A SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B and its application

Abstract

The present invention belongs to the field of detection technology, and specifically relates to a SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B. The preparation method of the SERS microfluidic chip comprises: (1) preparing an ordered gold nanobipyramid array with neat tip-to-tip arrangement by regulating gold nanobipyramids with hydrochloric acid; (2) coupling a thiol-modified VEGF aptamer and a thiol-modified PDGF-B aptamer to the ordered gold nanobipyramid array prepared in (1) through a gold-sulfur bond; (3) reacting complementary DNA 1 complementary to the VEGF aptamer and complementary DNA 2 complementary to the PDGF-B aptamer, which are labeled with Raman signal molecules, with the ordered gold nanobipyramid array prepared in (2) to prepare a functionalized SERS substrate; (4) preparing a SERS microfluidic chip from the functionalized SERS substrate. The SERS microfluidic chip can rapidly and highly sensitively detect VEGF and PDGF-B in serum within 20 minutes, with a detection limit as low as pg / mL.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and specifically relates to a SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B and its application. Background Art

[0002] Gastric cancer is one of the most common and devastating malignancies clinically, with the third highest mortality rate among all malignant tumors. The incidence of gastric cancer is influenced by numerous factors, and most patients are already in the advanced stages of the disease at the time of initial diagnosis. While traditional endoscopic and imaging techniques can improve gastric cancer detection rates, they are invasive and painful, and cannot meet the needs of all patients. With the deepening of research into the molecular mechanisms underlying gastric cancer development and progression, biomarkers, due to their specificity and non-invasive nature, have become a hot topic for early screening and auxiliary diagnosis of gastric cancer. In recent years, overexpression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-B (PDGF-B) has been shown to play a role in the development and progression of gastric cancer. VEGF is a major angiogenic factor that can induce lymphangiogenesis, promote vascular permeability and endothelial cell proliferation, and contribute to tumor proliferation, spread, and metastasis. PDGF-B, a connective tissue growth factor, promotes tumor growth through autocrine and paracrine effects and regulates the function of tumor stromal cells, making it closely associated with the development and progression of gastric cancer. Currently, methods for detecting VEGF and PDGF-B include electrochemical methods, fluorescence sensing, immunoassays, surface plasmon resonance, and colorimetry. However, these methods suffer from issues such as long detection times, fluorescence quenching, and poor anti-interference capabilities. Therefore, a rapid, sensitive, and specific method for detecting VEGF and PDGF-B is needed.

[0003] Surface-enhanced Raman scattering (SERS) is a method that significantly enhances Raman signals. Due to its unique fingerprint characteristics, non-destructiveness, resistance to photobleaching, and lack of fluorescence background interference, it is suitable for the simultaneous detection of multiple samples. It has made great progress in the fields of disease diagnosis, environmental monitoring, food safety, etc. The SERS enhancement mechanism is divided into electromagnetic enhancement (EM) and chemical enhancement (CM), among which the EM mechanism plays a dominant role in the entire SERS enhancement. EM, that is, the enhancement of Raman signals is achieved through the strong electromagnetic field generated by the local surface plasmon resonance (LSPR) excited by the rough surface of metal materials or the surface of metal nanoparticles.

[0004] Microfluidic chips, also known as "labs on a chip," are typically technology platforms that integrate routine operations such as sample processing, reaction, separation, and detection involved in chemical and biological experiments onto a small chip, achieving low-consumption, rapid, and efficient analysis. In addition, microfluidic chips have the advantages of high throughput and low cost. They have developed rapidly and are widely used in the field of biomedical analysis. Traditional microfluidic chips usually require external pumps and tubing, which are both complex and heavy. Introducing capillary pumps into the chip can increase the possibility of portability. Combining SERS with microfluidic chips can leverage the advantages of both technologies for sample detection. This not only achieves high-throughput and rapid detection using SERS, but also overcomes the disadvantage of the traditional SERS detection process being susceptible to environmental contamination. SERS microfluidic chips have outstanding performance in real-time detection and in vitro diagnosis, which is in line with the development trend of modern blood analysis technology.

[0005] However, most SERS microfluidic chips currently used for protein biomarker detection adopt a sandwich structure strategy, which has the disadvantages of high consumption and complex operation steps. Therefore, a SERS microfluidic chip with low consumption and simple operation was developed to detect gastric cancer biomarkers VEGF and PDGF-B. Summary of the Invention

[0006] In response to the above problems, one of the objectives of the present invention is to provide a SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B. The SERS microfluidic chip is a SERS microfluidic chip prepared by combining a gold nanobipyramid array and an aptamer based on competitive recognition of VEGF and PDGF-B. It has low consumption and simple operation steps.

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

[0008] On the one hand, the present invention provides a SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B, and the preparation method thereof comprises: (1) controlling gold nanobipyramids with hydrochloric acid to prepare an ordered gold nanobipyramid array with neatly arranged tip to tip; (2) coupling a thiol-modified VEGF aptamer and a thiol-modified PDGF-B aptamer to the ordered gold nanobipyramid array prepared in (1) through gold-sulfur bonds; (3) reacting complementary DNA 1 complementary to the VEGF aptamer and complementary DNA 2 complementary to the PDGF-B aptamer, which are labeled with Raman signal molecules, with the ordered gold nanobipyramid array prepared in (2) to prepare a functionalized SERS substrate; and (4) preparing a SERS microfluidic chip from the functionalized SERS substrate.

