A sers analysis platform for detecting early gastric cancer protein biomarkers and a preparation method and application thereof

Abstract

The application discloses a surface enhanced Raman scattering (SERS) analysis platform for detecting early gastric cancer protein biomarkers and 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@Pt nano-enzyme (Au@Pt NPs) nano-enzyme with a hairpin structure DNA1 (hpDNA1) to form two SERS probes; (2) modifying hpDNA2 on the surface of a special-shaped nano-pillar array (Au / SiNPA) as a capture substrate; and (3) assembling a 4*4 microarray chip as a SERS analysis platform. The SERS analysis platform has the advantages of simple operation, high throughput, low price, high sensitivity and high specificity, and has good repeatability and can be prepared on a large scale.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a SERS analysis platform for detecting protein biomarkers of early gastric cancer, its preparation method, and its application. Background Technology

[0002] Gastric cancer (GC) is a common malignant tumor that poses a significant threat to human health. Actively implementing secondary prevention of GC is crucial for effectively improving prognosis and reducing mortality. Notably, traditional serological tumor markers, such as carcinoembryonic antigen (CEA), carbohydrate antigen 72-4 (CA72-4), and carbohydrate antigen 19-9 (CA19-9), are insufficient for detecting GC due to their poor sensitivity and specificity. While gastroscopy with pathological analysis is considered the "gold standard" for diagnosing GC, this invasive procedure causes significant discomfort to patients and is therefore unsuitable for large-scale clinical screening. Therefore, it is necessary to explore potential biomarkers associated with GC to provide new insights for early diagnosis. Numerous 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, promoting the invasiveness of the primary tumor. Literature indicates that the area under the curve (AUC) for the combined detection of early GC by serum EFNA1 and MMP13 is improved (0.794) compared to single biomarkers. Currently, various methods exist for detecting EFNA1 and MMP13, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and electrochemical sensors. However, most of these methods suffer from high cost, time-consuming multiple assays, low sensitivity, and poor interference resistance. Therefore, developing a low-cost, simple, rapid, sensitive, and specific EFNA1 / MMP13 detection method is crucial to breaking with conventional approaches.

[0003] Nanozymes are nanomaterials with catalytic activity similar to natural enzymes. Compared to natural enzymes, nanozymes offer advantages such as high stability, tunable catalytic activity, and low cost, while avoiding the problem of easy inactivation of biological enzymes. Therefore, they show great potential in biosensing, therapy, and energy conversion. Generally, analytical research on nanozymes focuses primarily on visualizing the color changes of enzyme substrates after signal-catalyzed reactions. However, their practical application is limited by low sensitivity and color interference from the material itself and the sample matrix. To improve detection sensitivity and accuracy, researchers have employed electrochemical and fluorescence detection techniques in studies based on nanozyme-mediated catalysis platforms. These techniques offer rapid and sensitive detection, but also suffer from significant drawbacks such as electrode instability, susceptibility to fluorescence quenching, and complex operation. Surface-enhanced Raman scattering (SERS) is a powerful inelastic scattering spectroscopy technique that has rapidly developed in the field of biosensing due to its speed, non-destructive nature, and water insensitivity. SERS technology can easily acquire 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 low Raman scattering cross section of small protein molecules and their relatively weak interaction with the substrate, the sensitivity of label-free SERS detection strategies is insufficient.

[0004] Because the concentration of biomarkers in serum is extremely low during early GC, traditional detection methods cannot meet the stringent sensitivity requirements. Therefore, it is of great significance to develop a SERS analysis platform that is highly sensitive, specific, easy to operate, fast in detection, and has high throughput. Summary of the Invention

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

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

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

[0008] S1: hpDNA1 was modified onto the surface of Au@PtNPs nanozyme to form two SERS probes;

[0009] S2: hpDNA2 was modified onto the surface of an amorphous nanopillar array Au / SiNPA as a capture substrate;

[0010] S3: Assemble the capture substrate prepared in step S2 into a microarray chip as a 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 a SERS analysis platform for detecting protein biomarkers of early gastric cancer prepared by the above preparation method.

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

[0014] like Figure 1 As shown, this invention constructs a novel SERS analysis platform that effectively combines nanozymes and SERS technology. On one hand, nanozymes possess peroxidase-like (POD) activity, catalyzing the conversion of probe molecules into SERS-active reporter molecules, thereby generating a strong SERS signal. On the other hand, nanozymes, with their large surface area and numerous active sites, effectively increase the number of interacting signal molecules, thus improving detection sensitivity.

[0015] The bimetallic Au@Pt nanoparticles (Au@PtNPs) selected in this invention exhibit excellent POD-like activity and SERS enhancement effect. The enhanced catalytic activity of Au@PtNPs is mainly attributed to their porous structure and the formation of an electron-rich Pt shell, transforming the catalytic pathway from the generation of hydroxyl radicals to an electron transfer process. The selected SERS substrate is an irregularly shaped nanopillar array (Au / SiNPA) prepared by colloidal sphere template-assisted reactive ion etching (RIE), which possesses excellent uniformity and sensitivity.

