Surface enhanced Raman scattering method capable of realizing molecular-level detection of exosome

Abstract

The invention discloses a surface enhanced Raman scattering method capable of realizing molecular-level detection of exosomes. When the method is used for detecting the exosome, the process is simple and convenient, the cost is low, the detection sensitivity is high, and the method has large-scale popularization and application potential. The method has huge potential in high-sensitivity exosome quantitative analysis, and lays a foundation for application of the exosome in early disease diagnosis and biomarker detection.

Description

Technical Field

[0001] The present invention relates to a method for detecting exosomes, belonging to the field of biomedicine; in particular, it relates to a surface-enhanced Raman scattering method for molecular-level detection of exosomes. 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 that enables ultrasensitive Raman signal detection. Finding suitable surface-enhanced Raman scattering methods is crucial for improving the sensitivity and specificity of exosome Raman detection. Summary of the Invention

[0003] The present invention aims to provide a simple, efficient, and low-cost method for molecular-level detection and quantitative characterization of exosomes, providing a highly sensitive and specific method for studying exosomes using Raman spectroscopy. This method can be used to replace traditional, less sensitive and less specific methods for Raman spectroscopy of exosomes.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] In one aspect, the present invention provides a method for molecular-level detection of exosomes by surface-enhanced Raman scattering, comprising:

[0006] S1. Obtain the exosome sample to be tested;

[0007] S2. Raman detection of exosome samples using surface-enhanced Raman scattering substrate;

[0008] The surface enhanced Raman scattering substrate is an ordered gold cluster array on microneedles generated by an electrochemical method using microneedles as working electrodes.

[0009] In some embodiments, step S2 comprises:

[0010] (1) dropping the exosome solution onto the surface of the surface-enhanced Raman scattering substrate, and performing optical detection using a Raman spectrometer after natural drying;

[0011] Or (2) coupling the surface-enhanced Raman scattering substrate with an antibody or aptamer to obtain a functionalized substrate to specifically capture exosome membrane proteins, immobilizing the exosomes on the surface of the functionalized substrate, and then performing optical detection using a Raman spectrometer.

[0012] In some embodiments, the surface-enhanced Raman scattering substrate is prepared by the following method:

[0013] (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;

[0014] (2) etching the silicon wafer covered by the microsphere mask in step (1) to obtain a microneedle array;

[0015] (3) coating a titanium adhesion layer on the microneedle array and the surface thereof, and coating a gold film on the titanium adhesion layer;

[0016] (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.

[0017] The surface-enhanced Raman scattering substrate in the present invention is an ordered gold cluster array on microneedles generated by a specific electrochemical method using microneedles as working electrodes. The array can generate a strong local electromagnetic field, and the Raman signal of molecules in the electromagnetic field will be enhanced.

[0018] In the present invention, an ordered gold cluster array on a needle is used as a substrate to detect exosomes to obtain the Raman signal of the exosome sample. In the obtained Raman signal, the background (noise) signal is small enough and the fluctuation is stable so as not to interfere with the characteristic molecular signal of the exosome.

[0019] The method of the present invention also includes post-processing the extracted characteristic Raman signals, combining the organized and established database of exosome Raman signals of different concentrations to quantitatively characterize the exosome molecular components and exosome concentrations.

[0020] In some embodiments, in step (1), the microspheres are polystyrene (PS) microspheres. As a preferred embodiment, the diameter of the microspheres is 100 to 1000 nanometers, more preferably, the diameter of the microspheres is 900 nanometers.

[0021] In some embodiments, in step (2), O2 is used as an etching gas to etch the microspheres into smaller pieces. Preferably, the etching time is 40 seconds.

[0022] In some embodiments, SF6 and O2 are used as etching gases in step (2), preferably, the flow ratio of SF6 and O2 is 3:4; preferably, the etching time using SF6 and O2 as etching gases is 100-150s, more preferably 120s.

[0023] 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).

[0024] In some embodiments, the method further comprises, before step (3), ultrasonically removing the residual microspheres on the top of the microneedle array.

[0025] In some embodiments, in step (3), the thickness of the titanium adhesion layer on the surface of the microneedle array is 5 to 50 nm, and the thickness of the gold film on the surface of the microneedle array is 5 to 100 nm. As a preferred embodiment, the thickness of the titanium adhesion 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.

[0026] The gold clusters of the surface-enhanced Raman scattering substrate of the present invention use microneedles as working electrodes and are generated by a specific electrochemical method. The gold clusters are arranged in an orderly manner and have good morphological consistency.

[0027] 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.

[0028] 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.

[0029] In some embodiments, the gold cluster deposition time in step (4) is 30 seconds to 5 minutes, more preferably 2 minutes. In the present invention, the microneedles of the surface-enhanced Raman scattering substrate have good height uniformity and are arranged in an orderly manner.

[0030] In some embodiments, the height of the microneedles of the surface-enhanced Raman scattering substrate is 50 to 1000 nm.

[0031] In some embodiments, the taper of the microneedles of the surface-enhanced Raman scattering substrate is 1:0.1 to 1:10.