[0009] On the other hand, the present invention provides a method for detecting VEGF and PDGF-B in a sample to be detected using the above-mentioned SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B, comprising: (1) adding the sample to be detected to the sample addition port of the SERS microfluidic chip for detecting VEGF and PDGF-B, and reacting at 23°C-27°C; (2) performing SERS testing on the reaction area three times to obtain Raman molecular signals respectively; and obtaining the concentrations of VEGF and PDGF-B in the sample to be detected based on the working curve of the logarithmic values of the VEGF and PDGF-B concentrations and the SERS intensity change values of the Raman molecular signals.

[0010] The beneficial effects of the present invention include at least:

[0011] (1) The GNBPs array preparation method of the SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B provided by the present invention is simple, has the advantages of high uniformity and stability, and can be prepared in large quantities.

[0012] (2) The SERS microfluidic chip provided by the present invention for detecting gastric cancer markers VEGF and PDGF-B has good repeatability and can be prepared on a large scale.

[0013] (3) The SERS microfluidic chip provided by the present invention for detecting gastric cancer markers VEGF and PDGF-B, at room temperature, the aptamer recognizes the target and undergoes a conformational change, binding to the target with high specificity; the complementary DNA chain labeled with the Raman signal molecule is then competitively detached, thereby weakening the SERS signal with strong specificity; combined with the SERS effect of the ordered GNBPs array, it can be applied to the highly sensitive detection of a variety of targets.

[0014] (4) The SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B provided by the present invention can rapidly and highly sensitively detect VEGF and PDGF-B in serum within 20 minutes, with a detection limit as low as pg / mL level. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A is a schematic diagram of the preparation of gold nanobipyramids;

[0016] Figure 1B Schematic diagram of the detection of gastric cancer markers VEGF and PDGF-B based on a pump-free SERS microfluidic chip;

[0017] Figure 2A is a SEM photograph of the GNBPs prepared in Example 1;

[0018] Figure 2Bis a TEM image of the GNBPs prepared in Example 1;

[0019] Figure 2C is a high-resolution TEM image of the GNBPs prepared in Example 1;

[0020] Figure 2D is a SAED diffraction pattern photograph of the GNBPs prepared in Example 1;

[0021] Figure 2E is the UV-Vis-NIR spectrum of the GNBPs prepared in Example 1;

[0022] Figure 2F is the Raman spectrum of GNBPs labeled with 4-mercaptobenzoic acid in Example 1;

[0023] Figure 3A are the physical photos and SEM photos of the ordered GNBPs array prepared in Example 2;

[0024] Figure 3B is the SERS imaging image of the ordered GNBPs array prepared in Example 2;

[0025] Figure 3C is the SERS spectra of five points I, II, III, IV, and V randomly selected on the GNBPs array labeled with 4-mercaptobenzoic acid in Example 2;

[0026] Figure 3D is a Raman spectrum of the Raman signal molecule 4-mercaptobenzoic acid and the GNBPs array labeled with it in Example 2;

[0027] Figure 4A is the optimization of the concentration of complementary DNA chains (1 and 2) in Example 3;

[0028] Figure 4B This is the optimization of the incubation time in Example 3;

[0029] Figure 5A is a photograph of the SERS microfluidic chip in Example 3;

[0030] Figure 5B is a photograph of red ink flowing in the SERS microfluidic chip in Example 3;

[0031] Figure 5C is the SERS spectrum of regions I and II after the reaction in Example 3;

[0032] Figure 5D is the SERS spectrum of regions I and III after the reaction in Example 3;

[0033] Figure 6ASpectra of the SERS microfluidic chip detection chamber 1 at different storage times in Example 4;

[0034] Figure 6B Spectra of the SERS microfluidic chip detection chamber 2 at different storage times in Example 4;

[0035] Figure 6C The SERS microfluidic chip with different storage time in Example 4 detected VEGF and PDGF-B at 1363 cm -1 and 1174cm -1 A line graph of the characteristic peak intensity at ;

[0036] Figure 7A spectral graphs of the SERS microfluidic chip detection chamber 1 prepared in different batches in Example 4;

[0037] Figure 7B Spectra of the SERS microfluidic chip detection chamber 2 prepared in different batches in Example 4;

[0038] Figure 7C The SERS microfluidic chip prepared in different batches in Example 4 detects VEGF at 1363 cm -1 Histogram of the intensity at characteristic peaks;

[0039] Figure 7D The SERS microfluidic chip prepared in different batches in Example 4 detected PDGF-B at 1174 cm -1 Histogram of the intensity at characteristic peaks;

[0040] Figure 8A spectral graphs of different samples in the detection chamber 1 of the SERS microfluidic chip prepared in Example 4;

[0041] Figure 8B The SERS microfluidic chip prepared in Example 4 detects target VEGF and BSA, IgG, PCT, CEA and blank control at 1363 cm -1 Histogram of the characteristic peak intensity at ;

[0042] Figure 8C spectral graphs of different samples in the detection chamber 2 of the SERS microfluidic chip prepared in Example 4;

[0043] Figure 8D The SERS microfluidic chip prepared in Example 4 detects the target PDGF-B and BSA, IgG, PCT, CEA and the blank control at 1174 cm -1 The histogram of the characteristic peak intensity at .