[0016] This invention also utilizes catalytic hairpin self-assembly (CHA), a multifunctional isothermal enzyme-free amplification technique. Compared to other amplification strategies, such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and rolling circle amplification (RCA), CHA offers significant advantages including simple reaction conditions, high hybridization efficiency, low cost, low background signal, and good stability. Antibody-DNA conjugates, formed by the binding of specific antibodies to carefully designed oligonucleotide chains, can both specifically bind to target biomarkers and trigger CHA to amplify the signal; thus, applying CHA technology to SERS detection of protein biomarkers can further improve detection sensitivity.

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

[0018] The Au@Pt nanoparticle (Au@PtNPs) nanozyme prepared in this invention has a core-shell structure that combines good peroxidase (POD)-like activity and SERS-enhanced activity.

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

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

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

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

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

[0024] Figure 3 The images shown are: (a) SEM image of a monolayer PS colloidal sphere; (b) SEM image of SiNPA; (c) SEM image of Au / SiNPA and its locally magnified form; (d) SERS spectrum of 40 randomly selected points on Au / SiNPA; and (d) SERS spectrum at 1080 cm⁻¹. -1 The corresponding peak intensity (e) at 1592 cm⁻¹ and at 1592 cm⁻¹ -1 The corresponding peak intensity (f) at the position; SERS spectrum of 4-MBA labeled Au / SiNPA (g); pure 4-MBA (1×10⁻⁶) -1 M) and 4-MBA (1×10 -8 SERS spectrum (h) of Au / SiNPA labeled with M); 1592 cm⁻¹ of Au / SiNPA labeled with 4-MBA after storage in N₂ and air for 0–30 days. -1 The corresponding SERS intensity piecewise linear plot (i) is shown.

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

[0026] Figure 5 The following optimizations were made for the SERS nanotag volume (a), incubation time (b), incubation temperature (c), and pH of the reaction system (d) in this embodiment of the invention.

[0027] Figure 6 The following are examples of the selective evaluation of the SERS analysis platform used for the detection of EFNA1 and MMP13 in this embodiment of the invention (concentrations of CEA, AFP, CA19-9, CA72-4, MMP13, and EFNA1 were 1 mg / mL) (a); the effects of potential interfering substances such as small molecule glucose, uric acid, GSH, urea, AA, lactic acid, and cholesterol, as well as 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 the results at 1602 cm⁻¹. -1 The SERS intensity (d) of the corresponding characteristic peak at the location.

[0028] Figure 7The SERS 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 in this embodiment of the invention are shown in (a); the 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 are shown in (b); the logarithm of EFNA1 concentration and 1602 cm⁻¹ are also shown. -1 Linear curve of characteristic peak intensity at 1602 cm⁻¹ (c); logarithm of MMP13 concentration versus 1602 cm⁻¹ -1 The linear curve (d) of the characteristic peak intensity at the location.

[0029] Figure 8 The images show a top view and a side view (a) of the SERS microarray chip in an embodiment of the present invention; the average SERS spectra of EFNA1 and MMP13 in the serum of 20 healthy individuals and 20 patients with early-stage GC (b); and the image at 1602 cm⁻¹. -1 The intensity of the characteristic peak (c). Detailed Implementation

[0030] 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, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0032] This invention provides a surface-enhanced Raman scattering (SERS) analysis platform for detecting protein biomarkers in early gastric cancer. The preparation method includes: (1) modifying the surface of Au@Pt nanoparticles (Au@Pt NPs) nanozymes with hairpin-structured DNA1 (hpDNA1) to form two SERS probes; (2) modifying the surface of a heteromorphic nanopillar array (Au / SiNPA) with hpDNA2 as a capture substrate; and (3) assembling a 4×4 microarray chip as the SERS analysis platform. hpDNA1 includes hpDNA1-1 and hpDNA1-2, whose sequences are shown in Table 1; hpDNA2 includes hpDNA2-1 and hpDNA2-2, whose sequences are shown in Table 1.

[0033] In the detection process, this invention triggers CHA amplification by designing antibody-DNA conjugates and generating antigen@antibody-DNA conjugates that specifically bind to the target protein. This allows more Au@PtNPs nanozymes to attach to the Au / SiNPA surface, implying higher catalytic efficiency for the conversion of 3,3',5,5'-tetramethylbenzidine (TMB) to SERS-active oxidized TMB (ox-TMB). The close proximity of the SERS nanotags to the trapping substrate significantly amplifies the plasmonic coupling effect, generating more "hot spots" to further amplify the ox-TMB signal. Using this strategy, EFNA1 and MMP13 can be successfully detected with limits of detection (LOD) as low as pg / mL. This SERS analysis platform exhibits good specificity, anti-interference, and stability, and has been successfully applied to the serum detection of early gastric cancer, with results consistent with ELISA results. 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.

[0034] In some specific embodiments, the preparation method of the above-mentioned 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 thoroughly, while maintaining the temperature in the reaction flask at no less than 80°C. Then, adding AA and trisodium citrate dropwise to the mixed solution until the reaction is complete, thereby obtaining the Au@PtNPs nanozyme.

