A SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF and its application

Abstract

This invention relates to the field of biomarker detection technology, specifically to a SERS microfluidic chip for detecting gastric cancer biomarkers CEA and VEGF, and its applications. This SERS microfluidic chip successfully achieved quantitative determination of CEA and VEGF with a good linear relationship to concentration through evaluation of the 4-MBA frequency shift; furthermore, based on its dual-detection-region design, simultaneous detection of CEA and VEGF can be easily achieved with only one Raman reporter gene; the detection limits of this platform in physiological culture medium are as low as 0.38 pg / mL for CEA and 0.82 pg / mL for VEGF.

Description

Technical Field

[0001] This invention relates to the field of biomarker detection technology, specifically to a SERS microfluidic chip for detecting gastric cancer biomarkers CEA and VEGF and its applications. Background Technology

[0002] Gastric cancer (GC) is the fourth most common malignant tumor worldwide and the second leading cause of cancer-related deaths. Early-stage gastric cancer only invades the mucosa or submucosa, while late-stage gastric cancer is insensitive to existing treatments and has a poor prognosis. However, gastric cancer is insidious in its development, with most patients being diagnosed at an advanced stage. Although endoscopy combined with histopathological analysis is the gold standard for clinical diagnosis of gastric cancer, the subjectivity of pathological diagnosis and the uncertainty of endoscopic localization often lead to misdiagnosis of small lesions in early-stage GC. Currently, several serum tumor biomarkers, such as carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF), have been found to be associated with gastric cancer.

[0003] To achieve sensitive detection of CEA and VEGF, a series of methods have been developed, including enzyme-linked fluorescence spectrometry (ELISA), fluorescence immunoassay (FISA), colorimetric immunoassay (CIMA), and chemiluminescence immunoassay (CIA). While these methods offer high specificity and sensitivity, their high cost, time-consuming nature, and labor-intensive nature can be key limitations in certain environments. Therefore, developing a simple, rapid, and ultrasensitive detection method remains a challenge. Compared to traditional methods, SERS technology stands out as one of the most powerful analytical techniques due to its superior sensitivity, high selectivity, non-destructive nature, reliable detection, and speed. SERS can utilize strong localized electromagnetic fields (i.e., "hot spots") existing in nanoscale junctions or gaps, which allows the Raman detection sensitivity of some systems to be reduced even to the single-molecule level, making SERS a promising method for identifying tumor biomarkers.

[0004] In recent years, SERS-based microfluidic technology (microfluidic chips are centimeter-sized devices containing microchannels, valves, and chambers) has been considered a powerful and promising analytical platform due to its advantages such as low sample consumption, short reaction time, high detection efficiency, and high portability. Therefore, microfluidic chips can serve as an integrated platform to make SERS detection more reproducible, efficient, safe, and environmentally friendly.

[0005] Currently, indirect detection of protein biomarkers based on SERS mainly relies on Raman-active reporter molecules for indirect detection, utilizing SERS "hotspots" generated by sandwich structures. While this analytical strategy has achieved significant results in the quantitative analysis of protein biomarkers, non-specific adsorption induced by multiple immunoreaction steps remains an unresolved issue. On the one hand, this analytical process involves only a single immunoreaction step, significantly reducing the effectiveness of non-specific adsorption; on the other hand, similar to refractive index methods, it requires measuring the shift in a strong signal rather than addressing peaks above noise, thus reducing the spectral resolution requirements. However, the application of frequency-shift-based SERS analysis and microfluidic chips in biochemical clinical testing is still in its early stages. Traditional microfluidic chips generally rely on external pumps for microfluidic control, severely reducing portability and greatly limiting practical applications. Furthermore, most Raman frequency-shift-based sensors use microcontact printing to prepare ordered domains of the Raman reporter, enabling simultaneous analysis of multiple samples. Microcontact printing, of course, is not without limitations, primarily hindered by the mechanical instability of the stamp's embossed area. Summary of the Invention

[0006] To address the above problems, one of the objectives of this invention is to provide a SERS microfluidic chip for detecting gastric cancer biomarkers CEA and VEGF. This SERS microfluidic chip successfully achieved quantitative determination of CEA and VEGF with a good linear relationship with concentration by evaluating the 4-MBA frequency shift. Furthermore, based on the dual detection zone design, only one Raman reporter gene is needed to easily achieve simultaneous detection of CEA and VEGF, and the detection limit is low.

[0007] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0008] The present invention provides a SERS microfluidic chip for detecting gastric cancer biomarkers CEA and VEGF. The preparation method includes: (1) assembling hexagonal gold nanosheets into a closely packed array, transferring the array to a hydrophilically treated indium tin oxide glass surface to obtain a SERS substrate; (2) modifying the surface of the SERS substrate with 4-mercaptobenzoic acid, and coupling the 4-mercaptobenzoic acid with CEA antibody and VEGF antibody respectively to obtain a functionalized SERS substrate; (3) preparing a SERS microfluidic chip from the functionalized SERS substrate.

[0009] Another aspect of the present invention provides a method for detecting gastric cancer biomarkers CEA and VEGF, comprising: plotting a working curve of the half-logarithmic values ​​of CEA and VEGF concentrations and the absolute value of the peak shift of the SERS frequency (|ΔRaman shift|); adding the sample to be tested into the inlet of the SERS microfluidic chip for detecting gastric cancer biomarkers CEA and VEGF as described in any one of claims 1 to 8, and then performing a hybridization reaction at a constant temperature of 35℃-39℃; removing the SERS microfluidic chip, cleaning the reaction area, and performing SERS testing to detect a 4-MBA signal; and determining the concentrations of CEA and VEGF in the sample to be tested based on the working curve.

