GC patients

[0110]

[0111]

[0112] It should be noted that in this text, the term "including", "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent in such a process, method, article or device. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or device including that element.

[0113] The serial numbers of the above embodiments of the present invention are only for description and do not represent the superiority or inferiority of the embodiments.

[0114] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A preparation method of a SERS analysis platform for detecting protein biomarkers of early gastric cancer, characterized in that, The preparation method includes the following steps: S1: Modify hpDNA1 onto the surface of Au@PtNPs nanozyme to form two SERS probes; S2: Modify hpDNA2 onto the surface of the heteromorphic nano-column array Au / SiNPA as a capture substrate; S3: Assemble the capture substrate prepared in step S2 into a microarray chip as a SERS analysis platform; The hpDNA1 includes hpDNA1-1 and hpDNA1-2. The sequence of hpDNA1-1 is as shown in SEQ ID NO:3, and the sequence of hpDNA1-2 is as shown in SEQ ID NO:5; the hpDNA2 includes hpDNA2-2 and hpDNA2-2. The sequence of hpDNA2-2 is as shown in SEQ ID NO:4, and the sequence of hpDNA2-2 is as shown in SEQ ID NO:

6.

2. The preparation method of the SERS analysis platform for detecting early gastric cancer protein biomarkers according to claim 1, wherein The preparation method of the Au@PtNPs nanozyme includes: Take gold sol, add deionized water, AA solution and trisodium citrate to form solution A, where the diameter of the gold seeds in the gold sol is 16 - 18 nm; Add the HAuCl4 solution to solution A and react in an environment of 90 - 100 °C for more than 30 minutes to obtain solution B; Add the H2PtCl6 solution to solution B and mix well, while keeping the temperature in the reaction flask not lower than 80 °C; subsequently, add the AA solution and trisodium citrate dropwise to the mixed solution until the reaction is complete, and the Au@PtNPs nanozyme is obtained.

3. The preparation method of the SERS analysis platform for detecting early gastric cancer protein biomarkers according to claim 1 or 2, characterized in that, The step of modifying hpDNA1 onto the surface of Au@Pt NPs nanozyme to form two SERS probes includes: After activating hpDNA1, incubate it with Au@PtNPs nanozyme for more than 12 h, and disperse the obtained mixture in a bovine serum albumin solution, followed by centrifugal purification to collect two SERS nanolabels: Au@Pt NPs@hp1-1 and Au@PtNPs@hp1-2.

4. The preparation method of the SERS analysis platform for detecting early gastric cancer protein biomarkers according to claim 3, characterized in that, When performing centrifugal purification, the centrifugal speed is 10000 - 12000 rpm, and the centrifugal time is 10 - 12 min.

5. The preparation method of the SERS analysis platform for detecting early gastric cancer protein biomarkers according to claim 1, characterized in that, The preparation method of the Au / SiNPA includes: Obtain a monolayer densely arranged PS microsphere array by the gas-liquid interface self-assembly method, and perform ion etching on its surface using three etching gases SF6, O2 and Ar; perform heat treatment on the obtained intermediate product to remove the residual PS microspheres to obtain SiNCA; then deposit gold nanoparticles on the surface of SiNCA by ion sputtering process to obtain Au / SiNCA.

6. The preparation method of the SERS analysis platform for detecting early gastric cancer protein biomarkers according to claim 5, characterized in that, The sputtering time of the ion sputtering process is 5 - 8 min, the current is 30 - 35 mA, and the deposition rate is 0.5 - 0.7 nm / s.

7. The preparation method of the SERS analysis platform for detecting early gastric cancer protein biomarkers according to claim 1, wherein The step of modifying hpDNA2 onto the surface of the heteromorphic nano-column array Au / SiNPA includes: Drop the activated hpDNA2 solution onto the Au / SiNPA array and incubate for more than 12 h to prepare two capture substrates: Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2.

8. The preparation method of the SERS analysis platform for detecting early gastric cancer protein biomarkers according to claim 7, characterized in that, Step S3 specifically includes: embedding Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2 into the ITO glass substrate, aligning and bonding them with the PDMS microporous array interlayer through oxygen plasma technology, and covering the surface with a PDMS cover slip; The microarray chip is a 4×4 microarray chip; the PDMS microporous array interlayer is a 4×4 PDMS microporous array interlayer; The size of the microarray chip is 40 mm × 40 mm, and it contains 16 micropores.

9. A SERS analysis platform for detecting early gastric cancer protein biomarkers prepared by the preparation method according to any one of claims 1-9.

10. Use of the SERS analysis platform according to claim 10 for simultaneously detecting early gastric cancer protein biomarkers EFNA1 and MMP13 for purposes other than disease diagnosis and treatment.

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