SERS (Surface Enhanced Raman Scattering) detection platform for detecting gastric cancer biomarkers

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) detection platform for detecting gastric cancer biomarkers, and belongs to the technical field of materials. Comprising the following steps: (1) sequentially carrying out reactive ion etching and physical etching on a single-layer polystyrene microsphere (PS microsphere) colloid template to obtain a pure Si nano crown array SiNCA; (2) depositing gold nanoparticles on the surface of the SiNCA to obtain a gold-plated silicon nano crown array Au / SiNCA; and (3) adding a signal molecule 3-MPBA into the Au / SiNCA, and incubating, so as to obtain the SERS detection substrate material 3-MPBA (at) Au / SiNCA. The SERS microporous array chip prepared by the invention has the advantages of high sensitivity, strong specificity, simplicity in operation, high flux and the like.

Description

Technical Field

[0001] The present invention relates to the field of material technology, and in particular to a SERS detection platform for detecting gastric cancer biomarkers. Background Art

[0002] Gastric cancer (GC) is the fifth most common malignant tumor in the world and ranks third in the incidence and mortality of common malignant tumors in my country. About 90% of patients are already in the advanced stage when they are discovered, and the overall five-year survival rate is only 35.1%. Surgical resection is currently the most effective means of treating GC, and early detection and diagnosis are key measures to improve the prognosis of GC patients. CT, endoscopic ultrasound and other imaging tests cause patients to suffer mainly from radiation damage, high costs and high false positive rates in clinical applications. Gastroscopy and tissue biopsy are more invasive and have many complications such as bleeding and secondary infection, which are not suitable for universal screening of early GC. Therefore, there is an urgent need to develop a low-cost, efficient, non-invasive and reliable method for diagnosing early GC.

[0003] Compared with traditional serological screening, salivary tumor marker screening has the obvious advantages of being simple, easy and non-invasive, and has great potential applications in stage-specific monitoring and screening of tumors. It has been reported that the presence of D-amino acids (D-AAs) is associated with certain neurological, tumor and kidney diseases, especially closely related to early GC. Specifically, the levels of D-proline (D-Pro) and D-alanine (D-Ala) in the saliva of patients diagnosed with early GC (126.3-285.3mM and 46.1-114.5mM, respectively) are significantly higher than those in healthy individuals (7.3-18.3mM and 5.4-7.8mM). Therefore, accurate D-Pro and D-Ala monitoring can help in the early diagnosis of GC and can serve as an important reference value for efficacy evaluation. In the past 25 years, the advancement of analytical methods such as colorimetry, electrochemiluminescence and biosensors has made it possible to detect D-AAs in food and biological samples. Nevertheless, these methods still face problems such as expensive analytical equipment, complicated sample pretreatment, or high requirements for expertise, which make in situ monitoring of D-AAs difficult. Therefore, an effective and sensitive method for detecting D-AAs is necessary for early screening by GC.

[0004] Surface enhanced Raman scattering (SERS) is an inelastic scattering spectroscopy technique based on molecular vibrations. It can provide unique "fingerprint information" for each molecule and can quickly identify the structure, molecular composition and conformation information of the substance. Due to its advantages of being fast, non-destructive and not interfered by water, SERS has been widely used in drug, biological analysis and labeling research. "Hotspots", that is, spatial localizations with extremely strong electromagnetic fields, play an important role in SERS enhancement. The SERS signals of a small number of analyte molecules that fall into the "hotspots" account for a high proportion of the entire measured SERS signal. However, it is still a challenge to fabricate metal substrates with uniform and dense hotspots for sensitive and reproducible SERS detection. Studies have found that large-scale ordered nanostructures can produce uniform "hotspots" for SERS signal enhancement, while achieving excellent signal reproducibility and accurate SERS quantitative detection. Gold-coated silicon nanocrown arrays (Au / SiNCA) are a high-performance SERS substrate prepared by depositing Au on Si nanocrown arrays assisted by colloidal template etching. They have become ideal SERS substrates due to their high uniformity and excellent sensitivity.

[0005] However, since D-AAs and L-AAs enantiomers have the same molecular structure and similar Raman scattering signals, it is still challenging to directly distinguish and detect amino acid enantiomers using SERS. Summary of the invention

[0006] The purpose of the present invention is to provide a SERS detection platform for detecting gastric cancer biomarkers to solve the problems existing in the above-mentioned prior art and to achieve high-throughput and quantitative detection of gastric cancer biomarkers D-Pro and D-Ala.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a method for preparing a SERS detection substrate material 3-MPBA@Au / SiNCA for detecting gastric cancer biomarkers, comprising the following steps:

[0009] (1) The single-layer polystyrene microsphere (PS microsphere) colloidal template is subjected to reactive ion etching and physical etching in sequence to obtain a pure Si nanocrown array SiNCA;

[0010] (2) depositing gold nanoparticles on the surface of the SiNCA to obtain a gold-coated silicon nanocrown array Au / SiNCA;

[0011] (3) adding the signal molecule 3-MPBA to the Au / SiNCA for incubation to obtain the SERS detection substrate material 3-MPBA@Au / SiNCA.