[0044] Figure 9AThis is a SERS spectrum diagram of the SERS microfluidic chip prepared in Example 5 used to detect different concentrations of VEGF dispersed in serum;

[0045] Figure 9B The different concentrations of VEGF in Example 5 were detected at 1363 cm -1 The linear relationship curve between the change value of the characteristic peak intensity (Raman intensity) and the logarithm of VEGF concentration;

[0046] Figure 9C This is a SERS spectrum diagram of the SERS microfluidic chip prepared in Example 5 used to detect different concentrations of PDGF-B dispersed in serum;

[0047] Figure 9D The different concentrations of PDGF-B in Example 5 were detected at 1363 cm -1 The linear relationship curve between the change value of the characteristic peak intensity (Raman intensity) and the logarithmic value of PDGF-B concentration;

[0048] Figure 10A is the average SERS spectra of the serum of healthy subjects and gastric cancer patients in Example 5;

[0049] Figure 10B is the average SERS spectra of the serum of healthy subjects and gastric cancer patients in Example 5;

[0050] Figure 10C The average SERS spectrum of the serum of healthy people and gastric cancer patients in Example 5 is at 1363 cm -1 and 1174cm -1 The histogram of the characteristic peak intensity at . DETAILED DESCRIPTION

[0051] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0052] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0053] The embodiment of the present invention provides a SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B, and the preparation method thereof comprises: (1) controlling gold nanobipyramids with hydrochloric acid to prepare an ordered gold nanobipyramid array with neatly arranged tip to tip; (2) coupling a thiol-modified VEGF aptamer and a thiol-modified PDGF-B aptamer to the ordered gold nanobipyramid array prepared in (1) through a gold-sulfur bond; (3) reacting complementary DNA 1 complementary to the VEGF aptamer and complementary DNA 2 complementary to the PDGF-B aptamer, which are labeled with Raman signal molecules, with the ordered gold nanobipyramid array prepared in (2) to prepare a functionalized SERS substrate; and (4) preparing a SERS microfluidic chip from the functionalized SERS substrate.

[0054] It should be noted that compared with other nanoparticles, anisotropic gold nanobipyramids (GNBPs) have better chemical stability and higher local electric field enhancement at the tip. Even more attractive is that GNBPs can be assembled into an excellent surface-enhanced Raman scattering substrate in a tip-to-tip form. The ordered tip-to-tip assembly can fully utilize the controllable morphology, spatial arrangement and directional properties of GNBPs to couple external electromagnetic fields, thereby confining light in nanoscale gaps. This leads to a significant enhancement of the SERS signal. When assembled in close proximity, coupling occurs on the local surface plasmon resonance of individual nanoparticles, forming "hot spots" with enhanced optical electric fields at the junctions of the nanoparticles. The uniformly distributed dual-tip structure and narrow gaps make the "hot spots" on the ordered GNBPs array more dense, resulting in better SERS performance. In addition, the ordered GNBPs arrays used in SERS detection also have the advantages of high enhancement coefficient, uniform signal and long-term stability.

[0055] In addition, the SERS microfluidic chip of the present invention has the advantages of high sensitivity, strong specificity, simple operation, portability, and fast detection speed.

[0056] In some specific embodiments, the method for preparing gold nanobipyramids in the above step (1) includes: mixing hexadecyltrimethylammonium chloride and citric acid; then using sodium borohydride to quickly reduce chloroauric acid to obtain a gold seed solution; and reacting the gold seed solution with a growth solution containing hexadecyltrimethylammonium bromide, chloroauric acid, silver nitrate, hydrochloric acid and ascorbic acid to prepare gold nanobipyramids.

[0057] In some specific embodiments, the above step (1) includes: centrifuging the GNBPs solution and ultrasonically treating it to obtain dispersed GNBPs particles; mixing and incubating the dispersed GNBPs particle solution, cysteine solution and water; then adding hydrochloric acid to promote assembly; centrifuging and drying to obtain an ordered gold nanobipyramid array with neatly arranged tip to tip.

[0058] In some specific embodiments, the above step (2) includes: incubating the thiol-modified VEGF aptamer solution and the thiol-modified PDGF-B aptamer solution with trichloroethyl phosphate solution at room temperature for activation; and reacting the activated two thiol-modified VEGF aptamers and PDGF-B with the ordered gold nanobipyramid array arranged in a point-to-point manner through Au-S bonds to prepare the ordered gold nanobipyramid array.

[0059] In some specific embodiments, the above step (3) includes: reacting complementary DNA 1 labeled with a Raman signal molecule with an ordered gold nanobipyramid array having a thiol-modified VEGF aptamer; at the same time, reacting complementary DNA 2 labeled with a Raman signal molecule with an ordered gold nanobipyramid array having a thiol-modified VEGF aptamer; and washing to obtain a functionalized SERS substrate.

[0060] In some specific embodiments, the Raman signal molecule for labeling complementary DNA 1 is Cy5, and the Raman signal molecule for labeling complementary DNA 2 is 5-FAM.

[0061] In some specific embodiments, the concentrations of the complementary DNA 1 solution and the DNA 2 solution labeled with the Raman signal molecule are both 1 μM.

