A SERS sensing platform for simultaneous and joint detection of exosomal proteins and its preparation method

Abstract

This invention belongs to the technical field of exosome protein detection, and provides a SERS sensing platform for simultaneous joint detection of exosome proteins and its preparation method, including the following steps: pretreatment of the initial sample by differential centrifugation; preparation of a magnetic bead-exosome complex, and labeling three proteins on the surface of exosomes with different SERS tags; in-situ deposition of silver nanoparticles on the immunomagnetic bead-exosome complex; preparation of a gold film array substrate for SERS detection, utilizing the synergistic enhancement effect of SERS through multi-level signal amplification to obtain the SERS signals corresponding to each protein on the exosome. This constructs a SERS sensing platform integrating separation, enrichment, and detection for highly sensitive detection of exosome proteins. It significantly improves the binding efficiency of exosomes to noble metal Raman probes, enhancing the feasibility of simultaneous joint detection of multiple proteins in the same sample. This method has high sensitivity and specificity, enabling high-throughput detection of exosome proteins.

Description

Technical Field

[0001] This invention belongs to the technical field of exosomal protein detection, specifically relating to a SERS sensing platform for simultaneous and combined detection of exosomal proteins and its preparation method. Background Technology

[0002] Exosomes are nanolipid vesicles secreted by cells, with a particle size of approximately 30-150 nm. Almost all cell types can secrete them, with tumor cells producing particularly high levels. Exosomes contain various bioactive molecules, including nucleic acids, lipids, and proteins, playing a crucial role in intercellular communication through information transfer. Tumor-derived exosomes, in particular, provide favorable conditions for tumor proliferation and metastasis by regulating the tumor microenvironment, promoting angiogenesis, assisting tumor cells in escaping immune surveillance, and enhancing tumor drug resistance. Exosomal proteins are closely related to tumor growth, invasion, and metastasis. For example, high-level expression of the exosomal protein CD97 in gastric cancer promotes angiogenesis and enhances the proliferation of gastric cancer cells; exosomal membrane proteins lectins α6β4 and α6β1 play key roles in lung metastasis, while lectin αvβ5 plays a crucial role in liver metastasis; and the SMAD3 protein in serum exosomes from liver cancer can promote cancer cell proliferation and lung metastasis. These findings all indicate that exosomal proteins play an important role in regulating tumor proliferation and metastasis. Compared with traditional tissue biopsy, exosome detection has the advantages of convenient, rapid, and minimally invasive sampling. Therefore, exosome proteins have the potential to become a new type of liquid biopsy target for tumors, providing novel biomarkers for early cancer diagnosis and metastasis monitoring.

[0003] Currently, methods for detecting exosome proteins include Western blotting, immunogold immunoassay, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. These methods not only require large sample volumes and have low sensitivity, but also necessitate complex sample pretreatment, making them difficult to implement and promote in clinical applications. Therefore, there is an urgent need to develop a detection method for exosome protein detection that is highly sensitive, specific, accurate, and requires no sample pretreatment.

[0004] Surface-enhanced Raman scattering (SERS) is a light scattering phenomenon that utilizes a rough metal surface to enhance the Raman signal of an analyte. This technique offers unique advantages such as high sensitivity, rich spectral information, no photobleaching required, no sample pretreatment needed, and the ability to perform in-situ, non-destructive testing. Furthermore, it boasts a rich source of Raman reporter molecules with narrow spectral peaks, allowing for the simultaneous labeling or encoding of multiple analytes, making it suitable for multi-component detection. In addition, some Raman reporter molecules contain specific functional groups, including cyano, alkynyl, and deuterated groups, which are present in the Raman silent region of 1800–2800 cm⁻¹. -1 These molecules can generate strong SERS signals. Using them as SERS tags can avoid background interference from complex biological endogenous substances, enabling the construction of background-free SERS sensing technology. Currently, this technology has been widely used in biomedical fields such as tumor cell identification, cell metabolite detection, tissue imaging, and nucleic acid or protein detection. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a SERS sensing platform for simultaneous joint detection of exosomal proteins and its preparation method. The SERS detection method is a multi-level signal amplification SERS sensing platform that integrates separation, enrichment and detection, enabling high-sensitivity and high-throughput joint detection of tumor exosomal proteins. This method is also used for the simultaneous joint detection and analysis of tumor-related proteins (EGFR, GPC-3, PD-L1) in exosomals derived from liver cancer cells.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for preparing a SERS sensing platform for simultaneous and joint detection of exosomal proteins, comprising the following steps:

[0007] (1) Pretreatment of samples by differential centrifugation: Collect cell supernatant and centrifuge at 300g for 10 min, 2000g for 10 min, and 10000g for 30 min to remove residual dead cells, cell debris, large vesicles and other impurities from the sample; all differential centrifugation steps are performed at 4℃; filter the supernatant obtained in the last step through a 0.22μm filter membrane and collect the filtrate for later use;

[0008] (2) Immunoglobin-magnetic beads capture exosomes: 1 mL of CD63 antibody-modified immunoglobin-magnetic beads with a concentration of 10 mg / mL was mixed with 50 mL of the pretreated sample obtained in step (1) and incubated at 4 °C for 2 h. The CD63 antibody specifically binds to the CD63 protein on the surface of the exosomes. The resulting mixture was separated by a magnet for 2 min to obtain the magnetic bead-exosome complex MB-Au@Exo, which was dispersed in 2.5 mL of 10 mM PBS.

[0009] (3) Simultaneous labeling of multiple exosomal proteins with different Raman tags: 1 mM of different Raman reporter molecules were coupled with 0.1 mg / mL of the corresponding antibody molecules as Raman tags. The Raman reporter molecules were 4-mercaptobenzonitrile, 4-nitrobenzenethiol and 4-ethynylbenzenethiol; the antibody molecules were EGFR antibody, GPC-3 antibody and PD-L1 antibody.

[0010] The Raman tags are: 4-mercaptobenzonitrile-EGFR antibody, 4-nitrobenzenethiol-GPC-3 antibody, and 4-ethynylbenzenethiol-PD-L1 antibody; then the three tags at the same concentration (250 μM) are simultaneously incubated with 1 mL of the magnetic bead-exosome complex obtained in step (2). After incubation at 4 °C for 1 h, the mixture is separated by a magnet for 1 min. The crude product is washed three times with 10 mM PBS and dispersed in 1 mL of 10 mM PBS.

[0011] (4) In-situ deposition of silver nanoparticles in immunomagnetic bead-exosome complex: 100 μM and 10 μL of distearate-PEG-SH (2k) were added to 1 mL of exosomes obtained in step (3), and the mixture was incubated together at 4 °C for 30 min. DSPE-PEG-SH was inserted into the lipid membrane of the exosomes to obtain thiol-coated exosomes. The mixture was then washed three times with 10 mM PBS and redispersed in 1 mL of PBS. Then, 10 mM dilute ammonia was added to adjust the pH of the system to 10. 10 μL of 100 mM silver nitrate solution and 10 μL of 250 mM ascorbic acid reducing agent were added, mixed evenly, and incubated at 37 °C for 1 h. Silver nanoparticles were deposited in situ on both the exosomes and the gold-shelled magnetic beads to obtain MB-Au@Exo@Ag.

[0012] (5) Preparation of gold film SERS array substrate: A positively charged anti-detachment glass slide with a size of 2cm×2.5cm was placed in 10mL of 3mM chloroauric acid solution and incubated for 5min. Then, 28% concentrated ammonia solution was added and the reaction was shaken for 1min. The slide was then washed 3 times with deionized water. The volume ratio of chloroauric acid solution to concentrated ammonia solution was 50:1. The slide was then placed in 1mM sodium borohydride solution and incubated for 1min to generate light red gold nanoseeds. The slide was then placed in an equal volume mixed growth solution of 1mL of 0.75mM chloroauric acid and 1mL of 0.75mM hydroxylamine hydrochloride and the reaction was shaken at room temperature for 10min to further form a large-area rough, island-shaped plasma gold film. Finally, the slide was assembled with the flexible PDMS array holes to obtain the gold film SERS array substrate.