[0012] The second technical solution of the present invention is the SERS detection substrate material 3-MPBA@Au / SiNCA prepared by the preparation method.

[0013] The third technical solution of the present invention is the use of the SERS detection substrate material 3-MPBA@Au / SiNCA in the preparation of products for detecting gastric cancer markers.

[0014] The fourth technical solution of the present invention is a product for detecting gastric cancer markers, comprising the SERS detection substrate material 3-MPBA@Au / SiNCA.

[0015] The fifth technical solution of the present invention is a SERS microwell array chip for detecting gastric cancer markers. A 4×4 microwell array chip is prepared by micro-nanotechnology and embedded with the SERS detection substrate material 3-MPBA@Au / SiNCA, that is, the SERS microwell array chip.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] The SERS microwell array chip prepared by the present invention has the advantages of high sensitivity, strong specificity, simple operation, high throughput, etc. The SERS microwell array chip prepared by the present invention has good selectivity, only requires one step of sample addition, and utilizes the signal molecule 3-MPBA to be oxidized to 3-HTP, which is reflected in the Raman spectrum as 882cm -1 A new SERS peak was observed at the location, and the principle that the characteristic peak intensity increased with the increase of H2O2 concentration, combined with the advantages of SERS technology, improved the sensitivity and sensitivity of the detection platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a SEM image of a single-layer PS colloidal sphere in an embodiment of the present invention.

[0020] Figure 2 This is a low magnification SEM image of Au / SiNCA in an embodiment of the present invention.

[0021] Figure 3 This is a high magnification SEM image of Au / SiNCA in an embodiment of the present invention.

[0022] Figure 41 and 2 are top and side views of the microwell array chip in an embodiment of the present invention.

[0023] Figure 5 The SERS spectra of 10 independently detected TPP-labeled Au / SiNCA in the embodiment of the present invention are shown.

[0024] Figure 6 998cm of 10 independently detected TPP-labeled Au / SiNCA in the embodiment of the present invention -1 The corresponding SERS intensity histogram at .

[0025] Figure 7 is TPP (1×10 -8 M) SERS spectra of Au / SiNCA modified with pure TPP (1×10 -1 M) SERS spectrum.

[0026] Figure 8 The TPP (10 -8 -10 -13 M) SERS spectrum of modified Au / SiNCA.

[0027] Fig. 9 The TPP (10 -8 -10 -13 M) 998cm of Au / SiNCA modified -1 The linear fitting equation of the corresponding SERS intensity at and the logarithm of TPP concentration.

[0028] Fig.10 These are the SERS spectra of 3-MPBA@Au / SiNCA, 3-MPBA+H2O2@Au / SiNCA, 3-HTP@Au / SiNCA and Au / SiNCA in the embodiments of the present invention.

[0029] Fig.11 This is the SERS mapping image of 3-MPBA@Au / SiNCA in the embodiment of the present invention.

[0030] Fig.12 I is the I of five different batches of 3-MPBA+H2O2@Au / SiNCA in the embodiment of the present invention 882 / I 998 Histogram of ratios.

[0031] Fig.13 I is the I of 3-MPBA+H2O2@Au / SiNCA recorded within 30 days in the embodiment of the present invention. 882 / I 998 Histogram of ratios.

[0032] Fig.14 is the concentration of 3-MPBA in the embodiment of the present invention and I 882 / I 998 relationship diagram.

[0033] Fig.15 is the incubation time of Au / SiNCA and 3-MPBA in the embodiment of the present invention. 882 / I 998 relationship diagram.

[0034] Fig.16 is the pH value of the reaction system in the embodiment of the present invention and I 882 / I 998 relationship diagram.

[0035] Fig.17 is the reaction temperature and I in the embodiment of the present invention 882 / I 998 relationship diagram.

[0036] Fig.18 The SERS micropore array chip in the embodiment of the present invention is added with H2O2 for different reaction times and I 882 / I 998 relationship diagram.

[0037] Fig.19 This is a SERS spectrum diagram obtained after adding 0-5mM H2O2 to the SERS microwell array chip in an embodiment of the present invention and reacting for 30 minutes.