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

[0011] (1) The SERS microfluidic chip preparation method for detecting gastric cancer markers CEA and VEGF provided by the present invention is simple, has good stability, and can be prepared in large quantities.

[0012] (2) The SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF provided by this invention exhibits good repeatability, requires only one sample addition step, eliminates non-specific adsorption induced by multiple reaction steps, and greatly increases convenience and specificity. It can be prepared on a large scale.

[0013] (3) The SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF provided by this invention has successfully achieved quantitative determination of CEA and VEGF with good linear relationship with concentration by evaluating the 4-MBA frequency shift; and based on its dual detection zone design, only one Raman reporter gene is needed to easily achieve simultaneous detection of CEA and VEGF; the detection limits of this platform for CEA and VEGF in physiological culture medium are as low as 0.38 pg / mL and 0.82 pg / mL, respectively.

[0014] (4) The SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF provided by this invention has the same detection accuracy as the conventional ELISA method. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the detection of gastric cancer markers CEA and VEGF based on SERS microfluidic chip. (a) shows the preparation and sample addition process of SERS microfluidic chip, and (b) shows the frequency shift analysis strategy.

[0016] Figure 2These are SEM images, TEM images, high-resolution TEM images, SAED diffraction patterns, EDX images of various elements, UV-vis-NIR spectra of GNSs, and Raman spectra of GNSs and 4-MBA labeled with the Raman signal molecule 4-MBA, prepared in Example 1; wherein, (a) is a SEM image of GNSs, (b) is a TEM image of GNSs, (c) is a high-resolution TEM image of GNSs, (d) is a SAED diffraction pattern of GNSs, (e) is an EDX image of various elements in GNSs, (f) is a UV-vis-NIR spectrum of GNSs; and (g) is a Raman spectrum of GNSs and 4-MBA labeled with the Raman signal molecule 4-MBA.

[0017] Figure 3a Here is a SEM image of the GNSs substrate prepared in Example 2; Inset: Photograph of the interface assembly of the GNSs substrate and the gold film;

[0018] Figure 3b These are SERS spectra of 20 randomly selected points on the GNSs substrate prepared in Example 2;

[0019] Figure 3c The Raman spectrum of the 4-MBA labeled GNSs prepared in Example 2 at 1080 cm⁻¹ -1 A histogram of SERS intensity at the characteristic peak;

[0020] Figure 3d This is a SERS image of the 4-MBA labeled GNSs substrate prepared in Example 2;

[0021] Figure 3e This is a near-field electric field intensity distribution diagram simulated by the finite-difference time-domain method on the GNSs substrate prepared in Example 2;

[0022] Figure 4a The Fourier transform infrared spectrum of the GNSs substrate prepared in Example 3;

[0023] Figure 4b The Fourier transform infrared spectrum of the 4-MBA labeled GNSs substrate prepared in Example 3;

[0024] Figure 4c The Fourier transform infrared spectrum of the GNSs substrate labeled with 4-MBA conjugated antibody 1 prepared in Example 3;

[0025] Figure 4d The Fourier transform infrared spectrum of the GNSs substrate labeled with 4-MBA conjugated antibody 2 prepared in Example 3;

[0026] Figure 5aThis is a schematic diagram of the structural composition of the SERS microfluidic chip prepared in Example 4;

[0027] Figure 5b These are photographs of the automatic ink flow over time in the microchannels prepared in Example 4;

[0028] Figure 5c These are the SERS spectra recorded on Region I and GNSs substrates prepared in Example 4;

[0029] Figure 5d It is the one prepared in Example 4 Figure 5c SERS spectrum at 1593 cm⁻¹ -1 A magnified view of the SERS peak at the location;

[0030] Figure 5e This is a characterization diagram of the stability of the hydrophilic treatment of the micro-nano channels prepared in Example 4 over time;

[0031] Figure 6a These are SERS spectra of different time regions (I) after CEA incubation in Example 4;

[0032] Figure 6b It is in Example 4 Figure 6a SERS spectrum at 1593 cm⁻¹ -1 A magnified view of the SERS peak at the location;

[0033] Figure 6c It is in Example 4 Figure 6b SERS spectrum at 1593 cm⁻¹ -1 The frequency shift of the peak as a function of incubation time;

[0034] Figure 6d These are SERS spectra of different time regions II after VEGF incubation in Example 4;

[0035] Figure 6e It is in Example 4 Figure 6d SERS spectrum at 1593 cm⁻¹ -1 A magnified view of the SERS peak at the location;

[0036] Figure 6f It is in Example 4 Figure 6f SERS spectrum at 1593 cm⁻¹ -1 The frequency shift of the peak as a function of incubation time;

[0037] Figure 7 This is a characterization diagram of the response of the SERS microfluidic chip prepared in Example 4 to flow rate;

[0038] Figure 8aThese are the blank control SERS spectra measured in region I of Example 5, and the SERS spectra measured in region I and region II after the addition of CEA solution;