[0035] In some preferred embodiments, the preparation method of the above-mentioned Au@PtNPs nanozyme may include: taking gold sol and 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 at 90-100°C for at least 30 minutes to obtain solution B. Adding H2PtCl6 solution (4-4.5 mL, 0.096 mM) to solution B and mixing thoroughly while maintaining the temperature in the reaction flask at no less 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 at least 30 minutes until the reaction is complete to obtain Au@PtNPs nanozyme.

[0036] In some specific embodiments, the gold seed diameter of the gold sol is 16-18 nm.

[0037] In some specific embodiments, the above-mentioned surface modification of Au@PtNPs nanozyme with hpDNA1 to form two SERS probes may include: activating hpDNA1, co-incubating it with Au@PtNPs nanozyme for more than 12 hours, and dispersing the resulting mixture in bovine serum albumin (BSA) solution. After centrifugation purification, two SERS nanolabels are collected: Au@PtNPs@hp1-1 and Au@PtNPs@hp1-2.

[0038] In some preferred embodiments, the above-described surface modification of Au@PtNPs nanozyme with hpDNA1 to form two SERS probes includes: activating hpDNA1, co-incubating it with Au@PtNPs nanozyme for at least 12 hours, and dispersing the resulting mixture in bovine serum albumin (BSA) solution (160-180 L, 1 wt%). After centrifugation and purification, two SERS nanolabels are collected: Au@PtNPs@hp1-1 and Au@PtNPs@hp1-2.

[0039] In some specific embodiments, the centrifugation speed is 10,000-12,000 rpm and the centrifugation time is 10-12 min.

[0040] In some specific embodiments, the preparation method of Au / SiNPA described above may include: obtaining a monolayer densely packed array of PS microspheres through a gas-liquid interface self-assembly method, and then ion etching its surface using three etching gases, SF6, O2, and Ar. The obtained intermediate product is then heat-treated to remove residual PS microspheres, thus obtaining SiNCA. Finally, gold nanoparticles are deposited on the SiNCA surface using ion sputtering technology to obtain Au / SiNCA.

[0041] In some preferred embodiments, the preparation method of Au / SiNPA described above may include:

[0042] (1) Using a PS microsphere suspension (500 nm) as a precursor, it was mixed with ethanol at a volume ratio of 1:1 to self-assemble at the gas-liquid interface to obtain an ordered monolayer PS microsphere array. (2) Then, reactive ion etching was used. 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). Then, Ar gas was used to perform physical etching on its ionized surface (gas flow rate 10-15 sccm, etching power 100 W) to obtain an ordered array of PS microspheres / silicon pillar hybrid structure. (3) After superstructure annealing (≥350℃, 30 min), a silicon pillar array (SiNCA) with a nanoplatform on the surface was obtained. Finally, using Au as the target material, gold nanoparticles were deposited on the SiNCA surface by ion sputtering technology to successfully prepare Au / SiNPA, whose structural unit is composed of irregularly shaped nanopillars. Next, TECP buffer-activated hpDNA2 (hpDNA2-1 and hpDNA2-2) were modified on the Au / SiNCA surface to prepare capture substrates: Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2.

[0043] In some embodiments of the present invention, after obtaining the above-mentioned monolayer PS microsphere array, the method further includes transferring the PS microsphere array using a hydrophilically treated silicon wafer. The present invention does not impose any particular limitation on the method of hydrophilically treating the silicon wafer; any technical solution well known to those skilled in the art can be selected.

[0044] In some specific embodiments, the sputtering time of the above-mentioned ion sputtering technology is 5-8 min, the current is 30-35 mA, and the deposition rate is 0.5-0.7 nm / s.

[0045] Preferably, the temperature for activating hpDNA2 using TECP buffer is 80-95°C, and the time is no less than 30 minutes.

[0046] In some specific embodiments, the above-described assembly of a 4×4 microarray chip as a SERS analysis platform includes: an ITO glass substrate embedding Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2 sandwiched with a 4×4 PDMS micropore array, aligned and bonded using oxygen plasma technology, and covered with a PDMS cover sheet. The chip size is 40mm×40mm, containing 16 micropores (8 square and 8 circular).

[0047] In some preferred embodiments, the above-described assembly of a 4×4 microarray chip as a SERS analysis platform may include:

[0048] (1) First, the PDMS elastomer material prepared by mixing polydimethylsiloxane (PDMS) prepolymer and curing agent at a ratio of 10:1 was poured into a mold and cured at 80-90℃ for 2-2.5h. (2) Next, the PDMS elastomer material was cut and perforated to prepare PDMS microporous array sandwich and PDMS cover sheet respectively, and ultrasonically cleaned and dried with deionized water and ethanol. (3) Subsequently, the prepared Au / SiNPA was cut into 16 parts of corresponding size and embedded into the ITO glass substrate by laser etching. (4) Then, the ITO glass substrate and 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 sheet was covered to successfully construct the microarray chip. (6) The chip consists of three parts: an ITO glass substrate with embedded Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2, a PDMS microporous array sandwich and a PDMS cover sheet. The chip measures 40mm x 40mm and contains 16 micro-holes (8 squares and 8 circles), with a diameter / side length of 4mm, a spacing of 6mm, and a height of 6mm.