[0038] Fig. 20 In the embodiment of the present invention, 882 / I 998 Linear relationship graph with H2O2 concentration.

[0039] Fig.21 This is a SERS spectrum diagram obtained after adding 0-2mM D-Pro to the SERS microwell array chip in an embodiment of the present invention and reacting for 30 minutes.

[0040] Fig. 22 In the embodiment of the present invention, 882 / I 998 Linear relationship graph with D-Pro concentration.

[0041] Fig.23 This is a SERS spectrum diagram obtained after adding 0-2mM D-Ala to the SERS microwell array chip in an embodiment of the present invention and reacting for 30 minutes.

[0042] Fig.24 In the embodiment of the present invention, 882 / I 998Linear relationship graph with D-Ala concentration.

[0043] Fig.25 is the I of the 1 mM sample that may interfere in saliva in the embodiment of the present invention 882 / I 998 Histogram.

[0044] Fig.26 I is the SERS detection of 1 mM sample when there are different proportions of D-AAs in the embodiment of the present invention. 882 / I 998 Histogram.

[0045] Fig. 27 1 is a gastroscopic image of a healthy volunteer in an embodiment of the present invention.

[0046] Fig.28 Pathological biopsy images of healthy volunteers in the embodiments of the present invention.

[0047] Fig.29 CT images of healthy volunteers in the embodiments of the present invention.

[0048] Fig.30 This is a gastroscopic image of a patient whose gastroscopy showed GC in an embodiment of the present invention.

[0049] Fig.31 This is a pathological biopsy image of a patient with GC indicated by gastroscopy in an embodiment of the present invention.

[0050] Fig.32 This is a CT image of a patient with GC diagnosed by gastroscopy in an embodiment of the present invention.

[0051] Fig.33 I is obtained by SERS detection of 10 clinical saliva samples in the embodiment of the present invention. 882 / I 998 Histogram (healthy individuals are numbered 1-5, gastric cancer patients are numbered 6-10).

[0052] Fig.34 Schematic diagram of detecting gastric cancer biomarkers D-Pro and D-Ala in an embodiment of the present invention, wherein a is a schematic diagram of the enzymatic cascade reaction, b is the synthesis process of Au / SiNCA, and c is the preparation and detection process of the SERS microwell array chip. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0055] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0056] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0057] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0058] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0059] The embodiment of the present invention provides a method for preparing a SERS detection substrate material 3-MPBA@Au / SiNCA for detecting gastric cancer biomarkers, comprising the following steps:

[0060] (1) The single-layer PS microsphere colloidal template is subjected to reactive ion etching and physical etching in sequence to obtain a pure Si nanocrown array SiNCA;

[0061] (2) depositing gold nanoparticles on the surface of the SiNCA to obtain a gold-coated silicon nanocrown array Au / SiNCA;

[0062] (3) adding the signal molecule 3-MPBA to the Au / SiNCA for incubation to obtain the SERS detection substrate material 3-MPBA@Au / SiNCA.

[0063] In some specific embodiments, the preparation method of the single-layer PS microsphere colloidal template is: using PS microspheres as precursors and silicon wafers as substrates, obtaining a single-layer densely arranged microsphere array through gas-liquid interface self-assembly, and obtaining the single-layer PS microsphere colloidal template through heating and drying;

[0064] The PS microspheres were prepared by diluting a PS suspension (5% wt) into ethanol to form a mixture with a volume ratio of 1:1;

[0065] The conditions of the reactive ion etching are: using O2 and SF6 as etching gases, the time is 110-130s, and the gas flow rates are 8-12sccm and 18-22sccm respectively;

[0066] The physical etching conditions are: using Ar as etching gas, the etching time is 55-65 s, and the gas flow rate is 18-22 sccm.

[0067] In some specific embodiments, the heating and drying conditions are treatment at 70° C. for 30 min.

[0068] In some specific embodiments, the physical etching is followed by rinsing and heat treatment steps; the rinsing step is performed using ethanol; and the heat treatment step is performed at 600° C. for 2 hours.

[0069] In some specific embodiments, the deposition rate of the gold nanoparticles on the surface of the SiNCA is 0.5 nm / s;

[0070] The particle size of the gold nanoparticles is 15-17 nm;

[0071] The concentration of the gold nanoparticles is 1×10 -9 ~1×10 -8 M.

[0072] In some specific embodiments, the gold nanoparticles are synthesized by sodium citrate reduction method.

[0073] In some specific embodiments, the concentration of the signal molecule 3-MPBA is 2.3-2.7 mM;

[0074] The incubation conditions include: pH 7.35-7.45, temperature 36.5-37.5° C., and time 110-130 min.