[0062] In some specific embodiments, the above step (4) includes: (1) evenly applying UV photoresist on a silicon wafer and developing it by UV exposure to obtain a mold; then mixing a polydimethylsiloxane base material and a curing agent in a weight ratio of 10:1, and degassing to remove bubbles, pouring the mixture into a mold, curing, peeling the PDMS replica from the mold, punching holes at the injection and outlet positions of the samples, and obtaining a PDMS cover sheet with microchannels; (2) using a laser to carve three sets of grooves at corresponding positions on an indium tin oxide glass, and cleaning the PDMS cover sheet and the ITO glass substrate; (3) exposing the PDMS cover sheet and the ITO glass substrate to oxygen plasma and performing a hydrophilic treatment; embedding the functionalized SERS substrate into the grooves of the ITO glass plate, and aligning and bonding the PDMS cover sheet with the ITO glass plate to obtain a SERS microfluidic chip for detecting VEGF and PDGF-B.

[0063] It should be noted that in the two detection chambers of the above-mentioned SERS microfluidic chip, the target triggers the conformational change of the aptamer and specifically binds to it. At the same time, the complementary DNA1 and complementary DNA2 labeled with Raman signal molecules (Cy5 and 5-FAM) will be competitively replaced, causing the signal of the Raman signal molecules to weaken. In addition, the SERS microfluidic chip is designed with a comb-like structure. The design of the comb-like structure eliminates the requirement for external heavy pumps and pipelines, greatly improving the portability of the SERS microfluidic chip. In addition, the multi-channel design enables the simultaneous detection of multiple samples, significantly improving the detection efficiency.

[0064] It should also be noted that the above-mentioned SERS microfluidic chip can be designed as a small chip with a length of 4 cm, a width of 4 cm, and a height of 0.6 cm, which is easy to carry. In addition, it has a fast reaction speed and simple operation; and has good repeatability, which can be prepared on a large scale.

[0065] Another embodiment of the present invention provides a method for detecting VEGF and PDGF-B in a sample to be detected using the above-mentioned SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B, comprising: (1) adding the sample to be detected to the sample addition port of the SERS microfluidic chip for detecting VEGF and PDGF-B, and reacting at 23°C-27°C; (2) performing SERS testing on the reaction area three times to obtain Raman molecular signals respectively; and obtaining the concentrations of VEGF and PDGF-B in the sample to be detected based on the working curve of the logarithmic values of the VEGF and PDGF-B concentrations and the SERS intensity change values of the Raman molecular signals.

[0066] In some specific embodiments, the reaction time in step (1) of the above detection method is ≥20 min.

[0067] Specifically, the competitive recognition and binding strategy based on aptamers in the present invention is a method in which the Raman signal weakens as the number of targets increases. At room temperature, the aptamer recognizes the target and undergoes a conformational change, binding to the target with high specificity; the complementary DNA chain labeled with the Raman signal molecule is then competitively detached, thereby weakening the SERS signal. It has strong specificity and, combined with the SERS effect of the ordered GNBPs array, can be applied to the highly sensitive detection of a variety of targets.

[0068] In some specific embodiments, the preparation of the working curve of the logarithmic value of the concentration of VEGF and PDGF-B and the change value of the SERS intensity can be carried out according to the following steps: dispersing the VEGF and PDGF-B standard samples in serum to prepare a mixed solution of VEGF and PDGF-B standard samples with different concentrations; adding the mixed solution of VEGF and PDGF-B standard solutions with different concentrations to the sample injection port of the pump-free SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B, and placing it at 25°C for reaction; then, washing the reaction area three times with PBS buffer and performing SERS test to detect the signals of Cy5 and 5-FAM; according to the fluorescence intensity of 1363cm -1 (Cy5) and 1174 cm -1 The characteristic signals of 5-FAM were used to draw working curves of the logarithmic values of VEGF and PDGF-B concentrations and the SERS intensity change values (Raman intensity).

[0069] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0070] In the following examples, the following instruments, equipment, and test conditions were used: Scanning electron microscopy (SEM) images were obtained using a Hitachi S-4800II field emission scanning electron microscope (SEM); Transmission electron microscopy (TEM) images were obtained using a Philips TECNAI 10 transmission electron microscope (TEM); Raman spectra were obtained using an Invia Reflex laser micro-Raman spectrometer (Renishaw, UK). The laser wavelength was 785 nm, the exposure time was 10 s, and the laser intensity was 15 mW. SERS imaging was obtained using a Thermo Fisher DXRxi micro-Raman imaging spectrometer (Thermo Fisher Scientific, USA).

[0071] In the following examples, the synthesis and use of SERS microfluidic chips are as follows Figure 1A and Figure 1B .

[0072] Example 1 Synthesis and Characterization of GNBPs

[0073] (1) Synthesis of GNBPs

[0074] (a) Preparation of gold seeds: Hexadecyltrimethylammonium chloride (39.2 mL, 50 mM) and citric acid (200 μL, 1 M) were added to a 100 mL flask; chloroauric acid (400 μL, 24 mM) was then rapidly reduced with freshly prepared sodium borohydride (200 μL, 25 mM); after stirring at 700 rpm for 2 min at room temperature, the mixture turned from light yellow to brown; the seed solution was stirred at 300 rpm for 90 min at 80°C; the color gradually changed from brown to red, indicating that gold seeds had formed.

[0075] (b) 2.5 mL of gold seed solution was added to a growth solution containing hexadecyltrimethylammonium bromide (100 mL, 100 mM), chloroauric acid (5 mL, 10 mM), silver nitrate (1 mL, 10 mM), hydrochloric acid (2 mL, 1 M), and ascorbic acid (0.8 mL, 100 mM) at 30°C for 120 min; the solution was concentrated by centrifugation (10 min, 10,000 rpm) and resuspended in 10 mM hexadecyltrimethylammonium bromide to obtain long-term stable GNBPs.