[0039] Figure 8b It is in Example 5 Figure 8a SERS spectrum at 1593 cm⁻¹ -1 A magnified view of the SERS peak at the location;

[0040] Figure 8c These are the blank control SERS spectra measured in region II of Example 5, and the SERS spectra measured in regions I and II after the addition of VEGF solution;

[0041] Figure 8d It is in Example 5 Figure 8c SERS spectrum at 1593 cm⁻¹ -1 A magnified view of the SERS peak at the location;

[0042] Figure 9a These are the SERS spectra of CEA, AFP, CA125 solutions and blank control measured in regions I and II in Example 6;

[0043] Figure 9b In Example 6 Figure 9a SERS spectra in Region I at 1593 cm⁻¹ -1 A magnified view of the SERS peak at the location;

[0044] Figure 9c In Example 6 Figure 9a The SERS spectrum in Region II is at 1593 cm⁻¹ -1 A magnified view of the SERS peak at the location;

[0045] Figure 9d These are reproducibility characterization diagrams of regions I and II of the microfluidic chip in Example 6;

[0046] Figure 10a The SERS spectra of different concentrations of CEA (1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL and 1 μg / mL) in Region I in Example 7 are shown.

[0047] Figure 10b It is in Example 7 Figure 10a SERS spectrum at 1593 cm⁻¹ -1 A magnified view of the SERS peak at the location;

[0048] Figure 10c In Example 7, at 1593cm -1 A semi-logarithmic plot of the absolute peak shift as a function of CEA concentration;

[0049] Figure 10d The images show the SERS spectra of different concentrations of VEGF (1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL and 1 μg / mL) in region I in Example 7.

[0050] Figure 10e It is in Example 7 Figure 10d SERS spectrum at 1593 cm⁻¹ -1 A magnified view of the SERS peak at the location;

[0051] Figure 10f In Example 7, at 1593cm -1 A semi-logarithmic plot of the absolute peak shift as a function of VEGF concentration;

[0052] Figure 11a This is the average SERS spectrum of serum samples from healthy individuals and gastric cancer patients measured in regions I and II in Example 7;

[0053] Figure 11b It is in Example 7 Figure 11a Central region I at 1593cm -1 Enlarged view of the SERS peak at the location;

[0054] Figure 11c It is in Example 7 Figure 11a Middle Zone II at 1593cm -1 Enlarged view of the SERS peak at the location;

[0055] Figure 12 This is the OD calibration curve for determining VEGF concentration by ELISA in Example 7. Detailed Implementation

[0056] The illustrated embodiments are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the illustrated embodiments. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description of the invention still fall within the protection scope of the present invention.

[0057] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0058] An embodiment of the present invention provides a SERS microfluidic chip for detecting gastric cancer biomarkers CEA and VEGF. The preparation method includes: (1) assembling hexagonal gold nanosheets into a closely packed array, transferring the array to a hydrophilically treated indium tin oxide glass surface to obtain a SERS substrate; (2) modifying the surface of the SERS substrate with 4-mercaptobenzoic acid, and coupling the 4-mercaptobenzoic acid with CEA antibody and VEGF antibody respectively to obtain a functionalized SERS substrate; (3) preparing the SERS microfluidic chip from the functionalized SERS substrate.

[0059] In some specific embodiments, the preparation method of hexagonal gold nanosheets in step (1) above includes: mixing polyvinylpyrrolidone aqueous solution with HAuCl4, then adding ascorbic acid and stirring, letting stand, and centrifuging to obtain hexagonal gold nanosheets.

[0060] In some specific embodiments, in step (1) above, hexagonal gold nanosheets are assembled into a closely packed array by an immiscible organic / water self-assembly method.

[0061] In some specific embodiments, the method of assembling hexagonal gold nanosheets into a closely packed array in step (1) above includes: redispersing the hexagonal gold nanosheet solution with ethanol and then mixing it with an immiscible cyclohexane / water system to obtain a gold film, i.e., an array.

[0062] In some specific embodiments, the above-mentioned hydrophilic treatment of indium tin oxide glass includes: placing clean indium tin oxide glass in a sulfuric acid-hydrogen peroxide solution for 1 hour under an 80°C water bath condition; the volume ratio of sulfuric acid to hydrogen peroxide in the sulfuric acid-hydrogen peroxide solution is 7:3.

[0063] In some specific embodiments, the volume ratio of the above-mentioned cyclohexane, water and ethanol-dispersed hexagonal gold nanosheet solution is 2:2:1.

[0064] In some specific embodiments, in step (2) above, the preparation of the functionalized SERS substrate includes: incubating the SERS substrate in a 4-mercaptobenzoic acid ethanol solution and washing it to obtain SERS substrate a; then incubating the SERS substrate a with a phosphate buffer containing N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, washing, and drying to activate the carboxyl group to obtain SERS substrate b; adding CEA antibody and VEGF antibody to SERS substrate b and washing it to obtain SERS substrate c; and incubating SERS substrate c with BSA solution to obtain the functionalized SERS substrate.

[0065] In some specific embodiments, the method of preparing a SERS microfluidic chip from a functionalized SERS substrate in step (3) above includes: drilling six holes in an upper PDMS cover plate with microchannels as inlet and outlet; then performing oxygen plasma hydrophilic treatment on the PDMS cover plate and the glass slide; then embedding the functionalized SERS substrate into the glass slide by laser etching, and assembling the glass slide and the PDMS cover plate together to obtain the SERS microfluidic chip.

