Surface enhanced Raman scattering substrate and preparation method thereof
Microneedle arrays were prepared through liquid-gas self-assembly and plasma etching technology, and the growth of gold clusters was combined with electrochemical methods, which solved the problem of insufficient sensitivity and specificity of traditional Raman spectroscopy detection, and achieved low-cost and efficient quantitative detection of exosomes.
Patent Information
- Application Number
- CN202510617951.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-14
- Publication Date
- 2025-08-15
AI Technical Summary
The prior art is difficult to detect exosomes with high sensitivity and specificity, and it is impossible to achieve quantitative characterization of exosomes. Traditional Raman spectroscopy methods require high concentration samples.
A microsphere mask was prepared by liquid-gas self-assembly technology, a microneedle array was formed by plasma etching, and a titanium adhesion layer and a gold film were applied to its surface. A gold cluster was grown on the needle tip of the microneedle array using electrochemical methods to form a surface-enhanced Raman scattering substrate.
It realizes high sensitivity and specificity detection of exosomes, and can perform quantitative analysis in low-concentration samples, reducing detection costs and improving signal amplification capabilities, and reducing background noise fluctuations.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to growing gold clusters by liquid-gas self-assembly, plasma etching and electrochemical methods, belongs to the field of micro-nano manufacturing, and particularly relates to a surface-enhanced Raman scattering substrate and a preparation method thereof. Background Art
[0002] Exosomes are small extracellular vesicles with a diameter of approximately 30 to 150 nm. They are potential biomarkers for a variety of diseases, including cancer, and their detection is crucial for early diagnosis and monitoring. Raman spectroscopy is a powerful analytical tool for detecting and characterizing exosomes. However, the weak Raman signal inherent in exosomes poses a challenge for direct detection. Surface-enhanced Raman scattering (SERS) has garnered widespread attention due to its ability to amplify Raman signals and is a non-destructive method for ultrasensitive Raman signal detection. The selection and functionalization of SERS substrates are crucial for improving detection sensitivity and specificity. Summary of the Invention
[0003] The present invention aims to provide a simple, efficient, and low-cost method for preparing SERS substrates, enabling the highly sensitive, specific, and quantitative study of exosomes using Raman spectroscopy. This method can replace conventional Raman spectroscopy SERS substrate preparation methods, which have low sensitivity, low specificity, and are unable to quantitatively characterize exosomes.
[0004] In order to achieve the above object, the present invention adopts the following technical solutions:
[0005] The present invention provides a method for preparing a surface-enhanced Raman scattering substrate, wherein the method comprises:
[0006] (1) Assembling a layer of microspheres as a mask on the surface of a silicon wafer by liquid-vapor self-assembly technology to obtain a silicon wafer covered with the microsphere mask;
[0007] (2) etching the silicon wafer covered by the microsphere mask in step (1) to obtain a microneedle array;
[0008] (3) coating a titanium adhesion layer on the surface of the microneedle array and the silicon wafer on which it is located, and coating a gold film on the titanium adhesion layer;
[0009] (4) Using an electrochemical method, gold clusters were grown with the tips of microneedle arrays as working electrodes to obtain a surface-enhanced Raman scattering substrate.
[0010] In some embodiments, in step (1), the microspheres are polystyrene (PS) microspheres, and the diameter of the microspheres is 100 to 1000 nanometers. As a preferred embodiment, the diameter of the microspheres is 900 nanometers.
[0011] In some embodiments, O2 is used as the etching gas in step (2) to etch the microspheres into smaller pieces. Preferably, the etching time is 40 seconds.
[0012] In some embodiments, SF6 and O2 are used as etching gases in step (2); a microneedle array is etched on the surface of the silicon wafer. As a preferred embodiment, the flow ratio of SF6 to O2 is 3:4; preferably, the etching time using SF6 and O2 as etching gases is 100-150 seconds, more preferably 120 seconds.
[0013] In some embodiments, in step (2), a plasma etching device is used to etch the silicon wafer covered by the microsphere mask in step (1).
[0014] In some embodiments, the method further comprises, before step (3), ultrasonically removing the residual microspheres on the top of the microneedle array.