[0076] (2) Characterization of GNBPs’ morphology and SERS effect

[0077] The morphology and structure of GNBPs were examined by SEM, TEM, high-resolution TEM, SAED imaging, and EDX elemental mapping. Figure 2A and Figure 2B As shown, the GNBPs are shaped like two overlapping pyramids with two symmetrical tips. Furthermore, the GNBPs exhibit a complete structure, uniform shape, and good dispersion. The average short diameter is approximately 20 nm, and the average long diameter is approximately 55 nm.

[0078] like Figure 2C As shown, the HRTEM image shows the lattice edges of GNBPs with a lattice spacing of 0.238 nm.

[0079] Figure 2D Shown are SAED patterns of GNBPs to understand their crystal structure. The anisotropic growth of GNBPs confirms the presence of polycrystalline properties, such as {111}, {200}, {220}, and {311} crystal planes with annular diffraction patterns.

[0080] Figure 2E The UV-Vis-NIR spectra of GNBPs are shown, with a strong absorption peak at 691 nm. The narrow half-maximum width indicates the uniformity of the GNBPs morphology. To demonstrate the SERS activity of GNBPs, the Raman spectra of 4-mercaptobenzoic acid and 4-mercaptobenzoic acid-labeled GNBPs were measured.

[0081] like Figure 2F As shown, GNBPs labeled with 4-mercaptobenzoic acid exhibited a strong Raman signal, while the Raman signal of pure 4-mercaptobenzoic acid was very weak, indicating that the enhancement of SERS signals by GNBPs was effective.

[0082] Example 2 Assembly and Characterization of GNBPs Arrays

[0083] (1) Assembly of GNBPs arrays

[0084] (a) The GNBPs solution was centrifuged twice (8000 rpm, 10 min) and redispersed in ultrapure water to remove the bulk of cetyltrimethylammonium bromide. Subsequently, the GNBPs particles were dispersed by sonication for 3 min.

[0085] (b) 1 mL of GNBPs solution, 50 μL of 1 mM cysteine solution (as an adhesion molecule), and 3.85 mL of ultrapure water were added to a 50 mL beaker and incubated at 30°C for 30 min. Hydrochloric acid (100 μL, 8 M) was then added to promote assembly, and the reaction was continued at 30°C for 15 min. Finally, a small amount of the solution was centrifuged at 8000 rpm for 5 min, and the concentrated fraction was dropped onto glass. After drying, an ordered GNBPs array was obtained.

[0086] (2) Characterization of GNBPs array morphology and SERS effect

[0087] The morphology of the GNBPs array substrate was examined by SEM. The uniformity of the SERS substrate was evaluated. 4-Mercaptobenzoic acid was used as the Raman signal molecule to evaluate the uniformity of the SERS substrate. One prepared substrate was taken and immersed in a 1×10 - 9 The dried 4-mercaptobenzoic acid labeled substrate was placed on a Raman spectrometer. The 4-mercaptobenzoic acid was detected at 1593 cm -1 The SERS imaging of the substrate was performed by measuring the intensity of the characteristic peak at the substrate, and the laser scanned the selected area on the substrate with a point-to-point interval of 50 mm and an exposure time of 10 s.

[0088] like Figure 3A As shown, the SEM image clearly shows an ordered array of GNBPs, arranged in a tip-to-tip configuration. This directed tip-to-tip assembly of GNBPs is achieved in the presence of cysteine and hydrochloric acid. Cysteine acts as an adhesion molecule, covalently attached to the ends of GNBPs and inducing tip-to-tip assembly through electrostatic interactions between GNBPs. A digital photograph of the ordered GNBPs array is shown in the upper left corner, showing that the gold film of GNBPs is evenly coated on the glass.

[0089] Figure 3B Shown is the SERS spectrum of an ordered GNBPs array, the surface of which was labeled with 4-mercaptobenzoic acid to evaluate the uniformity of the ordered GNBPs array; the color distribution in the SERS spectrum is basically uniform, with only a small amount of yellow and blue present, indicating that the array is very uniform.

[0090] Five detection positions were randomly selected in the array, and the corresponding spectra are shown in Figure 3C The Raman intensity is at 1593 cm -1 The relative standard deviation (RSD) was 6.89%, indicating the uniformity of the ordered GNBPs array.

[0091] Figure 3D The results show that the SERS enhancement is affected by the ordered GNBPs array. In order to prove the SERS enhancement effect of the ordered GNBPs array, 4-mercaptobenzoic acid was used to generate the SERS signal at 1593 cm -1 The enhancement factor is calculated by the characteristic peak intensity at . The calculation formula is: EF=(I SERS / C SERS ) / (I RS / C RS ). I SERS and C SERS Represents 4-mercaptobenzoic acid (10 -9 M) Signal intensity and concentration when bound to an ordered GNBPs array. RS and C RS Represents 4-mercaptobenzoic acid (10 -2 M) signal intensity and concentration. Calculated EF = 7.32 × 10 8 , which indicates that the ordered GNBPs array has an obvious SERS enhancement effect.

[0092] Example 3 Optimization of preparation of pump-free SERS microfluidic chip

[0093] (1) GNBPs were prepared in the same manner as in Example 1.