[0049] It should also be noted that the application of any of the SERS analysis platforms described above for detecting early gastric cancer protein biomarkers in the simultaneous detection of early gastric cancer protein biomarkers EFNA1 and MMP13 for non-disease diagnosis and treatment purposes includes the following steps:

[0050] (1) First, demonstrate the EFNA1 detection process. Incubate 5.0-5.5 mL of diluted serum sample with 3.0-3.5 mL of antibody-DNA conjugates (Ab1-1@DNA1-1 and Ab2-1@DNA2-1) at 37°C with shaking for 2-3 hours to form antigen@antibody-DNA conjugates through antigen-antibody reaction. Then, drop the conjugate solution into the microarray chip (square sample port), and simultaneously add the SERS nanolabel (Au@Pt NPs@hp1-1) into the microarray chip.

[0051] (2) The conjugate and SERS nanolabels triggered a CHA reaction in the reaction chamber, allowing an increasing number of Au@PtNPs nanozymes to attach to the SERS capture substrate. After the reaction was terminated, excess SERS nanolabels were removed by washing three times with PBS buffer and deionized water.

[0052] (3) Finally, add 15-20 mL of freshly prepared solution, TMB (1 mM), and 0.5 M acetate / sodium acetate buffer (H2O2), and dry before proceeding to the next step of SERS detection. The MMP13 detection process is the same as above, with the detection area being the circular sample port. SERS spectra were acquired in the reaction chamber using a Raman microscope with a 50× objective and a 785 nm laser with a power of 5 mW. Each spectrum was acquired within 10 seconds. Each sample was scanned at five random locations, with a 20 mm² spot size ranging from 400 to 1800 cm⁻¹, to produce an average SERS spectrum. Error bars indicate the standard deviation of the five measurements.

[0053] Preferably, the volume of SERS probe Au@PtNPs@hp1-1 added is 3.0-3.5L, and the volume of SERS probe Au@PtNPs@hp2-1 added is 4.0-4.5L.

[0054] Preferably, the temperature of the reaction system is 36-38℃ and the pH value is 4.2-4.3.

[0055] To better understand this invention, specific examples are provided below to further illustrate its content; however, the invention is not limited to these examples. A schematic diagram of the preparation process of the SERS analysis platform of this invention and its detection of early gastric cancer biomarkers EFNA1 and MMP13 is shown below. Figure 1 As shown.

[0056] Unless otherwise specified, all raw materials and reagents used in the examples can be obtained through commercial channels.

[0057] The instruments, equipment, and testing conditions used in the experiments of this invention are as follows:

[0058] Transmission electron microscopy (TEM) images were obtained using a TECNAI12 TEM manufactured by Philips, Netherlands; high-resolution transmission electron microscopy (HRTEM) images and selected area electron diffraction (SAED) analysis were obtained using a FEI field emission transmission electron microscope combined with X-ray energy dispersive spectroscopy (EDS); scanning electron microscopy (SEM) images were obtained using an S-4800 field emission scanning electron microscope manufactured by Hitachi, Japan; Raman spectroscopy was performed using an InviaReflex laser micro-Raman spectrometer manufactured by Renishaw, UK. The laser wavelength was 785 nm, the exposure time was 10 s, the laser intensity was 50 mW, and the objective lens was 50×; SERS imaging was performed using a DXRxi micro-Raman imaging spectrometer manufactured by Thermo Fisher Scientific, USA.

[0059] The following specific embodiments illustrate the solution proposed in this invention:

[0060] Example 1: Synthesis and Characterization of Au@PtNPs Nanozymes

[0061] 1. Synthesis of Au@PtNPs nanozymes

[0062] (1) Take gold sol (16-18nm) and add it to deionized water, AA solution (1.0-1.5mL, 28mM) and trisodium citrate solution (1.0-1.5mL, 10mM) to form solution A.

[0063] (2) Add HAuCl4 solution (0.20-0.35 mL, 24 mM) to solution A and react at 90-100℃ for more than 30 minutes to obtain solution B.

[0064] (3) Add H2PtCl6 solution (4-4.5 mL, 0.096 mM) to solution B and mix thoroughly, while keeping the temperature in the reaction flask no lower than 80℃. 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 to obtain Au@PtNPs nanozyme.