[0066] In some specific embodiments, the entire SERS microfluidic chip consists of three parallel channel units, each of which is mainly divided into an inlet, reaction zone I, reaction zone II, capillary pump, and outlet; the SERS microfluidic chip can be 4cm long, 4cm wide, and 0.5cm high.

[0067] It should be noted that the SERS microfluidic chip used in this embodiment of the invention for detecting gastric cancer biomarkers CEA and VEGF is a capillary-driven SERS microfluidic chip based on a Raman frequency shift strategy, used to detect CEA and VEGF, thereby achieving early gastric cancer diagnosis. Figure 1 As shown, the SERS microfluidic chip is designed with three sets of parallel channels. Each set of channels consists of five functional parts: an inlet, reaction zone I, reaction zone II, a capillary pump, and an outlet. Using this chip, simultaneous analysis can be achieved without relying on microcontact printing; through the design of the capillary pump and the hydrophilic treatment of the microchannels, automated fluid flow is realized.

[0068] Specifically, this platform is based on a hexagonal gold nanosheet (GNSs) array. First, the substrate is functionalized with 4-mercaptobenzoic acid (4-MBA) to generate SERS spectra suitable for the spectral changes induced by deformation. Through the application of carbodiimide crosslinking, the capturing antibody is modified by the carboxyl group of 4-MBA, forming a 4-MBA-antibody conjugate. After functionalization, the substrate is embedded into a glass slide via laser etching to prepare a SERS microfluidic chip. Serum samples are pipetted into the injection port, and CEA and VEGF are captured by the 4-MBA conjugate antibody in reaction zones I and II, respectively, through an immunoassay, performing sandwich immunoassay without relying on a secondary antibody. CEA and VEGF can exert nanomechanical deformation on the 4-MBA molecule, resulting in a visually recognizable Raman shift. Since the degree of 4-MBA deformation is affected by the number and molecular weight of the target, a correlation can be established between the spectral changes induced by deformation and the analyte concentration, realizing the conversion of the Raman shift into the expression levels of CEA and VEGF in gastric cancer patients and healthy individuals. Furthermore, the feature of multiple parallel channels enables the simultaneous analysis of multiple samples.

[0069] Another embodiment of the present invention provides a method for detecting gastric cancer biomarkers CEA and VEGF, comprising: plotting a working curve of the half-logarithmic values ​​of CEA and VEGF concentrations and the absolute value of the peak shift of the SERS frequency (|ΔRaman shift|); adding the sample to be tested into the inlet of the SERS microfluidic chip for detecting gastric cancer biomarkers CEA and VEGF as described in any one of claims 1 to 8, and then performing a hybridization reaction at a constant temperature of 35℃-39℃; removing the SERS microfluidic chip, cleaning the reaction area, and performing SERS testing to detect a 4-MBA signal; and determining the concentrations of CEA and VEGF in the sample to be tested based on the working curve.

[0070] In some specific embodiments, the hybridization reaction is carried out for 6 minutes.

[0071] In some specific implementations, the method for generating working curves of the half-logarithmic values ​​of CEA and VEGF concentrations and the absolute values ​​of SERS frequency peak shifts (|ΔRaman shift|) includes: dispersing CEA and VEGF standards in serum to prepare mixed solutions of CEA and VEGF standards at different concentrations (10... -16 M~10 -10M), and the blank control was also performed in serum without CEA and VEGF; 3 μL of mixed solutions of CEA and VEGF standard solutions of different concentrations and the blank control were added to the inlet of the SERS microfluidic chip used to detect gastric cancer markers CEA and VEGF, respectively, and then placed in a 37°C incubator for hybridization reaction; the inlet of the SERS microfluidic chip used to detect gastric cancer markers CEA and VEGF was removed, the reaction area was washed with phosphate buffer, and then SERS test was performed, and the 4-MBA signal was detected; according to 1593 cm -1 The characteristic signals of (4-MBA) were used to plot the semi-logarithmic values ​​of CEA and VEGF concentrations and the absolute value of the SERS frequency peak shift (|ΔRaman shift|).

[0072] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0073] In the following examples, scanning electron microscope (SEM) images were obtained using an S-4800II field emission scanning electron microscope manufactured by Hitachi, Japan; transmission electron microscope (TEM) images were obtained using a TECNAI G2F30 transmission electron microscope manufactured by Philips, Netherlands; ultraviolet-visible absorption spectra were recorded using a Shimadzu UV3600, Japan; and Raman spectroscopy and SERS imaging were obtained using an Invia Reflex laser micro Raman spectrometer manufactured by Renishaw, UK, with the following test conditions: laser wavelength 785 nm, exposure time 10 s, 3 accumulations, and laser intensity 5 mW.

[0074] Example 1: Synthesis and Characterization of Gold Nanosheets (GNSs)

[0075] (I) Synthesis of Gold Nanosheets (GNSs)

[0076] (1) Add 3.08g of polyvinylpyrrolidone to 60mL of deionized water, heat in a water bath (30℃) and stir at 800rpm until completely dissolved;

[0077] (2) 2.4 mL (1 wt%) of HAuCl4 was quickly added to the solution obtained in step (1), and the color turned pale yellow; then 144 μL of 0.2 M ascorbic acid was added, and the color turned colorless after 1 min.