[0013] (6) Detection of exosomal proteins based on SERS synergistic enhancement effect: 10 μL of MB-Au@Exo@Ag obtained in step (4) was added to the gold film SERS array obtained in step (5). After the sample was dried, it was detected by laser confocal Raman spectroscopy to obtain the Raman signal corresponding to the exosomal protein.

[0014] The immunogold-shelled magnetic beads described in step (2) serve as a capture probe, comprising a magnetic core of iron oxide with a particle size of 200 nm and a gold shell modified with CD63 antibody.

[0015] The specific preparation method of the immunogold-shelled magnetic beads is as follows: 1 mL of 20 mg / mL magnetic beads is added to 50 mL of aqueous solution, followed by 10 mg / mL and 5 mL of chloroauric acid solution and 60 mg / mL and 5 mL of sodium citrate solution, respectively. The mixture is refluxed at 100 °C for 30 min using a solvothermal method to obtain gold-coated magnetic beads. The beads are washed three times with ultrapure water and stored. Then, 20 mL of 1 mg / mL gold-shelled magnetic beads are modified with 2 mL of 0.2 mg / mL CD63 antibody in 10 mM PBS medium and incubated at 37 °C for 1 h. The immunogold-shelled magnetic beads are then separated by magnetism, washed three times with 10 mM PBS, and blocked with 1% bovine serum albumin.

[0016] The different Raman tags mentioned in step (3) include Raman reporter molecules and corresponding antibodies. The Raman reporter molecule has a thiol group at the end of its structure and has a benzene ring conjugated structure. N-succinimide-4-(maleimide-methyl)cyclohexane carboxylate SMCC is used as a crosslinking agent. The maleimide at one end of SMCC is coupled to the thiol group of the Raman reporter molecule through a click chemical reaction. The succinimide group at the other end of SMCC is coupled to the amino group in the antibody to obtain the Raman reporter molecule-antibody tag.

[0017] The specific preparation method is as follows: 1 mM Raman reporter molecule is mixed with 1.1 mM crosslinking agent N-succinimide-4-(maleimide-methyl)cyclohexanecarboxylate SMCC, and the mixture is stirred at room temperature for 1 h. The resulting intermediate product is incubated with 0.1 mg / mL of the corresponding antibody in PBS medium at 37 °C for 1 h. Then, the residual Raman reporter molecule, SMCC and intermediate product are removed using a 30 KD ultrafiltration tube.

[0018] The flexible PDMS array pores in step (5) are prepared by mixing polydimethylsiloxane PDMS with the matching curing agent at a mass ratio of 10:1, removing air bubbles from the mixture, pouring it into a glass mold with a mold size of 80cm×80cm, and incubating it in an oven at 100℃ for 2h; and punching holes in the prepared PDMS film with a pore diameter of 3mm.

[0019] The specific test conditions for using a laser confocal Raman spectrometer in step (6) are: 785nm laser, integration time of 10s, and laser power of 100%.

[0020] The present invention also provides a SERS sensing platform for simultaneous joint detection of exosomal proteins obtained by the preparation method described above.

[0021] This invention also provides the application of the SERS sensing platform in the simultaneous joint analysis and detection of tumor-associated proteins EGFR, GPC-3, and PD-L1.

[0022] In this invention, the thiol group, being an electron-donating group, has a strong affinity for metal ions and can effectively enrich silver nitrate molecules, generating silver nanoparticles in situ on exosomes as a SERS substrate. Simultaneously, silver nanoparticles are also generated on the surface of gold-shelled magnetic beads, forming a gold-silver composite substrate. This doped composite structure can interact with the nano-silver substrate on the exosome surface to generate numerous SERS "hot spots," exhibiting a strong synergistic Raman enhancement effect.