[0075] The embodiment of the present invention also provides a SERS detection substrate material 3-MPBA@Au / SiNCA prepared by the preparation method.

[0076] The embodiment of the present invention also provides the use of the SERS detection substrate material 3-MPBA@Au / SiNCA in preparing a product for detecting gastric cancer markers.

[0077] In some specific embodiments, the products include detection chips, test strips, reagents and kits.

[0078] In some specific embodiments, the gastric cancer markers include D-Pro and D-Ala.

[0079] The embodiment of the present invention further provides a product for detecting gastric cancer markers, including the SERS detection substrate material 3-MPBA@Au / SiNCA.

[0080] The embodiment of the present invention also provides a SERS microwell array chip for detecting gastric cancer markers. A 4×4 microwell array chip is prepared by micro-nanotechnology and embedded with the SERS detection substrate material 3-MPBA@Au / SiNCA, namely the SERS microwell array chip.

[0081] In some specific embodiments, the preparation method of the SERS microwell array chip includes: embedding the obtained 3-MPBA@Au / SiNCA into an ITO glass substrate by laser etching, and its pore size and distribution match the microwell array on the PDMS chip; then accurately aligning and bonding the PDMS interlayer containing 16 array microwells and the glass substrate inlaid with 3-MPBA@Au / SiNCA by oxygen plasma technology; finally, covering with a PDMS cover glass to obtain a 4×4 microwell array chip with a "sandwich" structure.

[0082] In some specific embodiments, the detection sample of the SERS microwell array chip is saliva.

[0083] 3-Mercaptophenylboronic acid (3-MPBA) is a classic H2O2-responsive Raman probe that can be converted into 3-hydroxythiophenol (3-HTP) by reacting with H2O2 produced by D-AAs catalyzed by D-amino acid oxidase (D-AAO), showing obvious spectral changes. With the help of this chiral selection strategy, SERS microwell array chips can easily achieve chiral differentiation of amino acids in complex samples.

[0084] The present invention manufactures a SERS microwell array chip for detecting gastric cancer (GC) biomarkers, and combines enzyme-mediated cascade reactions to achieve ultrasensitive and specific detection of D-Pro and D-Ala in the saliva of GC patients. First, Au / SiNCA with a periodic structure was prepared by a single-layer colloidal template method assisted reactive ion etching (RIE) process and magnetron sputtering gold nanoparticle technology. 3-Mercaptophenylboronic acid (3-MPBA) was modified on the surface of Au / SiNCA as a traditional H2O2-responsive probe molecule, called 3-MPBA@Au / SiNCA. Finally, the ITO glass embedded with 3-MPBA@Au / SiNCA was aligned with the designed PDMS cover plate to produce a high-throughput microwell array chip containing 16 SERS detection units.

[0085] On this basis, enzymatic reaction was used to measure D-AAs. The natural enzyme DAAO first catalyzed the D-AAs in saliva to generate H2O2, and then the reaction probe 3-MPBA was oxidized to 3-HTP, resulting in the Raman spectrum at 882 cm -1 In addition, with the increase of H2O2 concentration, the intensity of the SERS peak also gradually increases. Finally, according to the 882cm -1 (response peak) and 998cm -1 The intensity ratio between the two characteristic peaks (internal standard peak) was used to establish a ratiometric SERS sensor for detecting the content of D-Ala and D-Pro. The above microwell array chip was effectively used for high-throughput detection of D-AAs in saliva samples, and the final clinical results were in line with expectations. This ratiometric SERS detection method based on enzymatic reaction can diagnose and detect GC more simply and quickly.

[0086] The instruments and test conditions used in the present invention are as follows:

[0087] The scanning electron microscope (SEM) images were obtained by a S-800II field emission scanning electron microscope produced by Hitachi, Japan.

[0088] Raman spectroscopy and SERS imaging were measured by the Invia Reflex laser microscope Raman spectrometer produced by Renishaw, UK. The test conditions were a laser wavelength of 785nm, a laser intensity of 50mW, and a detection range of 600-1800cm -1 Spectra were processed using Renishaw software (WiRETM) for operation and data acquisition, and baseline correction was performed to suppress background noise.

[0089] Example 1

[0090] Preparation and characterization of Au / SiNCA and microwell array chips

[0091] 1) Using PS microspheres (PS microspheres are a mixture of 5% wt PS suspension diluted in ethanol to form a volume ratio of 1:1) as a precursor and a hydrophilic silicon wafer as a substrate (hydrophilic treatment method: cut the clean silicon wafer, immerse it in piranha solution (H2SO4:30% H2O2=7:3), then transfer it to a water bath and slowly heat it to 90°C, keep it for 1 hour, and then cool it to room temperature. Then rinse it repeatedly with ultrapure water and dry it to obtain a hydrophilic silicon wafer), a single-layer densely arranged microsphere array was obtained through the gas-liquid interface self-assembly method. After heating and drying (70°C, 30 minutes), the single-layer PS colloidal sphere template can be firmly fixed on the silicon wafer surface.