[0078] (3) Stir the solution obtained in step (2) at 800 rpm for 5 min and let it stand for 12 h. The color turns reddish-brown. Then, centrifuge (10000 rpm, 10 min) to purify it.

[0079] (II) Morphology of GNSs and Characterization of SERS Effect

[0080] The morphology and structure of GNSs were detected by SEM, TEM, high-resolution TEM, SAED imaging, and EDX elemental mapping. The results are as follows: Figure 2 As shown, SEM and TEM images indicate ( Figure 2 (a) Figure 2 (b) and Figure 2 (c) The prepared nanosheets are symmetrical hexagonal nanosheets with a diameter of 161 nm and good dispersibility; the corners of several triangular nanosheets are truncated, showing a tendency to transition to hexagonal nanosheets. In principle, under low cooling rates, the growth rate of pure metal crystals along the direction perpendicular to the atomically dense surface is the slowest; therefore, parallel advancement of the dense surface is more common, resulting in a stable and densest atomic surface. Due to the face-centered cubic (fcc) structure of Au crystals, Au generated by ascorbic acid reduction... 0 Small triangular nanosheets are formed by growth along the (111) surface; when a large number of triangular nanosheets are present in the solution, the contact area between the vertices of the triangular nanosheets and the solution is large, and the activity of the gold nanosheets is high, leading to an erosion reaction until relatively low-energy hexagonal nanosheets and truncated triangles and other symmetrical structures are formed; such as Figure 2 As shown in (c), the lattice spacing of 0.235 nm corresponds to the (111) plane of Au, and the diffraction rings ( Figure 2 (d) corresponds to surfaces (111), (200), (220), and (311).

[0081] In addition, the EDS spectrum also verified this. Figure 2 The elemental composition of GNSs in (e).

[0082] In addition, the UV-Vis absorption spectrum shows that GNSs has a visible peak at 930 nm. Figure 2 (f)).

[0083] In addition, the SERS activity of GNSs was also evaluated. Figure 2 (g)) The results showed that 4-MBA labeled GNSs (1×10 -6 The SERS intensity of M) is much higher than that of pure 4-MBA (1×10). -2 Therefore, the embodiments of the present invention successfully synthesized GNSs with satisfactory optical properties.

[0084] Example 2: Preparation and characterization of hexagonal gold nanosheets (GNSs) substrate

[0085] (I) Preparation of hexagonal gold nanosheets (GNSs) substrates

[0086] (1) Prepare hexagonal gold nanosheets (GNSs) in the same manner as in Example 1;

[0087] (2) Cut the ITO glass into small squares, pre-treat the small square ITO glass to remove organic matter from the surface of the small square ITO glass; use sulfuric acid-hydrogen peroxide solution to hydrophilize the clean small square ITO glass to increase the hydroxyl groups on the surface of the small square ITO glass to make it hydrophilic, and then dry it for later use.

[0088] (3) Add 10 mL of deionized water to a beaker, remove an equal amount of cyclohexane from the water layer, redisperse the GNSs solution in step (1) with ethanol, and inject 5 mL into the immiscible cyclohexane / water system; then, GNSs quickly self-assemble into a closely packed array, and a gold film appears on the cyclohexane / water interface.

[0089] (4) Transfer the gold film to the hydrophilic ITO glass surface prepared in step (2), and after drying, obtain a SERS substrate with uniform and dense GNSs arrangement.

[0090] (II) Morphology and SERS uniformity characterization of hexagonal gold nanosheet (GNSs) substrate

[0091] GNSs substrates were prepared via the above-mentioned cyclohexane / water interface self-assembly, such as... Figure 3a As shown, GNSs formed a closely packed metal film (inset), and SEM images demonstrate the uniformity of the GNSs film transferred to the ITO surface. To verify the uniformity, SERS spectra were acquired at 20 randomly selected points on the GNSs substrate, as shown in the image. Figure 3b As shown, there is almost no difference in the curve, indicating good homogeneity of the GNSs substrate. Figure 3c 1593cm -1 The peak intensity at [location] was [value], with a relative standard deviation (RSD) of 5.08%. Furthermore, SERS mapping was performed on the GNSs substrate to characterize consistency, such as [example data]. Figure 3d As shown, the colors in the images are basically similar, indicating that the GNSs substrate has good consistency.

[0092] In addition, according to Figure 2 a and Figure 2 Based on the geometric characteristics of GNSs shown in b, the electric field distribution of the GNSs substrate was simulated using the finite-difference time-domain method, and a simulation model was established. The simulation results show that due to electromagnetic coupling, a high-density "hot spot" is formed between adjacent GNSs, resulting in excellent SERS signal enhancement. The finite-difference time-domain simulation can explain the mechanism of the high-density "hot spot" generation on the GNSs substrate, providing strong theoretical guidance for the construction of high-sensitivity SERS substrates.

[0093] Example 3: Preparation and Characterization of Functionalized SERS Substrates

[0094] (I) Preparation of Functionalized SERS Substrates

[0095] (1) Prepare gold nanosheet substrates as in Example 2.

[0096] (2) The SERS substrate prepared in step (1) was placed in 4-MBA ethanol solution (10mM) and incubated for 1 h, and then washed with ethanol;

[0097] (3) The substrate obtained in step (2) was incubated with 50 μL of phosphate buffer containing N-hydroxysuccinimide (50 mM) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (200 mM) for 5 h, then washed with washing buffer and dried to activate the carboxyl group.