[0032] In some embodiments, the distance between adjacent microneedle axes of the surface-enhanced Raman scattering substrate is 50 to 1500 nm.

[0033] 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.

[0034] Another aspect of the present invention provides a kit or a device comprising the surface-enhanced Raman scattering substrate described in the above method.

[0035] The present invention also provides the use of the kit or device in preparing a product for detecting exosomes. In some embodiments, the product is a kit.

[0036] Beneficial effects of the present invention:

[0037] The present invention demonstrates a method for quantitatively characterizing exosome endonucleases and exosome concentrations based on surface-enhanced Raman scattering and establishing an exosome Raman signal database. This method can achieve molecular-level detection of exosomes with high sensitivity and specificity.

[0038] This paper proposes a method for detecting exosomes using surface-enhanced Raman scattering (SERS) and combining it with database technology for quantitative characterization. This method offers simple, low-cost, and highly sensitive detection, and has the potential for large-scale application. This exosome detection method, based on an ordered gold cluster array substrate, has significant potential for high-sensitivity quantitative analysis of exosomes, paving the way for their application in early disease diagnosis and biomarker detection.

[0039] The present invention discloses a method for molecular-level detection of exosomes using surface-enhanced Raman scattering (SERS). The method comprises: an ordered array of gold clusters attached to microneedles (SERS substrate), which generates a strong, locally enhanced electromagnetic field that significantly enhances the Raman signals of molecules within it; detection of exosomes using the ordered gold cluster array as a substrate; and post-processing the Raman signals to quantitatively characterize the exosome components. This method offers the advantages of low cost, rapid preparation, ease of operation, and high detection sensitivity and resolution, enabling highly sensitive and quantifiable Raman detection of exosomes at the molecular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The present invention shows the process of detecting and quantitatively characterizing exosomes using an ordered gold cluster array;

[0041] Figure 2 The surface-enhanced Raman scattering substrate diagram of the ordered gold cluster array on needles according to Example 1 of the present invention is shown;

[0042] Figure 3 A schematic diagram of Raman signal detection using the aforementioned substrate according to Example 1 of the present invention is shown;

[0043] Figure 4 The Raman signal spectra of exosomes at different concentrations obtained in Example 1 of the present invention are shown;

[0044] Figure 5 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

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The present invention provides a method for molecular-level Raman detection of exosomes using an ordered gold cluster array, comprising the following steps:

[0049] Exosomes were detected using an ordered gold cluster array generated by a microneedle as a working electrode. This substrate significantly enhanced the Raman signal.

[0050] Before detecting exosomes, the substrate surface needs to be functionalized with antibodies or ligands to fix the exosomes on the substrate;

[0051] Raman spectrometer is used to extract the characteristic signals of exosome molecules;

[0052] Establish a Raman signal database of different exosome concentrations based on the aforementioned substrates and summarize and analyze the corresponding data;

[0053] The exosome signals in the new samples were compared with the data in the database, and the exosome molecules and exosome concentrations were quantitatively characterized.

[0054] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0055] The following describes a method for molecular-level detection of exosomes based on surface-enhanced Raman scattering of ordered gold clusters on needles according to an embodiment of the present invention with reference to the accompanying drawings.

[0056] Example 1

[0057] Figure 1 The present invention provides a process for the molecular-level detection of exosomes based on surface-enhanced Raman scattering of ordered gold clusters on needles. Figure 2 and Figure 3 They are respectively a diagram of a surface-enhanced Raman scattering substrate of an ordered gold cluster array on needles according to Example 1 of the present invention and a schematic diagram of Raman signal detection using the aforementioned substrate. Figure 4 : The Raman signal spectra of exosomes at different concentrations obtained according to Example 1 of the present invention. The four can be combined for reference.

[0058] like Figure 1 As shown, the method for molecular-level detection of exosomes based on surface-enhanced Raman scattering of ordered gold clusters on needles includes the following steps:

[0059] Step 1: Using ordered gold clusters on microneedles as a surface-enhanced Raman scattering substrate; the base diameter of the microneedles is 800 nanometers; the diameter of the gold clusters is 800 nanometers; the height of the gold clusters on the microneedles is 400 nanometers; and the distance between the axes of the gold clusters on the microneedles can be 1000 nanometers. The height of the gold clusters on the microneedles refers to the height from the top of the gold clusters to the substrate surface.

[0060] The surface-enhanced Raman scattering substrate is based on self-assembly technology, plasma etching technology and electrochemical method to grow gold clusters to prepare the gold cluster array on the needle. The preparation method is as follows:

[0061] (1) Assembling a layer of microspheres as a mask on the surface of a silicon wafer using liquid-vapor self-assembly technology;

[0062] 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.

[0063] The present invention uses gel containing sodium dodecylbenzenesulfonate to accelerate the formation of tightly arranged single-layer PS microspheres.

[0064] (2) etching the silicon wafer covered with the microsphere mask using a plasma etching device;

[0065] 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 procedure to remove residual gases and ensure a contamination-free process. Next, the mask size was reduced in an O2 plasma environment to partially expose the substrate for 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) for 120 seconds to obtain the microneedle array.