[0015] In some embodiments, in step (3), the thickness of the titanium attachment layer on the surface of the microneedle array is 5 to 50 nm, and the thickness of the gold film on the titanium attachment layer is 5 to 100 nm. As a preferred embodiment, the thickness of the titanium attachment layer on the surface of the microneedle array is 10 nm, and the thickness of the gold film on the surface of the microneedle array is 20 nm.
[0016] In some embodiments, the electrochemical method is a constant potential polarization method. As a preferred embodiment, the potential of the constant potential polarization method is 0.1 mV to 1 mV, more preferably 0.6 mV.
[0017] In some embodiments, the electrolyte solution used in the constant potential polarization method is a chloroauric acid solution. As a preferred embodiment, the concentration of the chloroauric acid solution is 50 mM to 100 mM, more preferably 80 mM.
[0018] In some embodiments, the gold cluster deposition formation time in step (4) is 30 s to 5 min, more preferably 2 min.
[0019] Another aspect of the present invention provides a surface enhanced Raman scattering substrate, which is prepared by the method described above.
[0020] In the present invention, the microneedles of the surface-enhanced Raman scattering substrate have good height uniformity and are arranged in an orderly manner.
[0021] In some embodiments, the height of the microneedles of the surface-enhanced Raman scattering substrate is 50 to 1000 nm.
[0022] In some embodiments, the distance between adjacent microneedle axes of the surface-enhanced Raman scattering substrate is 50 to 1500 nm.
[0023] In some embodiments, the taper of the microneedles of the surface-enhanced Raman scattering substrate is 1:0.1 to 1:10.
[0024] In some embodiments, in the surface-enhanced Raman scattering substrate, the bottom diameter of the microneedle is 800 nanometers; the diameter of the gold cluster is 800 nanometers; the height of the gold cluster structure on the needle is 400 nanometers; and the distance between the axes of the gold clusters on the needle is 1000 nanometers.
[0025] In the present invention, the gold clusters on the microneedle array of the surface-enhanced Raman scattering substrate have good morphological consistency and are arranged neatly, which is consistent with the arrangement of the microneedle array.
[0026] Beneficial effects:
[0027] The present invention demonstrates a method for preparing a nanostructure that can be used as a SERS substrate. The SERS substrate can be used for the detection of exosomes with high sensitivity and specificity, and has the ability to quantify exosomes.
[0028] This paper proposes a method for preparing a SERS substrate based on self-assembly technology, plasma-etched microneedle arrays, and gold cluster growth using a specific electrochemical method. This method offers simplicity, low cost, strong Raman signal amplification, low background noise, and stable fluctuations, making it potentially suitable for large-scale application. This nanoneedle array-based SERS substrate has great potential for high-sensitivity and high-specificity exosome analysis, enabling quantitative characterization of exosomes. It is also applicable to scenarios involving small exosome sample quantities and low concentrations. This approach could pave the way for the application of exosomes in early disease diagnosis and biomarker detection.
[0029] The present invention uses self-assembly technology to create a microsphere mask on the surface of a silicon wafer; plasma etching equipment is used to prepare an ordered microneedle array with the aid of the microsphere mask; residual microspheres at the microneedle tips are ultrasonically removed; a titanium adhesion layer and a gold film are coated on the surface of the microneedle array; gold clusters are grown on the needle tips using an electrochemical method; and Raman detection is performed using this generated structure as a substrate. This method offers the advantages of low cost, rapid preparation, high Raman signal amplification, low background noise, and stable fluctuations, enabling efficient Raman detection of nanoscale materials. Existing methods that improve detection sensitivity by introducing random, non-arrayed, general nanoparticles for surface-enhanced Raman scattering (SERS) have poor signal stability and reproducibility, potentially affecting the properties of exosomes or introducing additional interference to the exosome signal. This technology, using arrayed microneedles and gold clusters grown on the needles using a specific electrochemical method, does not interfere with exosomes or the signals they generate. This technology can be used to efficiently, quickly and at low cost to produce substrates for surface-enhanced Raman scattering, achieving highly sensitive, highly specific and accurately quantitative Raman detection of biological structures including exosomes. Traditional Raman spectroscopy technology usually requires a sample concentration of ≥106 particles / μL to obtain an effective signal in exosome detection, while the exosome concentration in clinical body fluids (such as blood, urine, etc.) is generally around 10 2 The present invention successfully improves detection sensitivity by constructing arrayed nanoscale SERS hotspots, fully covering the concentration range of clinical samples and effectively breaking through the concentration barrier of traditional methods. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The present invention shows the process of preparing a SERS substrate based on self-assembly technology, plasma etching microneedle array technology and specific electrochemical method to grow gold clusters;