[0092] 2) The single-layer PS colloidal sphere template obtained in step 1) was subjected to reactive ion etching (RIE) for 2 minutes using O2 and SF6 as etching gases, with gas flow rates of 10 and 20 sccm, respectively, and then its ionized surface was physically etched for 1 minute using Ar gas (gas flow rate 20 sccm). The intermediate product was then rinsed with anhydrous ethanol and heat treated (600°C, 2 hours) to remove the residual PS microspheres, thus obtaining a pure Si nanocrown array (SiNCA).

[0093] 3) 15-17 nm AuNPs were synthesized by sodium citrate reduction method: 50 mL HAuCl4 solution (1 mM) was added to a 100 mL double-necked flask and heated to boiling. Then 8 mL sodium citrate solution (38 mM) was added dropwise to the double-necked flask, and then the reaction was continued under stirring (700 rpm, 15 minutes), and the final color of the solution changed from light yellow to wine red, obtaining a 15-17 nm AuNPs solution.

[0094] 4) The AuNPs (the concentration of gold nanoparticles was 1×10 -9 M) was deposited on the surface of SiNCA to obtain gold-coated silicon nanocrown arrays (Au / SiNCA).

[0095] 5) The signal molecule 3-mercaptophenylboronic acid (3-MPBA) solution (20 μL, 2.5 mM) was added dropwise to the Au / SiNCA obtained in step 4) and incubated (2 hours) at a pH of 7.4 and an incubation temperature of 37°C to obtain 3-MPBA@Au / SiNCA as a SERS substrate responsive to H2O2. Finally, it was rinsed with ethanol and deionized water, and then dried with N2.

[0096] The preparation steps of 3-HTP@Au / SiNCA are the same as above, and 3-HTP is m-hydroxyphenylethanol.

[0097] 6) Embed the 3-MPBA@Au / SiNCA obtained in step 5) into the ITO glass substrate by laser etching, and the pore size and distribution thereof need to match the micropore array on the PDMS chip.

[0098] 7) Then, the PDMS interlayer containing 16 array microwells and the glass substrate inlaid with 3-MPBA@Au / SiNCA obtained in step 6) were precisely aligned and bonded by oxygen plasma technology. Finally, a PDMS cover glass was covered to successfully construct a 4×4 microwell array chip with a "sandwich" structure.

[0099] Scanning electron microscopy (SEM) was used to characterize the morphology of the intermediate products and final products during the preparation of Au / SiNCA. Figure 1 As shown in the figure, during the preparation process, a uniform and compact layer of PS colloidal spheres with a diameter of 190-210 nm was first obtained through the gas-liquid interface self-assembly strategy and overall transfer. Then, by optimizing the RIE process conditions and sputtering gold nanoparticles (AuNPs) using a magnetron sputtering device, an Au / SiNCA array with a unique morphology was obtained. Figure 2 and 3 It can be clearly seen that the SERS substrate is composed of crown-shaped structural units, which are arranged in an orderly periodic manner with a period of 190-210nm, which is consistent with the diameter of the PS colloidal sphere. Theoretically, the unique crown structure of Au / SiNCA has an excellent specific surface area, which can provide abundant binding sites for the fixation of biomolecules, which is conducive to subsequent functionalization. Figure 4 The top and side views of the three-layer structured microwell array are shown.

[0100] The concentration used was 1×10 -8 The suitability of the array as a SERS substrate was evaluated by using TPP-labeled Au / SiNCA. Ten points on the array were randomly selected to collect Raman spectra, and the 998 cm -1 Intensity histogram of the characteristic peak at (phenyl N1 mode) Figure 5 and 6 The obtained Raman spectra have the same shape trend, but the intensity is slightly different, with an RSD value of only 6.22%, which shows the advantage of the high uniformity of the array ( Figure 7 ).

[0101] The enhancement factor (EF) of Au / SiNCA was calculated according to the following formula:

[0102] EF=(I SERS / C SERS ) / (I R / C R );

[0103] Among them, ISERS and I R The peak intensity (998 cm) of Au / SiNCA and TPP on silicon wafer -1 ), C SERS and C R 1×10 -8 M and 1×10 -1 M.

[0104] The final calculated EF value of Au / SiNCA is 1.68×10 9 The sensitivity of Au / SiNCA is crucial for the subsequent quantitative detection of H2O2.