[0098] (4) Add 50 μL of rabbit monoclonal anti-CEA antibody and rabbit monoclonal anti-VEGF antibody (20 μg / mL; purchased from Shanghai Sangon Biotech Co., Ltd.) and incubate with the substrate obtained in step (3) for 12 h, then wash with washing buffer; after incubating with BSA solution (10 mg / mL) for 1 h, functionalized SERS substrate is obtained.

[0099] (II) Characterization of Functionalized SERS Substrates

[0100] The preparation of functionalized substrates was characterized by Fourier transform infrared spectroscopy. Clearly, no obvious characteristic peaks were observed on the unfunctionalized SERS substrate. Figure 4a );in addition, Figure 4b The Fourier transform infrared spectrum of the 4-MBA labeled GNSs substrate is shown at 1639 cm⁻¹. -1 Only one prominent peak appeared at 1251 cm⁻¹, which is due to the extension of C=O amide I from the carboxyl group of 4-MBA; after modification with anti-CEA antibody, the peak was observed at 1251 cm⁻¹. -1 1557cm -1 and 1636cm -1 The presence of peaks indicating CN stretching, NH amide II stretching, and C=O amide I stretching suggests that the anti-CEA antibody was successfully conjugated to 4-MBA (see [link to relevant documentation]). Figure 4c Similarly, GNSs substrates modified with anti-VEGF antibodies showed a 1251 cm⁻¹ appearance. -1 1557cm -1 1636cm -1 Peak (see) Figure 4d ).

[0101] The above evidence suggests that the functionalized substrate has been successfully produced.

[0102] Example 4: Optimized Fabrication of SERS Microfluidic Chips

[0103] (1) Prepare functionalized SERS substrates as in Example 3;

[0104] (2) The parameters of the microfluidic chip were designed using AutoCAD. A negative SU-8 photoresist mold was fabricated on a clean silicon wafer using a high-resolution photomask. Subsequently, air bubbles were removed from the mixture of polydimethylsiloxane (PDMS) precursor and thermal initiator (mixing ratio of 10:1) using a vacuum pump. The mixture was then poured onto the prepared template using a casting method and stored in an oven (70°C) for 2 hours. The mold was then removed and cooled to room temperature to obtain an upper PDMS cover sheet with microchannels.

[0105] (3) Drill 6 holes (d=4mm) on the PDMS cover sheet obtained in step (2) as inlet and outlet; then, perform oxygen plasma hydrophilic treatment (100s) on the PDMS cover sheet and glass slide to reduce the hydrophilicity angle of the fluid; immediately embed the functionalized SERS substrate into the glass slide by laser etching, assemble the glass slide and PDMS cover sheet together to obtain the SERS microfluidic chip based on the Raman frequency shift strategy;

[0106] (4) The entire chip consists of three parallel channel units. Each channel unit is mainly divided into an inlet, reaction zone I, reaction zone II, capillary pump and outlet. The microfluidic chip is 4cm long, 4cm wide and 0.5cm high.

[0107] (5) Figure 5a As shown in the structural diagram, the proposed SERS microfluidic chip consists of three sets of parallel channels. Each set of parallel channels comprises five functional parts: an inlet, reaction zone I, reaction zone II, a capillary pump, and an outlet. A capillary pump was designed to achieve automatic fluid flow within the microchannels. Furthermore, oxygen plasma was used to hydrophilize the channels, significantly reducing the hydrophilic angle. To verify the hydrophilicity effect, blue ink was used. The results show that the ink can flow out of the channel independently of the external pump within 32 seconds (see...). Figure 5b No leakage occurred during the flow process, indicating that the chip has good sealing performance. Furthermore, its hydrophilic stability was also evaluated, such as... Figure 5e As shown, the chip effluent time was measured after different storage times, demonstrating that the proposed SERS microfluidic chip has excellent stability and can be used in practical applications. Since PDMS material possesses its own Raman signal, investigating whether PDMS affects SERS results is crucial for ensuring detection sensitivity. Figure 5c The SERS spectra measured on the GNSs substrate after incubation with CEA and the spectra measured in Region I were recorded; such as Figure 5dAs shown, there is no significant Raman frequency shift between the spectra, indicating that the PDMS coating does not interfere with the SERS results. This demonstrates that the SERS microfluidic chip was successfully established.

[0108] (6) To obtain the best analytical performance, optimal conditions for sensor function are proposed; such as... Figure 6a As shown, the SERS spectrum was measured as a function of incubation time. In CEA solutions incubated for different times, due to the CC respiration mode, the SERS spectrum at 1593 cm⁻¹ was [data missing]. -1 The peak position gradually changes, and its significant spectral changes are evident in... Figure 6b The difference is more pronounced in the middle; as the incubation time increases, 1593cm -1 The peak shifts upwards, and the absolute value of the frequency peak shift (|ΔRaman shift|) reaches its maximum at 6 minutes, after which it gradually shifts downwards. Figure 6c This unusual time-dependent frequency shift is caused by the aggregation process of CEA in solution. As previously mentioned, the Raman frequency shift mechanism is due to the change in mechanical force caused by the binding of the target to the Raman reporter. Initially, the anti-CEA antibody interacts with the CEA monomers, and due to the relatively light weight and small size of the CEA protein, the frequency shift is small; subsequently, as CEA gradually aggregates with other monomers, its mass and size increase, leading to a larger frequency shift; then, the frequency shift gradually decreases after reaching a maximum at 6 min, due to the separation of CEA oligomers from anti-CEA, and the self-interaction occupying the interaction sites on the CEA oligomers; a similar Raman frequency shift phenomenon can also be observed in VEGF analysis, such as... Figure 6d , Figure 6e and Figure 6f As shown, the maximum absolute value of the frequency peak shift (|ΔRaman shift|) can be reached in 5 minutes. Therefore, 6 minutes is selected as the optimal reaction time. Furthermore, by varying the channel aspect ratio, the maximum detection efficiency is achieved at an average volumetric flow rate of approximately 25 μL / min. Figure 7 ).