[0065] 2. Morphology, POD-like activity, and SERS properties of Au@PtNPs nanozymes

[0066] like Figure 2 As shown in image a, the TEM image reveals that Au@PtNPs are highly uniform, regular spheres with a height of 52 nm. Numerous smaller, granular platinum atom clusters (approximately 4 nm) are uniformly distributed on the surface of the Au core, assembling them into a porous outer layer, as shown in the HRTEM image. Figure 2 b). Lattice fringes of a typical particle with a crystal spacing of 0.226 nm ( Figure 2 c) Corresponds well to the Pt(111) surface. In the selected area electron diffraction (SAED) pattern ( Figure 2 In d), four distinct diffraction rings match the (111), (200), (220), and (311) crystal planes, reflecting its polycrystalline nature. Furthermore, energy-dispersive X-ray (EDX) spectroscopy confirmed the elemental composition of Au@PtNPs, with Au and Pt elements being the dominant elements. Figure 2 f). To further elucidate the elemental distribution of Au@PtNPs, high-angle annular dark-field scanning TEM (HAADF-STEM) images and corresponding elemental maps were obtained. Figure 2 e) The Pt distribution around the Au core forms a metallic shell, indicating that the core-shell structured Au@PtNP was successfully synthesized.

[0067] Peroxidase (POD)-like activity is one of the important properties of Au@PtNPs, which can be verified by classical colorimetric methods. Figure 2 g). Using TMB as the colorimetric substrate, experimental results showed that the color rapidly turned blue only when TMB, Au@PtNPs, and H2O2 were present simultaneously in the solution. This clearly indicates that Au@PtNPs exhibit good POD-like activity. Furthermore, the steady-state kinetics of Au@PtNPs were analyzed. Figure 2 As shown in h, the Michaelis-Menten curves and their corresponding double reciprocal plots were obtained. The Vt of Au@PtNPs catalyzing TMB was... max and K m They are 0.853 mM·s -1 With a concentration of 0.570 mM, it is superior to the natural enzyme HRP, which can be attributed to its porous structure enhancing TMB adsorption. The enhancement factor (EF) is an important indicator of SERS enhancement capacity. Figure 2 As shown in i, Au@PtNPs significantly enhance the Raman signal of ox-TMB, and through formula (I) SERS / C SERS ) / (I Raman / C Raman )Calculate EF as 4.05 × 10 6 Among them, ISERS and I Raman These are from ox-TMB (1×10) -6 Au@PtNPs labeled with M) and pure ox-TMB (1×10⁻⁶) -1 1602cm obtained from M) -1 The intensity at the peak. In summary, this embodiment successfully prepared a bifunctional Au@PtNPs nanozyme with strong POD-like activity and SERS performance.

[0068] Example 2: Preparation and Characterization of Au / SiNPA

[0069] 1. Preparation of Au / SiNPA

[0070] (1) Using PS microsphere suspension (500 nm) as a precursor, it was mixed with ethanol at a volume ratio of 1:1 and self-assembled at the gas-liquid interface to obtain an ordered monolayer PS microsphere array.

[0071] (2) Then, reactive ion etching is used. First, SF6 and O2 are used as etching gases to perform ion etching on the obtained monolayer PS colloidal sphere template for 6-8 minutes (gas flow rate 20-25 sccm / 10-15 sccm, etching power 100W). Then, Ar gas is used to perform physical etching on its ionized surface (gas flow rate 10-15 sccm, etching power 100W) to obtain an ordered array of PS microsphere / silicon pillar hybrid structure.

[0072] (3) After superstructure annealing (≥350℃, 30min), a silicon pillar array (SiNCA) with a nanoplatform on the surface was obtained. Finally, using Au as the target material, Au / SiNPA (deposition rate of 0.5-0.7nm / s) was successfully prepared by ion sputtering at a current of 30-35mA for 5-8min. Its structural unit is composed of irregularly shaped nanopillars.

[0073] 2. Morphology and SERS effect characterization of Au / SiNPA

[0074] like Figure 3 As shown in Figure a, this embodiment employs a gas-liquid interface self-assembly method and overall transfer to successfully form a uniform and dense layer of PS colloidal spheres (500 nm in diameter) on the surface of a hydrophilic silicon wafer. Figure 3 As shown in b, by optimizing the etching process and annealing operation, a silicon pillar array (SiNPA) with nanoplatforms on the surface was prepared, in which the silicon pillars exhibited 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 heterogeneous nanopillars with a height of 290 nm, a period of 500 nm, and uniformly arranged in an ordered pattern. Figure 3 The bottom right corner shows a magnified view of Au / SiNPA, revealing that the rough array surface is characterized by many prominent near-spherical particles with abundant nano-intervals, which can generate a large number of SERS "hot spots".

[0075] Subsequently, SERS detection was performed on 40 randomly selected points on the 4-MBA labeled Au / SiNPA to evaluate the uniformity of the array. Figure 3 d). The results show that the overall spectral shape exhibits a consistent trend with only slight intensity differences. Statistically, the values ​​at 1080 and 1592 cm⁻¹ are... -1 The relative standard deviation (RSD) values ​​of the 4-MBA characteristic peak intensity were only 5.84% and 5.48%, where the red horizontal line represents the average intensity and the area between the two blue lines represents the range of deviation. Figure 3 e and f). The results show that Au / SiNPA exhibits satisfactory uniformity and signal reproducibility. Furthermore, Figure 3 g shows the SERS spectrum of 4-MBA labeled Au / SiNPA in a 50×50mm area, which shows an overall uniform green (color represents SERS signal intensity), further demonstrating the significant uniformity of the array. Figure 3 h showed pure 4-MBA (1×10) -1 M) and 4-MBA (1×10 -8The SERS spectrum of Au / SiNPA labeled with M) was calculated to have an EF value of 1.09 × 10⁻⁶. 9 The results show that Au / SiNPA has a significant SERS enhancement capability, consistent with the previous hypothesis.