[0105] from Figure 8 It can be seen that different concentrations of TPP (10 -8 -10 -13 The spectrum of the TPP concentration (M) shows a significant change, with the logarithm of the TPP concentration being proportional to the 998 cm -1 There is a strong linear correlation between the intensities of the characteristic peaks at Fig. 9 ).

[0106] The linear equation is:

[0107] y=6182.6x+82187.9,R 2 =0.986.

[0108] Therefore, the limit of detection (LOD) can be calculated to be 73.4 fM, which reveals an excellent SERS signal.

[0109] Example 2

[0110] Verification of sensor principle and performance evaluation of 3-MPBA@Au / SiNCA

[0111] Control experiments were performed to verify the feasibility of the established H2O2 and D-AAs SERS detection method.

[0112] 1) SERS spectral analysis was performed on the modified 3-MPBA@Au / SiNCA, 3-MPBA+H2O2@Au / SiNCA (3-MPBA@Au / SiNCA treated with 1 mM H2O2 for 30 min), 3-HTP@Au / SiNCA and Au / SiNCA chip.

[0113] like Fig.10 As shown in Figure 2, the SERS spectrum of Au / SiNCA is smooth, without obvious characteristic peaks, and has good cleanliness. The spectrum of 3-MPBA@Au / SiNCA has peaks at 785, 995, 1076, and 1554 cm -1There is an obvious characteristic peak at 882cm, indicating that the signal molecule 3-MPBA has been successfully modified on the surface of Au / SiNCA array through Au-S bond. After H2O2 treatment, the SERS spectrum of 3-MPBA@Au / SiNCA is very consistent with the SERS spectrum of 3-HTP, indicating that the conversion of borate ester to phenol is in line with expectations. -1 The new peak at is related to the stretching vibration of the benzene ring of 3-HTP.

[0114] 2) The SERS spectrum of 3-MPBA@Au / SiNCA shows an overall uniform green color ( Fig.11 ), indicating that 3-MPBA was uniformly modified on the surface of the SERS substrate. To verify the reliability of this SERS substrate for detecting H2O2, five batches of 3-MPBA@Au / SiNCA were synthesized at different times and treated with 1 mM H2O2 respectively.

[0115] like Fig.12 As shown, in the SERS measurements of five different batches of Au / SiNCA, I 882 / I 998 The specific intensity of the substrates was basically unchanged, with an RSD of only 2.12%. In addition, the same batch of prepared SERS substrates were placed for different days and then treated with H2O2. The SERS detection results showed that 882 / I 998 There is only a slight change over time ( Fig.13 ). Therefore, the SERS sensing platform prepared by the present invention is successfully prepared and has good uniformity, repeatability and stability.

[0116] Example 3

[0117] Optimization of experimental conditions

[0118] After evaluating the performance of 3-MPBA@Au / SiNCA, several key properties of the prepared SERS microwell array chip were optimized, including the concentration of 3-MPBA, the incubation time of Au / SiNCA and 3-MPBA, and the pH value and temperature of the reaction system.

[0119] like Fig.14 As shown in Figure 2, with the increase of 3-MPBA concentration, I 882 / I 998 When the concentration of 3-MPBA reaches 2.5 mM, a strong SERS signal can be obtained. In order to save costs, 2.5 mM is taken as the optimal modification concentration. Fig.15 When Au / SiNCA was co-incubated with 3-MPBA for 2 h, I 882 / I 998It gradually increases from 0.121 to a maximum of 0.514, so the optimal incubation time of 3-MPBA is 2 hours. Too long incubation time may cause the AuNPs sputtered and deposited on the Au / SiNCA surface to detach, resulting in unstable SERS performance of the substrate. 882 / I 998 It reached its peak at pH = 7.4 and 37°C, and decreased significantly with increasing pH and temperature ( Fig.16 and 17 ). The results showed that pH = 7.4 and 37 ° C were the optimal pH value and temperature of the reaction system.

[0120] Example 4

[0121] Quantitative SERS detection of D-AAs

[0122] 1) Prepare a SERS microwell array chip as in Example 1.

[0123] 2) Under the optimal optimized conditions, a H2O2 (500 mM) solution prepared in phosphate buffer was added dropwise to the 3-MPBA@Au / SiNCA integrated microwell array chip, and SERS detection was performed after the reaction.