[0066] (3) Ultrasonic removal of residual microspheres on the top of the microneedle array.

[0067] 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.

[0068] (4) Coating a titanium adhesion layer and a gold film on the microneedle array and its surface.

[0069] 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;

[0070] 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.

[0071] (5) Gold clusters were grown using a specific electrochemical method with the microneedle array tips as working electrodes.

[0072] The gold clusters were grown using the potentiostatic polarization method at a potential of 0.6 mV;

[0073] The concentration of chloroauric acid solution is 80 mM;

[0074] The formation time of gold cluster deposition is 2 min.

[0075] Step 2: Perform Raman detection on the exosomes on the aforementioned substrate.

[0076] Raman spectroscopy targets exosomes, which were extracted using the following method: 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.

[0077] The substrate surface is coupled with antibodies or aptamers to specifically capture exosomal membrane proteins;

[0078] Immobilize exosomes on the functionalized substrate surface.

[0079] Step 3: Optical detection was performed using a Raman spectrometer (LabRAM HR Evolution, HORIBA, Kyoto, Japan) with a laser excitation wavelength of 785 nm. The measured Raman spectra were processed using the LabSpec 6.3 software that comes with the instrument.

[0080] Step 4: Establish a Raman signal database of different exosome concentrations based on the ordered gold cluster substrate and perform summary analysis.

[0081] In step 5, the new exosome sample is subjected to Raman detection on the ordered gold cluster substrate and the exosome molecules and exosome concentration are quantitatively characterized by combining the existing data in the database.

[0082] result:

[0083] like Figure 2 and Figure 3 1 is a schematic diagram of a surface-enhanced Raman scattering substrate of an ordered gold cluster array on needles according to an embodiment of the present invention and a schematic diagram of Raman signal detection using the aforementioned substrate. Figure 4 Figure 2 shows the Raman signal patterns of exosomes at different concentrations obtained in the examples of the present invention. Depending on the needs of different exosome Raman detection, the ordered microneedle array can use different gold cluster sizes, and the spacing, height, and taper of the microneedles can also be selected to have different values. Figure 5 The Raman spectra of exosome detection on the microneedle array substrate without gold clusters and the microneedle array substrate with gold clusters are shown.

[0084] Compared with the microneedle array without gold clusters, the SERS substrate of the present application has a higher resolution for detecting exosomes.

[0085] 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.

[0086] 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.

[0087] 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 molecular-level detection of exosomes by surface-enhanced Raman scattering, comprising: S1. Obtain the exosome sample to be tested; S2. Raman detection of exosome samples using surface-enhanced Raman scattering substrate; The surface enhanced Raman scattering substrate is an ordered gold cluster array on microneedles generated by an electrochemical method using microneedles as working electrodes.

2. The method according to claim 1, wherein Step S2 includes: (1) dropping the exosome solution onto the surface of the surface-enhanced Raman scattering substrate, and performing optical detection using a Raman spectrometer after natural drying; Or (2) coupling the surface-enhanced Raman scattering substrate with an antibody or aptamer to obtain a functionalized substrate to specifically capture exosome membrane proteins, immobilizing the exosomes on the surface of the functionalized substrate, and then performing optical detection using a Raman spectrometer.

3. The method according to claim 1, wherein The surface enhanced Raman scattering substrate is prepared by the following method: (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) 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 microneedle array and the surface thereof, and coating a gold film on the titanium adhesion layer; (4) Using an electrochemical method to grow gold clusters with the microneedle array tips as working electrodes to obtain a surface-enhanced Raman scattering substrate; Preferably, in step (1), the microspheres are polystyrene microspheres; preferably, the diameter of the microspheres is 100 to 1000 nanometers, more preferably, the diameter of the microspheres is 900 nanometers.

4. The method according to claim 3, wherein: In step (2), O2 is used as the etching gas to etch the microspheres into smaller pieces.

5. The method according to claim 3, wherein In step (2), SF6 and O2 are used as etching gases. Preferably, the flow ratio of SF6 and O2 is 3:4; preferably, the etching time using SF6 and O2 as etching gases is 100-150s, more preferably 120s.

6. The method according to claim 3, wherein: The method further comprises, before step (3), ultrasonically removing the residual microspheres on the top of the microneedle array.

7. The method according to claim 3, wherein: 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 surface of the microneedle array 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.

8. The method according to claim 3, wherein: The electrochemical method is a constant potential polarization method. Preferably, the potential of the constant potential polarization method is 0.1mV to 1mV, more preferably 0.6mV; 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; Preferably, the height of the microneedles of the surface-enhanced Raman scattering substrate is 50 to 1000 nm; preferably, the taper of the microneedles of the surface-enhanced Raman scattering substrate is 1:0.1 to 1:10; preferably, the distance between the axes of adjacent microneedles of the surface-enhanced Raman scattering substrate is 50 to 1500 nm.

9. A kit or device comprising the surface-enhanced Raman scattering substrate according to any one of claims 1 to 8.

10. Use of the kit or device according to claim 9 in preparing a product for detecting exosomes.

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