[0076] Finally, the prepared 4-MBA-labeled Au / SiNPA was placed in air and N2 for different numbers of days to evaluate the array's stability. (1592 cm⁻¹) -1 The characteristic peak intensity remains high 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 is confirmed that it can meet the requirements of subsequent practical applications.

[0077] Example 3: Feasibility assessment of the CHA nucleic acid amplification strategy

[0078] The feasibility of the CHA nucleic acid amplification strategy must be evaluated before it can be applied to quantify EFNA1 and MMP13. Therefore, agarose gel electrophoresis was used. Figure 4 The amplification process was analyzed. In this embodiment, refDNA (see Table 1) was innovatively used to mimic the complex structure of Ab1-1@EFNA1@Ab2-1, allowing coupling between DNA1-1 and DNA2-1 to form a co-conjugate complex (DNA1-1-refDNA-DNA2-1) that triggers the CHA reaction. Figure 4 In lane a, lanes 2 to 4 clearly show individual bands of DNA1-1, DNA2-1, and refDNA, respectively, demonstrating that the experimental materials in this embodiment 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, demonstrating the generation of DNA1-1-refDNA-DNA2-1.

[0079] exist Figure 4In lane b, lanes 2 and 3 clearly show bright bands of individual hpDNA1-1 and hpDNA2-1, respectively, confirming the integrity of the hairpin structure. Lane 4 contains a mixture of DNA1-1, DNA2-1, hpDNA1-1, and hpDNA2-1, but no new bands were observed, indicating that CHA nucleic acid amplification is not spontaneous without a trigger. However, after adding the DNA1-1+refDNA+DNA2-1 co-conjugate complex as a trigger in lane 5, a new band appeared, marking the successful initiation of CHA nucleic acid amplification. Simultaneously, the DNA1-1-refDNA-DNA2-1 band is clearly visible in lane 5, further confirming that target-triggered CHA nucleic acid amplification proceeds effectively.

[0080] Table 1. Nucleotide sequences used in the experiment.

[0081]

[0082] Example 4: Assembly and Parameter Optimization of the SERS Analysis Platform

[0083] 1. Assembly of the SERS analysis platform

[0084] (1) First, the PDMS elastomer material prepared by mixing polydimethylsiloxane (PDMS) prepolymer and curing agent in a ratio of 10:1 is poured into a mold and cured at 80-90℃ for 2-2.5h.

[0085] (2) Next, the PDMS elastomer material was cut and perforated to prepare the PDMS microporous array sandwich and PDMS cover sheet respectively, and ultrasonically cleaned and dried with deionized water and ethanol.

[0086] (3) The prepared Au / SiNPA was then cut into 16 parts of corresponding size and embedded into the ITO glass substrate by laser etching.

[0087] (4) Then, the ITO glass substrate and the PDMS microporous array interlayer are aligned and bonded by oxygen plasma technology to ensure a tight connection between the two.

[0088] (5) Finally, a PDMS cover sheet was applied to successfully construct the microarray chip.

[0089] (6) The chip consists of three parts: an ITO glass substrate with embedded Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2, a PDMS micro-hole array interlayer, and a PDMS cover sheet. The chip has a size of 40mm×40mm and contains 16 micro-holes (8 squares and 8 circles), with a diameter / side length of 4mm, a spacing of 6mm, and a height of 6mm.

[0090] 2. Parameter optimization of the SERS analysis platform

[0091] Experimental parameters such as SERS nanotag volume, incubation time, reaction system temperature, and pH were optimized. First, the sensing capability of the SERS analysis platform under different SERS nanotag volumes was evaluated. Figure 5 The results showed that the SERS signal enhanced when the volume of the EFNA1 SERS nanotag increased from 2 mL to 3 mL, but subsequently weakened with further increases in nanotag volume. This is because the increased frequency of molecular collisions in solution limits the assembly efficiency of the SERS nanotags. Similarly, the optimal volume for the MMP13 SERS nanotag was 4 mL. This volume difference may be related to the spatial blocking effect caused by the length of hpDNA, affecting assembly affinity and hybridization efficiency. Next, as... Figure 5 As 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 of the reaction system significantly affect the POD-like activity of the SERS nanotags, which in turn affects the catalytic efficiency of TMB. Figure 5 c and Figure 5 As shown in d, the intensity of the SERS signal first increases and then decreases with increasing temperature and pH value. Therefore, the optimal temperature is 37℃ and the optimal pH value is 4.25.

[0092] Example 5: Specificity, robustness, and reproducibility of the SERS analysis platform

[0093] 1. Assemble the SERS analysis platform as in Example 4.