[0124] exist Fig.18 middle, I 882 / I 998 The value of changes with time (minutes), and the signal tends to be stable after 30 minutes. Therefore, the optimal reaction time is 30 minutes. Fig.19 The SERS spectra obtained after the microwell array chip reacted with different concentrations of H2O2 are shown. It can be clearly observed that with the increase of H2O2 concentration, the 882cm -1 The peak value at becomes higher. Fig. 20 Draw I 882 / I 998 The relationship curve with H2O2 concentration shows that in the range of 10-500mM, I 882 / I 998 The value of has a good linear relationship with the H2O2 concentration. The linear equation is y = 0.768x + 0.133, R 2 =0.986, and the detection limit (LOD) was calculated to be 7.7 mM based on the three-fold signal-to-noise ratio.

[0125] 3) Similar to H2O2 detection, I 882 There is a clear correlation with the H2O2 produced by the oxidation of D-Pro by D-amino acid oxidase (DAAO). Fig.21 As shown in Figure 2, with the increase of D-Pro concentration, the 882 cm -1 The peak gradually increases, and 998cm -1 The peak height remains basically unchanged.882 / I 998 The correlation curve with D-Pro concentration is as follows Fig. 22 As shown, surprisingly, there is a good linear relationship between the two in the concentration range of 20-500mM. The corresponding linear regression equation of D-Pro is y=0.656x+0.121, and the correlation coefficient R 2 =0.993, LOD is 10.1 mM. Fig.23 The SERS spectra obtained by using a microwell array chip to detect different concentrations of D-Ala. Fig.24 As shown, within the concentration range of 20-500 mM, D-Ala and I 882 / I 998 There is also a good linear relationship between the concentrations of . The corresponding regression equation is y = 0.618x + 0.117, R 2 =0.997, LOD as low as 13.7mM.

[0126] 4) Table 1 details the performance of different methods for detecting D-AAs. The data show that the detection performance (including linear range, LOD) of the SERS method of the present invention for D-Ala and D-Pro is comparable to that of fluorescence, colorimetry and electrochemical methods, but it omits the complicated enzyme fixation procedure and high temperature synthesis conditions, and the sample pretreatment is simple, which has significant advantages.

[0127] Table 1 Comparison of the present invention with other methods for detecting D-Pro (or D-Ala)

[0128]

[0129] Example 5

[0130] 1) Prepare a SERS microwell array chip as in Example 1.

[0131] 2) Since the testing of clinical saliva samples is more complicated than that in laboratory settings, it is crucial to evaluate the specificity and other performance of this SERS microwell array chip in detecting D-Pro and D-Ala. L-proline (L-Pro), L-alanine (L-Ala), cysteine ​​(Cys), glutathione (GSH), glycine, valine, serine, threonine and some inorganic salts in saliva samples may confuse the detection results of D-Pro and D-Ala, thereby leading to a false positive diagnosis of GC. However, as Fig.25 As shown, these introduced interferences cannot react with the native enzyme DAAO to trigger the H2O2-responsive probe, thus failing to generate the H2O2-responsive probe at 882 cm -1Therefore, this SERS microwell array chip has excellent specificity for D-Pro and D-Ala and does not react to potential interference. In order to study the possibility of selectively detecting specific amino acids in the presence of two D-amino acids, experiments were conducted using mixtures of D-Pro and D-Ala in different ratios (the total concentration of D-Pro and D-Ala was 1 mM). The test showed that I 882 / I 998 The molar ratio of the two amino acids does not change much ( Fig.26 ), which indicates that specific detection of one amino acid cannot be achieved. Given that both D-Pro and D-Ala are present in actual saliva samples and their linear regression equations show a high degree of similarity, this SERS method has the ability to measure the total concentration of D-Pro and D-Ala.

[0132] Example 6

[0133] SERS microwell array chip for clinical sample analysis

[0134] 1) Prepare a SERS microwell array chip as in Example 1.

[0135] 2) Provide the gastroscopy, CT and pathological biopsy information of a healthy volunteer and a GC patient. Gastroscopy and CT examinations are commonly used screening tools for gastrointestinal cancer in my country. They are of great significance for early qualitative diagnosis, guidance of subsequent treatment and prognosis evaluation. Pathological biopsy, as the gold standard for tumor diagnosis, plays an irreplaceable role. Gastroscopy shows that the gastric mucosa of healthy people is smooth and well expanded, without obvious ulcers or neoplasms ( Fig. 27 ), and no abnormalities were found in the biopsy results ( Fig.28 Gastroscopy revealed that GC patients had extensive ulcers in the cardia and gastric body, which appeared as sunken yellow-brown lichens with granulomatous necrosis, slightly hard texture, and easy bleeding when touched ( Fig.30 ). Pathological biopsy showed dysplastic glandular epithelium, which was irregular glandular tubular with invasive growth ( Fig.31 ), diagnosed as ulcerative adenocarcinoma of the cardia. CT examination of healthy people showed no abnormality ( Fig.29 ), while CT examination of GC patients showed local thickening of the gastric wall accompanied by hepatogastric nodules ( Fig.32 ).