[0094] (2) Preparation of functionalized SERS substrate

[0095] (a) Thiol-modified VEGF aptamer solution (50 μL, 10 μM) and thiol-modified PDGF-B aptamer solution (50 μL, 10 μM) were activated with freshly prepared trichloroethyl phosphate (TCEP) solution (5 μL, 50 mM) at room temperature for 60 min.

[0096] (b) The two activated thiol-modified aptamers were reacted with the ordered GNBPs array prepared in Example 1 via Au-S bonds at 37°C for 12 h; the excess thiol-modified aptamers were washed three times with phosphate buffered saline (PBS) and removed.

[0097] (c) Cy5-labeled complementary DNA 1 (50 μL, 1 μM) was reacted with the ordered GNBPs array linked to the thiol-modified VEGF aptamer obtained in step (b) for 12 h. Simultaneously, 5-FAM-labeled complementary DNA 2 (50 μL, 1 μM) was reacted with the ordered GNBPs array linked to the thiol-modified PDGF-B aptamer obtained in step 2.2) for 12 h.

[0098] (d) Wash three times with PBS to remove excess reactants and dry at room temperature to obtain a functionalized SERS substrate.

[0099] (3) Preparation of pump-free SERS microfluidic chip

[0100] (a) A microfluidic chip design is created using a CAD program. Ultraviolet (UV) photoresist is evenly applied to a silicon wafer and developed by UV exposure to create a mold. Subsequently, a polydimethylsiloxane (PDMS) substrate and a curing agent are mixed in a 10:1 ratio (weight ratio) and degassed to remove bubbles. The mixture is poured into a mold and cured at 90°C for 1 hour. The PDMS replica is peeled from the mold, and holes are punched at the injection and outlet locations to create a PDMS cover slip with microchannels.

[0101] (b) Three sets of grooves were laser-etched at corresponding locations on an indium tin oxide (ITO) glass substrate. The prepared PDMS cover slip and ITO glass substrate were cleaned with an isopropyl alcohol and acetone solution under ultrasonication for 5 minutes. Subsequently, they were rinsed three times with deionized water and dried.

[0102] (c) exposing the PDMS cover sheet and ITO glass plate obtained in step (b) to oxygen plasma for 90 seconds, hydrophilizing them with an appropriate amount of polyethylene glycol (PEG), and heating them at 150°C for 30 minutes; washing them three times with deionized water to remove excess reagents, and drying them;

[0103] (d) The functionalized SERS substrate prepared in step (2) was embedded in the grooves of the ITO glass plate (detection chambers 1 and 2). Subsequently, the PDMS cover sheet was aligned and bonded to the ITO glass plate to obtain a pump-free SERS microfluidic chip based on the competitive recognition and binding strategy of aptamers; the entire chip consists of three parallel channel units, each of which is mainly divided into a sample inlet, detection chamber 1, detection chamber 2, a comb structure area, and a sample outlet.

[0104] (4) Determination of the concentration of the optimal complementary DNA chains (1 and 2)

[0105] (a) To screen the optimal concentration of complementary DNA chains (1 and 2), different concentrations of complementary DNA chains (1 and 2) (200nM, 400nM, 600nM, 800nM and 1000nM) were incubated with the ordered GNBPs array of thiol-modified VEGF aptamers obtained in step 2.2) for 12 hours. Figure 4A As shown in Figure 3, when the concentration of complementary DNA reaches 1000 nM, the corresponding SERS signal no longer increases significantly. This is because the Cy5 and 5-FAM on the surface of the ordered GNBPs array gradually increase to a saturated state. Therefore, 1000 nM was selected as the optimal concentration of complementary DNA chains (1 and 2);

[0106] (5) Screening of optimal incubation time

[0107] To screen out the optimal incubation time, 10 -7 pg / mL concentration of VEGF and PDGF-B, incubated for different time periods (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min), as shown in Figure 5. Figure 4B As shown, before the reaction time reaches 20 min, the 1363 cm -1 and 1174cm -1 The Raman intensity of the two characteristic peaks gradually decreased; after 20 minutes, there was no significant change in intensity; after the reaction time reached 20 minutes, the competitive binding of VEGF and PDGF-B with the corresponding aptamers reached saturation, resulting in the shedding of Cy5-labeled complementary DNA1 and 5-FAM-labeled complementary DNA2. Among them, the VEGF aptamer recognized VEGF, formed a G-quadruplex conformation with binding ability, and specifically bound to VEGF; the PDGF-B aptamer recognized PDGF-B, formed a hairpin conformation with binding ability, and specifically bound to PDGF-B. Due to the change in the spatial conformation of the aptamer, complementary DNA1 and complementary DNA2 can no longer bind to it. Therefore, 20 minutes was selected as the optimal incubation time. The nucleotide sequences used in the experiment are shown in Table 1.

[0108] Table 1 Nucleotide sequences used in the experiments

[0109]

[0110]

[0111] (6) Verification of the airtightness and hydrophilicity of the SERS microfluidic chip

[0112] Red ink was used to verify the airtightness and hydrophilicity of the SERS microfluidic chip. The flow process is completely controlled by the capillary driving force and is not subject to human interference. Figure 5A A digital photo of a real chip is shown. Three regions in the chip were selected as SERS signal detection areas to exclude interference from the Raman signal of PDMS. Figure 5B The flow process of red ink in the chip is shown. The whole process lasted 30s, and there was no leakage of red ink during this process. It can be seen that the chip has major hydrophilicity and airtightness, and does not require any external pump connection. Figure 5 (C and D) shows the Raman spectra of regions I, II and III. In region I, the SERS signal is generated only by PDMS. The signals in regions II and III are generated in the presence of VEGF (10 -7 g / mL) and PDGF-B (10 -7 The SERS signal in region I is very weak compared to regions II and III, indicating that the detection of VEGF and PDGF-B is not affected by the weak Raman intensity of PDMS.