[0094] 2. The established SERS analysis platform was exposed to multiple clinical tumor markers and serum interfering agents at the same concentration (1 mg / mL) to examine its selectivity and anti-interference ability. The results of the selectivity experiment are as follows: Figure 6 As shown in Figure a, 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) appeared weaker and similar to the blank spectrum. Furthermore, as... Figure 6 As shown in b, this invention measured a mixed solution of key interfering substances and the target protein. It was found that even when the concentration of these interfering substances reached 10 times that of the target protein, they had almost no effect on the SERS intensity of the target protein. These results demonstrate that the platform possesses strong anti-interference capabilities and specificity, making it perfectly suitable for the accurate identification of biomarkers in complex samples.

[0095] In addition, such as Figure 6As shown in c and 6d, this invention also recorded the SERS spectra of five SERS analysis platforms prepared in different batches for detecting EFNA1. The results showed that there were no significant differences in the Raman spectra and the intensity of the main characteristic peaks among the different platforms, indicating that the platform has good reproducibility.

[0096] Example 6: Quantitative Analysis of Two Target Protein Biomarkers (EFNA1 and MMP13)

[0097] 1. Assemble the SERS analysis platform as in Example 4.

[0098] 2. Under optimized experimental conditions, the sensitivity of the SERS analysis platform for detecting and analyzing different concentrations of target proteins (EFNA1 and MMP13) in serum was investigated. Figure 7 The SerS spectra recorded in a and 7c clearly show that the intensity of the SERS spectrum gradually increases with increasing EFNA1 and MMP13 concentrations. Notably, the SERS signal intensity of ox-TMB indirectly reflects the concentration of the target protein.

[0099] With ox-TMB at 1602cm -1 A linear curve was plotted between the characteristic peak intensity and the logarithm of the target protein concentration to obtain the corresponding linear regression equation. Figure 7 (b and 7d). The regression equation for FENA1 is y = 4059.28x + 51059.74, and the coefficient of determination (R²) is... 2 The coefficient of performance (COP) was 0.984, and the linear range was 1 pg / mL - 1 mg / mL. The regression equation for MMP13 was y = 3702.48x + 46785.73, with an R-value of 0.984. 2 The linear range was 0.982, and the linearity was 1 pg / mL–1 mg / mL. Therefore, the nanozyme-mediated catalytic signal amplification combined with CHA nucleic acid amplification strategy used in this SERS analysis platform can effectively quantify EFNA1 and MMP13. Furthermore, the limits of detection (LODs) for EFNA1 and MMP13 were calculated to be 0.75 and 0.84 pg / mL, respectively (S / N = 3). Compared with other assays (see Table 2), this protocol enables rapid and accurate quantification of EFNA1 and MMP13 in complex serum environments.

[0100] Table 2 Comparison of the detection method in this experiment with other reported methods

[0101]

[0102] Example 7: SERS analysis platform for clinical sample analysis

[0103] 1. Assemble the SERS analysis platform as in Example 4.

[0104] 2. Twenty serum samples from healthy individuals and 20 serum samples from patients with early-stage GC were tested using the SERS analysis platform. Figure 8 The image clearly shows the top and side views of the microarray chip with a three-layer structure, ensuring that SERS detection can be performed under contamination-free conditions. The chip contains 16 detection units, with a finely arranged layout of square and circular micropores distinguishing the EFNA1 and MMP13 detection regions, enabling high-throughput detection of the target protein. Figure 8 As shown in b, when EFNA1 and MMP13 were detected, the total intensity of the mean SERS spectrum in patients with early-stage GC was higher than that in healthy individuals. The 1602 cm⁻¹ height was evaluated using the Student's t-test. -1 Significance of characteristic peak intensity ( Figure 8 c), which revealed significant differences in the expression levels of EFNA1 and MMP13 in the serum of patients with early GC and healthy individuals.

[0105] Furthermore, by using 1602cm -1 The SERS intensity was substituted into the linear regression equation obtained in human serum to calculate the concentrations (ng / mL) of EFNA1 and MMP13. Subsequently, the ELISA method was established as the gold standard for detecting the expression levels of EFNA1 and MMP13 in clinical samples, and the results were compared with those of the SERS analysis platform (Tables 3-4). The relative deviations (RD) of the results from both methods were within acceptable ranges. The linear fitting results showed a high correlation between the two detection methods for EFNA1 and MMP13, with R0... 2 The values ​​were 0.973 and 0.981, respectively, indicating good consistency between them. Although the combination of gastroscopy and biopsy can diagnose early GC, its invasiveness and complications make it unsuitable as a large-scale screening tool. Therefore, the SERS analysis platform constructed in this invention is expected to better meet the needs of clinical early GC screening, providing an efficient and low-risk solution.

[0106] Table 3. SERS and ELISA results of EFNA1 and MMP13 concentrations in serum of healthy individuals.

[0107]

[0108] Table 4. SERS and ELISA results of serum EFNA1 and MMP13 concentrations in patients with early-stage GC.