[0136] 3) In order to test the effectiveness of this SERS microwell array chip, 100-fold diluted healthy human saliva samples were treated with different concentrations of D-Pro. The results are shown in Table 2, and the recovery rates ranged from 97.60% to 105.08%. In order to study the feasibility of clinical application of this chip, experiments were conducted on clinical samples No. 1-10 (No. 6-10 were from GC patients). Fig.33It can be clearly seen that GC patients have a -1 The characteristic peak intensity at was significantly higher than that of healthy people. According to the linear fitting equation of D-Ala, the total concentrations of D-Pro and D-Ala in saliva samples 6-10 were calculated to be 304.88, 278.96, 335.37, 388.72 and 361.28 mM, respectively, which is consistent with the total concentration range of these two amino acids in the saliva of GC patients (174.2-399.8 mM).

[0137] Table 2 Analysis results of recovery test

[0138]

[0139]

[0140] In summary, the preparation method of the SERS micropore array chip of the present invention includes preparing a pure Si nano-crown array (SiNCA) with a crown structure by a single-layer colloidal template method assisted by reactive ion etching (RIE) process, and depositing a dense layer of gold nanoparticles (AuNPs) on its surface by magnetron sputtering, that is, obtaining a gold-plated silicon nano-crown array (Au / SiNCA) with a crown structure. The present invention uses 3-MPBA as a Raman probe to perform surface modification on the obtained Au / SiNCA, constructs a SERS analysis platform that responds to H2O2, and uses a micropore array chip equipped with 16 detection units as a SERS micropore array chip to achieve high-throughput, high-sensitivity, high-specificity and high-reliability quantitative detection of D-AAs (D-Pro and D-Ala), providing a new technical channel for detecting gastric cancer.

[0141] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a SERS detection substrate material 3-MPBA@Au / SiNCA for detecting gastric cancer biomarkers, characterized in that: The following steps are involved: (1) The single-layer PS microsphere colloidal template is subjected to reactive ion etching and physical etching in sequence to obtain a pure Si nanocrown array SiNCA; (2) depositing gold nanoparticles on the surface of the SiNCA to obtain a gold-coated silicon nanocrown array Au / SiNCA; (3) adding the signal molecule 3-MPBA to the Au / SiNCA for incubation to obtain the SERS detection substrate material 3-MPBA@Au / SiNCA.

2. The preparation method according to claim 1, characterized in that: The preparation method of the single-layer PS microsphere colloidal template is as follows: using PS microspheres as precursors and silicon wafers as substrates, obtaining a single-layer densely arranged microsphere array through gas-liquid interface self-assembly, and obtaining the PS microsphere colloidal template through heating and drying; The PS microspheres are prepared by diluting 5% wt PS suspension into ethanol to form a mixture with a volume ratio of 1:1; The conditions of the reactive ion etching are: using O2 and SF6 as etching gases, the time is 110-130s, and the gas flow rates are 8-12sccm and 18-22sccm respectively; The physical etching conditions are: using Ar as etching gas, the etching time is 55-65 min, and the gas flow rate is 18-22 sccm.

3. The preparation method according to claim 1, characterized in that: The deposition rate of the gold nanoparticles on the surface of the SiNCA is 0.5 nm / s; The particle size of the gold nanoparticles is 15-17 nm; The concentration of the gold nanoparticles is 1×10 -9 ~1×10 -8 M.

4. The preparation method according to claim 1, characterized in that: The concentration of the signal molecule 3-MPBA is 2.3-2.7 mM; The incubation conditions include: pH 7.35-7.45, temperature 36.5-37.5° C., and time 110-130 min.

5. The SERS detection substrate material 3-MPBA@Au / SiNCA prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the SERS detection substrate material 3-MPBA@Au / SiNCA as claimed in claim 5 in the preparation of products for detecting gastric cancer markers.

7. The use according to claim 6, characterized in that: The products include detection chips, test strips, reagents and test kits.

8. The use according to claim 6, characterized in that: The gastric cancer markers include D-Pro and D-Ala.

9. A product for detecting gastric cancer markers, characterized in that: It includes the SERS detection substrate material 3-MPBA@Au / SiNCA as described in claim 5.

10. A SERS microwell array chip for detecting gastric cancer markers, characterized in that: A 4×4 microwell array chip is prepared by micro-nano technology and embedded with the SERS detection substrate material 3-MPBA@Au / SiNCA described in claim 5, which is the SERS microwell array chip.

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