[0113] Example 4 Stability, Reproducibility and Specificity of Pump-Free SERS Microfluidic Chip

[0114] (1) A pump-free SERS microfluidic chip was prepared in the same manner as in Example 3.

[0115] (2) The prepared SERS microfluidic chip was stored at room temperature for different days (0 days, 5 days, 10 days, 15 days and 20 days) and the 10 -7 SERS spectra of VEGF and PDGF-B solutions were measured to evaluate their stability. Chips prepared from five different batches were used to detect 10 -7 SERS spectra of VEGF and PDGF-B solutions were obtained by randomly selecting 10 locations in the test area for SERS detection and obtaining the average SERS spectra to evaluate the reproducibility of the microfluidic chip. Several interfering substances (10 -7 g / mL), including BSA, IgG, PCT, and CEA, were incubated with the pump-free SERS microfluidic chip respectively and reacted at 25°C for 20 min. Subsequently, the reaction area was gently washed three times with PBS buffer and then SERS test was performed to evaluate the specificity of the pump-free SERS microfluidic chip.

[0116] Figure 6A and Figure 6B It shows that when the chip is placed at room temperature for 20 days, VEGF (10 -7 g / mL) and PDGF-B (10 -7g / mL) for detection, and SERS spectra in detection chambers 1 and 2.

[0117] Figure 6C The five measurements show the -1 and 1174cm -1 The corresponding Raman intensity line graph at . During the 20-day storage period, the intensity of the characteristic Raman peak did not change significantly. This demonstrates the excellent stability of the pump-free SERS microfluidic chip. Five different batches of chips were fabricated using the same detection method.

[0118] like Figure 7A and 7B As shown, five batches of chips were used to detect VEGF (10 -7 g / mL) and PDGF-B (10 -7 g / mL), the corresponding spectra had no obvious differences from each other.

[0119] Figure 7C and Figure 7D 1363cm respectively -1 and 1174cm -1 Peak intensity histograms at . The RSD values were 5.94% and 6.37%, respectively, with a variation of less than 10%. The superior reproducibility of the pump-free SERS microfluidic chip was confirmed. Due to the complex composition of serum, the pump-free SERS microfluidic chip required high specificity. BSA, IgG, CEA, and PCT were used as interfering substances due to their similarity to the target.

[0120] Figure 8A and Figure 8C Raman spectra of VEGF, PDGF-B, and related interfering substances are shown;

[0121] Figure 8B and Figure 8D Shows that they are at 1363cm -1 and 1174cm -1 Intensity histogram of characteristic peaks.

[0122] In summary, it can be seen that when VEGF exists in detection chamber 1 and PDGF-B exists in detection chamber 2, 1363 cm -1 and 1174cm -1 The SERS peak intensity of the sample was lower than that of the blank control. However, when the interfering substance was present, the corresponding SERS peak did not show significant differences compared to the blank control. This indicates that the interfering substance did not significantly alter the SERS signal intensity. Clearly, the pump-free SERS microfluidic chip exhibits high specificity and anti-interference properties.

[0123] Example 5 Pump-free SERS microfluidic chip for detection of VEGF and PDGF-B in clinical samples

[0124] (1) A pump-free SERS microfluidic chip was prepared in the same manner as in Example 3.

[0125] (2) Disperse VEGF and PDGF-B into serum to obtain a concentration of 10 -12 g / mL, 10 -11 g / mL, 10 -10 g / mL, 10 -9 g / mL, 10 -8 g / mL, 10 -7 g / mL of VEGF and PDGF-B mixed solution, 50 μL of different concentrations of VEGF and PDGF-B mixed solution were added dropwise to the sample port of the pump-free SERS microfluidic chip, and then placed at 25 ° C for 20 minutes. Subsequently, the reaction area was washed three times with PBS buffer and then SERS detection was performed to detect the signals of Cy5 and 5-FAM. According to the Cy5 and 5-FAM at 1363 cm -1 and 1174cm -1 The change value of the characteristic peak signal intensity (Raman intensity) was used to draw a standard curve of the logarithm of VEGF and PDGF-B concentration and the change value of SERS signal intensity (Raman intensity).

[0126] The results show that in Figure 9A In the experiment, as the VEGF concentration increased, the SERS intensity gradually weakened. There was a good linear relationship between the SERS intensity change value (Raman intensity) and the logarithm of VEGF concentration ( Figure 9B ).exist Figure 9C In the experiment, as the concentration of PDGF-B increased, the SERS intensity gradually weakened. There was a good linear relationship between the SERS intensity change value (Raman intensity) and the logarithm of PDGF-B concentration ( Figure 9D The linear regression equation of VEGF is y=2869.27x+35765.54, R 2 =0.984, the detection limit is 0.342pg / mL. The linear regression equation of PDGF-B is y=2713.47x+34125.95, R 2 =0.988, and the detection limit was 0.265 pg / mL.

[0127] (3) The serum of 30 healthy subjects and 30 gastric cancer patients was added to the sample port of the pump-free SERS microfluidic chip, and the reaction was carried out at 25°C for 20 minutes. Subsequently, the reaction area was washed three times with PBS buffer and then SERS detection was performed.