[0109]

[0110]

[0111] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0112] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0113] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a SERS analytical platform for detecting early gastric cancer protein biomarkers, characterized by, The early gastric cancer protein biomarker comprises EFNA1 and MMP13; the preparation method comprises the following steps: S1: hpDNA1-1 and hpDNA1-2 are modified to the surface of Au@Pt NPs nanoscale enzyme respectively, and two SERS probes for EFNA1 and MMP13 respectively are formed; S2: hpDNA2-1 and hpDNA2-2 are modified to the surface of the heteromorphic nano-pillar array Au / SiNPA respectively, and two capture substrates for EFNA1 and MMP13 respectively are formed; S3: the two capture substrates prepared in step S2 are assembled into independent microarray chips respectively, and are used as SERS analysis platforms for detecting EFNA1 and MMP13 respectively; S4: 3,3',5,5'-tetramethylbenzidine solution and hydrogen peroxide solution are prepared and used for detection when EFNA1 or MMP13 is detected; The sequence of the hpDNA1-1 is shown as SEQ ID NO: 3, and the sequence of the hpDNA1-2 is shown as SEQ ID NO: 5; the sequence of the hpDNA2-1 is shown as SEQ ID NO: 4, and the sequence of the hpDNA2-2 is shown as SEQ ID NO: 6; The SERS analysis platform further comprises antibody-DNA conjugates, and the antibody-DNA conjugates comprise Ab1-1@DNA1-1 and Ab2-1@DNA2-1; the sequence of the DNA1-1 is shown as SEQ ID NO: 1, and the sequence of the DNA2-1 is shown as SEQ ID NO:

2.

2. The method for preparing a SERS analysis platform for detecting an early gastric cancer protein biomarker according to claim 1, characterized in that, The preparation method of the Au@Pt NPs nanoscale enzyme comprises: A gold sol is taken, deionized water, ascorbic acid solution and trisodium citrate are added to form solution A, wherein the diameter of the gold seed in the gold sol is 16-18 nm; HAuCl4 solution is added to solution A and reacted at 90-100 DEG C for more than 30 minutes to obtain solution B; H2PtCl6 solution is added to solution B and mixed thoroughly, while the temperature in the reaction bottle is kept not lower than 80 DEG C; then, ascorbic acid solution and trisodium citrate are added dropwise to the mixed solution until the reaction is completed, and the Au@Pt NPs nanoscale enzyme is obtained.

3. The method for preparing the SERS analysis platform for detecting protein biomarkers of early gastric cancer according to claim 1 or 2, characterized in that, The step of modifying the hpDNA1 to the surface of the Au@Pt NPs nanoscale enzyme to form two SERS probes comprises: After the hpDNA1 is activated, it is incubated with the Au@Pt NPs nanoscale enzyme for more than 12 hours, and the obtained mixture is dispersed in a bovine serum albumin solution, centrifuged and purified, and two SERS nanolabels, Au@Pt NPs@hp1-1 and Au@Pt NPs@hp1-2, are collected.

4. The method for preparing the SERS analysis platform for detecting early gastric cancer protein biomarkers according to claim 3, characterized in that, When centrifuged and purified, the centrifugal speed is 10,000-12,000 rpm, and the centrifugal time is 10-12 min.

5. The method for preparing 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 comprises: The PS microsphere array in single layer and dense arrangement is obtained by a gas-liquid interface self-assembly method, and the surface thereof is ion etched by using three kinds of etching gases SF6, O2 and Ar; the intermediate product obtained is subjected to heat treatment to remove residual PS microspheres, so as to obtain SiNCA; gold nanoparticles are deposited on the surface of the SiNCA by an ion sputtering process, so as to obtain Au / SiNCA.

6. The method for preparing 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 method for preparing the SERS analysis platform for detecting protein biomarkers of early gastric cancer according to claim 1, characterized in that, The step of modifying the hpDNA2 to the surface of the heteromorphic nano-pillar array Au / SiNPA includes: dropping the activated hpDNA2 solution onto the Au / SiNPA array and incubating for more than 12 h, so as to prepare two kinds of capture substrates: Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2. 8.The method for preparing a SERS analysis platform for detecting an early gastric cancer protein biomarker according to claim 7, characterized in that, The step S3 specifically includes: embedding the Au / SiNPA@hp2-1 and Au / SiNPA@hp2-2 into an ITO glass substrate, aligning and bonding with a PDMS microwell array sandwich through oxygen plasma technology, and covering the surface with a PDMS cover sheet. The microarray chip is a 4×4 microarray chip, and the PDMS microwell array sandwich is a 4×4 PDMS microwell array sandwich. The microarray chip has a size of 40 mm × 40 mm and contains 16 microwells.

9. A SERS analysis platform for detecting early gastric cancer protein biomarkers, which is prepared by the preparation method of any one of claims 1-8.

10. Application of the SERS analysis platform of claim 9 in simultaneously detecting early gastric cancer protein biomarkers EFNA1 and MMP13 for non-disease diagnosis and treatment purposes.

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