[0109] Example 5 Qualitative Analysis of CEA and VEGF

[0110] (1) A pump-free SERS microfluidic chip was prepared in the same manner as in Example 4;

[0111] (2) To determine whether the SERS microfluidic chip can effectively identify CEA and VEGF, samples containing CEA (10) were placed in the atmosphere. -10 M) and VEGF(10 -10 A solution containing only CEA (10) -10 A solution containing only VEGF (10) -10The solution containing M) and the solution without CEA and VEGF were added to the sample port of the chip, and the SERS spectrum was detected after reacting in a constant temperature incubator at 37℃ for 6 min.

[0112] (3) After successfully establishing the SERS microfluidic chip and optimizing the experimental parameters, a qualitative analysis of CEA and VEGF was performed. Figure 8a The blank SERS spectrum (recorded on a GNSs substrate) and the SERS spectra measured in regions I and II after incubation with CEA are shown; clearly, when only CEA is present in the sample solution, the 4-MBA peak in region I shifts as expected, while the 4-MBA peak in region II does not shift. Figure 8b ); Figure 8c The blank SERS spectrum (recorded on a GNSs substrate) and the SERS spectra measured in regions I and II after incubation with VEGF are shown; when VEGF is present only, the 4-MBA peak in region II shifts, while the 4-MBA peak in region I does not shift. Figure 8d Therefore, the proposed SERS microfluidic chip achieves excellent qualitative analysis performance without significant cross-interference between the two biomarker detection channels.

[0113] Example 6: Specificity and reproducibility 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) Specificity and reproducibility are two main aspects related to analytical performance.

[0116] To assess this, it will be in 10 -10 CEA (and VEGF) at M concentration and 10 -9 After incubation with different interfering substances (AFP and CA125) at M concentration, SERS spectra were recorded in Region I (and Region II). Figure 9a After incubation with CEA, 4-MBA reached 1593 cm. -1 The expected changes in frequency peaks were clear, but no frequency shift was observed (see [link]). Figure 9b ); detected in control experiments conducted at higher concentrations in the same buffer. Similarly, as Figure 9c As shown, only in the presence of VEGF can the 1593 cm of 4-MBA be observed. -1 A noticeable frequency shift only appears at the peak. Therefore, SERS microfluidic chips have strong specificity, and can only respond to the target, even in complex samples.

[0117] In addition, the effects of SERS microfluidic chips on 4-MBA1593 cm⁻¹ excitation wavelength at 785 nm were investigated. -1 Reproducibility of the SERS signal of the peak; comparing the chip with different concentrations of CEA and VEGF (10 -16 M, 10 -14 M, 10 -12 M and 10 -10 After incubation with M), the 1593 cm⁻¹ value was measured in 20 mM phosphate buffer. -1 The absolute frequency shift of the peak; SERS spectra were recorded on parallel chips prepared in three different batches, such as... Figure 9d As shown, the SERS response has high repeatability, with a calculated average relative standard deviation (RSD) of 4.31%, indicating that the SERS microfluidic chip has high reproducibility.

[0118] Example 7: SERS microfluidic chip for detecting CEA and VEGF in clinical samples

[0119] (1) A SERS microfluidic chip was prepared in the same manner as in Example 3;

[0120] (2) CEA and VEGF were dispersed in serum. To study the sensitivity of the SERS microfluidic chip in detecting CEA and VEGF, samples were collected at 1000 μL of serum. -16 M~10 -10 SERS spectra were measured within the M range after incubation with CEA and VEGF solutions.

[0121] The results are as follows Figure 10a and Figure 10b As shown, with the increase of CEA concentration, the concentration at 1593 cm⁻¹ in region I can be clearly observed. -1 The expected change in peak frequency was observed; the blank control was also performed in CEA-free phosphate buffer, and no peak frequency shift was detected. Figure 10c As shown, the absolute value of the frequency peak shift shows a linear relationship with the semi-logarithmic plot of CEA concentration. Similarly, in region II, the 1593 cm⁻¹ of 4-MBA... -1 The frequency peaks changed with increasing VEGF concentration (see...). Figure 10d and Figure 10e The absolute value of the Raman frequency peak shift also showed a linear relationship with the semi-logarithmic plot of VEGF concentration. Figure 10f Additionally, it should be noted that the limiting factor is not resolution, but accuracy; for Raman instruments, the root mean square (RMS) change in the peak position of any single point in the graph between 5 repetitions is only 0.022 cm. -1 Considering a noise level three times higher to clearly distinguish differences, this instrument can differentiate as small as 0.066 cm. -1 Peak shift. In this embodiment of the invention, 0.15cm -1The safety value is defined as the minimum measurable displacement. Therefore, the limits of detection (LOD) for CEA and VEGF are 0.38 pg / mL and 0.82 pg / mL, respectively, indicating that the chip can be used for quantitative detection.