[0031] Figure 2 The PS microsphere mask prepared by the liquid-vapor self-assembly method in Example 1 is shown;
[0032] Figure 3 The ordered microneedle array obtained after plasma etching on the silicon wafer surface and removal of microsphere residues in Example 1 is shown;
[0033] Figure 4 The gold clusters grown on the microneedle array tips as working electrodes in Example 1 are shown;
[0034] Figure 5 shows an electron micrograph of gold clusters grown on the ordered microneedle array in Example 1;
[0035] Figure 6The Raman signal spectra of exosomes at different concentrations obtained by Raman detection of exosomes on a substrate using the SERS substrate of the present invention are shown;
[0036] Figure 7 The Raman spectra of exosome detection on the microneedle array substrate without gold clusters and the microneedle array substrate with gold clusters are shown. DETAILED DESCRIPTION
[0037] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.
[0038] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.
[0039] Unless otherwise specified, experimental methods, detection methods, and preparation methods not described in detail in the present invention all adopt conventional techniques in this technical field.
[0040] The present invention provides a method for preparing a SERS substrate by growing gold clusters based on self-assembly technology, plasma etching technology, and a specific electrochemical method, including the following steps:
[0041] Efficient preparation of densely packed single-layer microsphere masks via liquid-vapor self-assembly method;
[0042] The microspheres used in this method are monodisperse polystyrene microspheres that have been hydrophobically treated;
[0043] Among them, the diameter of the polystyrene microspheres can be 100 to 1000 nm;
[0044] In the mask preparation process, a gel containing sodium dodecylbenzenesulfonate is used to accelerate the formation of tightly arranged single-layer microspheres.
[0045] Ordered microneedle arrays were prepared by plasma etching;
[0046] The plasma etching process gas may be SF6 and O2, and the flow ratio of SF6 to O2 is 3:4.
[0047] Ultrasonic removal of residual microspheres on the surface of the microneedle array;
[0048] Among them, the sample needs to be placed in ultrapure water during ultrasound;
[0049] The ultrasound duration can be 5 to 10 minutes.
[0050] A titanium adhesion layer is coated on the surface of the microneedle array and the silicon wafer on which it is located, and a gold film is coated on the titanium adhesion layer;
[0051] The thickness of the titanium adhesion layer on the microneedle and the silicon wafer on which it is located is a value between 5 and 50 nm;
[0052] The thickness of the gold film on the surface of the titanium adhesion layer is a value between 5 and 100 nm;
[0053] The titanium adhesion layer and the gold film are both achieved by surface sputtering process.
[0054] A specific electrochemical method was used to grow gold clusters on the tips of nanoneedle arrays using the microneedle tips as working electrodes;
[0055] Wherein, the specific electrochemical method is a constant potential polarization method, and the working potential is a value between 0.1mV and 1mV;
[0056] The electrolyte solution used in the constant potential polarization method is chloroauric acid solution with a solution concentration of 50 mM to 100 mM.
[0057] Raman detection of exosomes was performed based on the substrate generated in the previous steps.
[0058] Among them, before detection, it is necessary to modify the coupled antibody or aptamer on the substrate, and drop the sample on the substrate to fix the exosomes in the sample.
[0059] Among them, Raman detection needs to be performed using a confocal Raman spectrometer.
[0060] The following describes a method for preparing an on-needle gold cluster array as a SERS substrate based on self-assembly technology, plasma etching technology, and gold cluster growth technology using a specific electrochemical method according to an embodiment of the present invention with reference to the accompanying drawings.
[0061] Example 1
[0062] Figure 1 The present invention provides a process for preparing a gold cluster array on a needle as a SERS substrate based on self-assembly technology, plasma etching technology and a specific electrochemical method for growing gold clusters. Figure 2 and Figure 3 They are respectively the PS microsphere mask image of Example 1 of the present invention and the ordered microneedle array image after plasma etching and removal of microsphere residues. Figure 4Figure 2 shows the growth of gold clusters using the microneedle tip as the working electrode. The four figures can be combined for reference.