[0128] The results show that Figure 10A and 10B Figure 5. Average SERS spectra of serum from healthy individuals and gastric cancer patients. Each spectrum represents the average result of 30 different clinical serum samples. Figure 10C For these SERS spectra at 1363 cm -1 and 1174cm -1 The intensities of these two characteristic peaks were substituted into the linear regression equation in 3) to calculate the values of VEGF and PDGF-B. The expression levels of VEGF and PDGF-B in the above clinical serum samples were detected by ELISA kits. The test results of ELISA were highly consistent with the SERS results (Table 2).

[0129] Table 2 SERS and ELISA test results of clinical samples

[0130]

[0131]

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B, characterized in that: The preparation method comprises the following steps: (1) controlling gold nanobipyramids with hydrochloric acid to prepare an ordered gold nanobipyramid array with neat tip-to-tip arrangement; (2) coupling a thiol-modified VEGF aptamer and a thiol-modified PDGF-B aptamer to (1) through a gold-sulfur bond to prepare an ordered gold nanobipyramid array coupled with a thiol-modified VEGF aptamer and a thiol-modified PDGF-B aptamer; (3) reacting complementary DNA 1 and complementary DNA 2 labeled with a Raman signal molecule and complementary to the VEGF aptamer with the ordered gold nanobipyramid array prepared in (2) to prepare a functionalized SERS substrate; (4) preparing a SERS microfluidic chip from the functionalized SERS substrate; In step (1), the preparation method of the gold nanobipyramid array includes: mixing hexadecyltrimethylammonium chloride and citric acid; then using sodium borohydride to quickly reduce chloroauric acid to obtain a gold seed solution; reacting the gold seed solution with a growth solution containing hexadecyltrimethylammonium bromide, chloroauric acid, silver nitrate, hydrochloric acid and ascorbic acid to prepare gold nanobipyramids; centrifuging the gold nanobipyramid solution and ultrasonically treating it to obtain dispersed gold nanobipyramid particles; mixing the dispersed gold nanobipyramid particle solution, cysteine solution and water and incubating them; then adding hydrochloric acid to promote assembly; centrifuging and drying to obtain an ordered gold nanobipyramid array with neatly arranged tip to tip; Step (4) comprises: evenly applying UV photoresist on a silicon wafer, developing by ultraviolet exposure, and obtaining a mold; subsequently mixing a polydimethylsiloxane base material and a curing agent in a weight ratio of 10:1, and performing a degassing treatment to remove bubbles, pouring the mixture into a mold, curing, peeling the PDMS replica from the mold, punching holes at the injection and outlet sample positions, and obtaining a PDMS cover sheet with microchannels; laser engraving three groups of grooves at corresponding positions on an indium tin oxide glass, and cleaning the PDMS cover sheet and the ITO glass substrate; exposing the PDMS cover sheet and the ITO glass substrate to oxygen plasma and performing a hydrophilic treatment; embedding a functionalized SERS substrate into the groove of the ITO glass plate, and aligning and bonding the PDMS cover sheet and the ITO glass plate to obtain a SERS microfluidic chip for detecting VEGF and PDGF-B.

2. The SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B according to claim 1, characterized in that: Step (2) comprises: incubating and activating the thiol-modified VEGF aptamer solution and the thiol-modified PDGF-B aptamer solution with trichloroethyl phosphate solution at room temperature; and reacting the activated two thiol-modified VEGF aptamers and PDGF-B aptamers with an ordered gold nanobipyramid array arranged in a point-to-point manner through Au-S bonds to prepare an ordered gold nanobipyramid array coupled with the thiol-modified VEGF aptamer and the thiol-modified PDGF-B aptamer.

3. The SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B according to claim 1 or 2, characterized in that: Step (3) includes: reacting complementary DNA 1 labeled with a Raman signal molecule with an ordered gold nanobipyramid array having a thiol-modified VEGF aptamer; at the same time, reacting complementary DNA 2 labeled with a Raman signal molecule with an ordered gold nanobipyramid array having a thiol-modified VEGF aptamer; and washing to obtain a functionalized SERS substrate.

4. The SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B according to claim 3, characterized in that: The Raman signal molecule for labeling complementary DNA 1 is Cy5, and the Raman signal molecule for labeling complementary DNA 2 is 5-FAM.

5. The SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B according to claim 3, characterized in that: The concentrations of the complementary DNA 1 solution and the DNA 2 solution labeled with Raman signal molecules are both 1 μM.

6. A method for detecting VEGF and PDGF-B in a sample using the SERS microfluidic chip for detecting gastric cancer markers VEGF and PDGF-B according to any one of claims 1 to 5, characterized in that: include: (1) The sample to be tested was added to the sample injection port of the SERS microfluidic chip for detecting VEGF and PDGF-B, and the reaction was carried out at 23°C-27°C; (2) The reaction area was washed three times with PBS buffer and then a SERS test was performed to obtain Raman molecular signals respectively; the concentrations of VEGF and PDGF-B in the sample to be tested were obtained by using the working curve of the Raman molecular signals based on the logarithmic values of the VEGF and PDGF-B concentrations and the SERS intensity change values.

7. The method for detecting VEGF and PDGF-B in a sample to be detected according to claim 6, characterized in that: The reaction time in step (1) of the above detection method is ≥20 min.

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