[0122] (3) Figure 11a As shown, serum samples were added to the chip's sample inlet, and after reacting in a 37°C incubator for 6 minutes, SERS spectra were recorded in regions I and II. Figure 11b and Figure 11c The frequency peak shifts shown indicate that the mean expression levels of CEA and VEGF in the serum of gastric cancer patients were 18.4 ng / mL and 2.39 ng / mL, respectively. In healthy individuals, the mean expression levels of CEA and VEGF in serum were 3.06 ng / mL and 0.14 ng / mL, respectively. Furthermore, to verify the accuracy of the results, an ELISA assay was used, and the relative errors between the mean results of the two methods were within a satisfactory range (Table 1).

[0123] Table 1 Comparison of mean SERS of real samples and ELISA method

[0124]

[0125] In addition, Table 2 lists all the results for both methods, demonstrating the accuracy of the SERS results.

[0126] Table 2. Results of serum SERS and ELISA measurements in healthy individuals.

[0127]

[0128]

[0129] Furthermore, its sensitivity was studied using samples containing different concentrations of VEGF (1, 10, 100, 300, 500, 700, 900, and 1000 pg / mL), such as... Figure 12 As shown, the optical density (OD) calibration curves indicate that ELISA exhibits good analytical performance in the range of 100–1000 pg / mL. Low concentrations (below 100 pg / mL) cannot accurately measure VEGF. Compared to ELISA, the SERS microfluidic chip demonstrates satisfactory sensing performance for low-concentration targets. Therefore, these results demonstrate the potential application of frequency-shift-based SERS microfluidic chips for trace biomarker detection in clinical settings.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF, characterized in that, The preparation method includes: (1) assembling hexagonal gold nanosheets into a closely packed array, transferring the array to a hydrophilically treated indium tin oxide glass surface to obtain a SERS substrate; (2) modifying the surface of the SERS substrate with 4-mercaptobenzoic acid, and coupling it with CEA antibody and VEGF antibody respectively to obtain a functionalized SERS substrate; (3) preparing a SERS microfluidic chip from the functionalized SERS substrate. In step (1), hexagonal gold nanosheets are assembled into a closely packed array by an immiscible organic / water self-assembly method. The method of assembling hexagonal gold nanosheets into a closely packed array in step (1) includes: redispersing the hexagonal gold nanosheet solution with ethanol and then mixing it with an immiscible cyclohexane / water system to obtain a gold film, i.e., an array.

2. The SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF according to claim 1, characterized in that, In step (1), the preparation method of hexagonal gold nanosheets includes: mixing polyvinylpyrrolidone aqueous solution with HAuCl4, then adding ascorbic acid and stirring, letting stand, and centrifuging to obtain hexagonal gold nanosheets.

3. The SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF according to claim 1 or 2, characterized in that, The hydrophilic treatment of indium tin oxide glass involves placing clean indium tin oxide glass in a sulfuric acid-hydrogen peroxide solution for 1 hour under an 80°C water bath condition; the volume ratio of sulfuric acid to hydrogen peroxide in the sulfuric acid-hydrogen peroxide solution is 7:

3.

4. The SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF according to claim 1 or 2, characterized in that, The volume ratio of cyclohexane, water, and ethanol-dispersed hexagonal gold nanosheet solution was 2:2:

1.

5. The SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF according to claim 1 or 2, characterized in that, In step (2), the preparation of the functionalized SERS substrate includes: incubating the SERS substrate in 4-mercaptobenzoic acid ethanol solution and washing to obtain SERS substrate a; then incubating the SERS substrate a with phosphate buffer containing N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, washing, and drying to activate the carboxyl group to obtain SERS substrate b; adding CEA antibody and VEGF antibody to SERS substrate b and washing to obtain SERS substrate c; and incubating SERS substrate c with BSA solution to obtain the functionalized SERS substrate.

6. The SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF according to claim 1 or 2, characterized in that, In step (3), the method for preparing a SERS microfluidic chip from a functionalized SERS substrate includes: drilling six holes in an upper PDMS cover plate with microchannels as inlet and outlet; then performing oxygen plasma hydrophilic treatment on the PDMS cover plate and the glass slide; then embedding the functionalized SERS substrate into the glass slide by laser etching, and assembling the glass slide and the PDMS cover plate together to obtain the SERS microfluidic chip.

7. A method for detecting gastric cancer markers CEA and VEGF, characterized in that, include: Plot a working curve for the half-logarithmic values ​​of CEA and VEGF concentrations and the absolute values ​​of SERS frequency peak shifts; add the sample to be tested into the inlet of the SERS microfluidic chip for detecting gastric cancer markers CEA and VEGF as described in any one of claims 1 to 6, and then carry out a hybridization reaction at a constant temperature of 35℃-39℃; remove the SERS microfluidic chip, clean the reaction area, and perform SERS testing to detect the 4-MBA signal; determine the concentrations of CEA and VEGF in the sample to be tested based on the working curve.

8. The method for detecting gastric cancer markers CEA and VEGF according to claim 7, characterized in that, The hybridization reaction took 6 minutes.

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