[0063] like Figure 1 As shown, the method for preparing a gold cluster array on a needle as a SERS substrate based on self-assembly technology, plasma etching technology and electrochemical method for growing gold clusters includes the following steps:
[0064] (1) Assembling a layer of microspheres as a mask on the surface of a silicon wafer using liquid-vapor self-assembly technology;
[0065] The silicon wafer was ultrasonically cleaned in acetone, ethanol, and deionized water for 5 minutes and then air-dried. Next, a colloidal solution of hydrophobic monodisperse polystyrene nanoparticles (PS microspheres) (900 nm diameter, 2.5% w / v, Baseline Chromtech Research Center, Tianjin, China) was added to deionized water. A hydrogel containing sodium dodecyl sulfate (SDS) was inserted into the water surface to densely pack the PS microspheres. The substrate was then removed from the solution, resulting in a substrate with a monolayer of PS microspheres assembled as a mask.
[0066] The present invention uses gel containing sodium dodecylbenzenesulfonate to accelerate the formation of tightly arranged single-layer PS microspheres.
[0067] (2) etching the silicon wafer covered with the microsphere mask using a plasma etching device;
[0068] After the PS-coated silicon surface dried naturally, the sample was processed using an inductively coupled plasma system (GSE-C200 RIE, NAURA, Beijing, China). The system first ran a predefined cleaning program to remove residual gases and ensure a contamination-free process. Next, the mask microspheres were initially reduced in size in an O2 plasma environment, partially exposing the substrate, with an etching time of 40 seconds. Subsequently, the PS microspheres and silicon substrate were plasma-etched in the same system using a specific SF6 and O2 gas combination (flow ratio 3:4) to obtain the microneedle array. The etching time was 120 seconds.
[0069] (3) Ultrasonic removal of residual microspheres on the top of the microneedle array.
[0070] The ultrasonic cleaning process needs to be carried out in ultrapure water, with an ultrasonic power of 180w, a frequency of 40KHz, and an ultrasonic duration of 10 minutes.
[0071] (4) Coating a titanium adhesion layer and a gold film on the microneedle array and its surface.
[0072] A titanium adhesion layer with a thickness of 10 nm was prepared on the surface of the microneedles and the silicon wafer on which they were placed by a surface sputtering process;
[0073] A gold film with a thickness of 20 nm is prepared on the surface of the titanium adhesion layer by using a surface sputtering process.
[0074] (5) Gold clusters were grown using a specific electrochemical method with the microneedle array tips as working electrodes.
[0075] The gold clusters were grown using the potentiostatic polarization method at a potential of 0.6 mV;
[0076] The concentration of chloroauric acid solution is 80 mM;
[0077] The formation time of gold cluster deposition is 2 min.
[0078] A SERS substrate was obtained, whose parameters were: the bottom diameter of the microneedle was 800 nanometers; the diameter of the gold cluster was 800 nanometers; the height of the gold cluster structure on the needle was 400 nanometers; and the distance between the axes of the gold clusters on the needle was 1000 nanometers.
[0079] The height of the gold cluster structure on the needle refers to the height from the top of the gold cluster to the substrate surface.
[0080] (6) Raman detection of exosomes was performed on the prepared SERS substrate.
[0081] Exosome extraction: Human breast cancer cell line MCF-7 (Cell Resource Center, Peking Union Medical College, China) was cultured in DMEM (1% penicillin-streptomycin, 1% insulin, and 10% fetal bovine serum) at 37°C in a humidified incubator with 5% CO2. When the cells reached approximately 90% confluency, the medium was changed to serum-free medium. After 24 hours, the cell culture supernatant was collected and centrifuged at 2000 × g for 30 minutes at 4°C to remove cells and debris. Exosomes were then extracted using a commercial exosome isolation kit (Thermo Fisher Scientific, USA) and stored at -80°C for long-term storage.
[0082] Raman spectroscopy: Antibodies or aptamers were conjugated to the substrate surface to specifically capture exosome membrane proteins. Exosomes were then immobilized on the functionalized substrate surface. Optical detection was performed using a Raman spectrometer (LabRAM HR Evolution, HORIBA, Kyoto, Japan) at a 785 nm laser excitation wavelength. The resulting Raman spectra were processed using the instrument's accompanying LabSpec 6.3 software.
[0083] result:
[0084] like Figure 2 and Figure 3 1 and 2 are diagrams showing a self-assembled PS microsphere mask and a microneedle array according to an embodiment of the present invention. Figure 4Figure 2 shows the electrochemical growth of gold clusters using the microneedle tip as the working electrode. Depending on the needs of Raman detection, the microspheres can have different diameters, and the height and taper of the microneedles can also vary.
[0085] Figure 5 This is an electron microscope image of gold clusters growing on an ordered microneedle array.
[0086] The SERS substrate prepared in this application can realize Raman detection of exosomes. Figure 6 The Raman signal spectra of exosomes at different concentrations obtained by Raman detection of exosomes on a substrate using the SERS substrate of the present invention are shown;
[0087] Figure 7 The Raman spectra of exosome detection on the microneedle array substrate without gold clusters and the microneedle array substrate with gold clusters are shown.
[0088] Compared with the microneedle array without gold clusters, the SERS substrate of the present application has a higher resolution for detecting exosomes.
[0089] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.
[0090] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.
[0091] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.
Claims
1. A method for preparing a surface-enhanced Raman scattering substrate, wherein: The method includes: (1) Assembling a layer of microspheres as a mask on the surface of a silicon wafer by liquid-vapor self-assembly technology to obtain a silicon wafer covered with the microsphere mask; (2) plasma etching the silicon wafer covered by the microsphere mask in step (1) to obtain a microneedle array; (3) coating a titanium adhesion layer on the surface of the microneedle array and the silicon wafer on which it is located, and coating a gold film on the titanium adhesion layer; (4) Using an electrochemical method, gold clusters were grown with the tips of microneedle arrays as working electrodes to obtain a surface-enhanced Raman scattering substrate.
2. The method according to claim 1, wherein In step (1), the microspheres are polystyrene microspheres. Preferably, the diameter of the microspheres is 100 to 1000 nanometers, and more preferably, the diameter of the microspheres is 900 nanometers.
3. The method according to claim 1, wherein In step (2), O2 is used as the etching gas to etch the microspheres into smaller pieces.
4. The method according to claim 1, wherein In step (2), SF6 and O2 are used as etching gases. Preferably, the flow ratio of SF6 and O2 is 3:
4.
5. The method according to claim 1, wherein The method further comprises, before step (3), ultrasonically removing the residual microspheres on the top of the microneedle array.
6. The method according to any one of claims 1 to 5, wherein: In step (3), the thickness of the titanium attachment layer on the surface of the microneedle array and the silicon wafer on which it is located is 5 to 50 nm, and the thickness of the gold film on the titanium attachment layer is 5 to 100 nm. Preferably, the thickness of the titanium attachment layer on the surface of the microneedle array is 10 nm, and the thickness of the gold film on the surface of the microneedle array is 20 nm. Preferably, the electrochemical method is a constant potential polarization method. Preferably, the potential of the constant potential polarization method is 0.1 mV to 1 mV, more preferably 0.6 mV; Preferably, the electrolyte solution used in the constant potential polarization method is a chloroauric acid solution. Preferably, the concentration of the chloroauric acid solution is 50 mM to 100 mM, more preferably 80 mM. Preferably, the gold cluster deposition formation time in step (4) is 30s to 5min, more preferably 2min.
7. A surface-enhanced Raman scattering substrate, prepared by the method according to any one of claims 1 to 6.
8. The surface-enhanced Raman scattering substrate according to claim 7, wherein: The height of the microneedles of the surface enhanced Raman scattering substrate is 50-1000 nm.
9. The surface-enhanced Raman scattering substrate according to claim 7, wherein: The taper of the microneedles of the surface enhanced Raman scattering substrate is 1:0.1 to 1:
10.
10. The surface-enhanced Raman scattering substrate according to claim 7, wherein: The distance between adjacent microneedle axes of the surface enhanced Raman scattering substrate is 50 to 1500 nm.