[0023] A large number of "hot spots" can be generated between the silver substrate on the exosome, the gold-silver composite substrate on the magnetic beads, and the solid-phase plasma gold film. This synergistic effect is conducive to forming a multi-level signal amplification SERS sensing platform, which significantly improves the detection sensitivity of the method.

[0024] This invention combines magnetic separation technology with multi-stage signal amplification SERS sensing technology to propose an exosomal protein analysis platform integrating separation, enrichment, and detection. Unlike traditional SERS techniques that label noble metal colloidal particles, this invention employs in-situ simultaneous deposition of silver nanoparticles as the SERS substrate on an exosome-magnetic bead complex. This significantly improves the problem of low labeling efficiency of noble metal colloidal particles due to the small size of exosomes and steric hindrance, which hinders the simultaneous joint detection of multiple proteins. Furthermore, by combining a rough gold film substrate, a sensing platform with a synergistic SERS enhancement effect is constructed, further improving the feasibility of detecting low-abundance targets. This method exhibits high sensitivity and specificity, enabling multi-channel and high-throughput detection of exosomal membrane proteins.

[0025] Compared with existing detection technologies, the present invention has the following advantages and beneficial effects:

[0026] High detection sensitivity: Exosomes exhibit high heterogeneity, with significant differences in the expression levels of various proteins on the exosome membrane. This method constructs a multi-stage signal amplification SERS sensing platform, which can generate a strong synergistic SERS enhancement effect, enabling the simultaneous detection of multiple proteins. Compared with traditional SERS technology for labeling noble metal colloidal particles, this method significantly improves the sensitivity of the detection method.

[0027] Fast separation speed: The method collects the target product through magnetic separation and removes residual impurities at each step throughout the experimental process, which greatly improves the detection efficiency.

[0028] High specificity: This method uses differential centrifugation and filtration membrane to remove impurities, captures exosomes with antibody-modified immunomagnetic beads, and labels exosomal proteins with antibody-modified Raman reporter molecules, which makes the method highly specific.

[0029] High scalability: This method is suitable for the simultaneous detection of multiple membrane proteins in exosomes from different cell or body fluid sources, requiring only the replacement of the specific antibodies corresponding to each protein.

[0030] Simultaneous joint detection of multiple proteins in exosomes was achieved: multiple specific Raman tags were pre-labeled on exosomes, and then silver nanoparticles were directly deposited on them in situ. Compared with the traditional method of mixing different kinds of noble metal Raman probes and then incubating them with exosomes, the steric hindrance effect can be significantly reduced, which is conducive to the simultaneous detection of proteins in multiple channels. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of a SERS sensing platform based on simultaneous joint detection of exosomal proteins in this invention.

[0032] Figure 2 This is a schematic diagram illustrating the use of immunogold-shelled magnetic beads to capture exosomes in this invention;

[0033] Figure 3 This is a structural characterization diagram of the gold-shelled magnetic bead-exosome complex in this invention;

[0034] Figure 4 This is a structural characterization diagram of the magnetic bead-exosome complex coated with silver nanoparticles in this invention;

[0035] Figure 5 The SERS spectra of the three Raman reporter molecules screened in this invention are shown.

[0036] Figure 6 This is a structural characterization of the plasma-coated gold film rough substrate in this invention;

[0037] Figure 7 This is a working curve of the analysis of exosomal proteins derived from liver cancer cells based on the simultaneous joint detection SERS sensing platform in this invention;

[0038] Figure 8 This is an interference experiment for detecting exosomal proteins based on the SERS sensing platform in this invention;

[0039] Figure 9The results of this invention, based on the SERS sensing platform, detected three exosomal proteins (GPC-3, EGFR, and PD-L1) from different cell lines.

[0040] Figure 10 To further verify the SERS detection results in this invention, the dot immunoblotting method is used. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0043] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0045] The CD63 antibody (ab134045), EGFR antibody (ab40815), GPC-3 antibody (ab216606), and PD-L1 antibody (ab243877) used in this invention were purchased from Abcam. The positively charged anti-detachment glass slides used were purchased from Huake, catalog number HK2143-46052. The distearate phosphatidylethanolamine-polyethylene glycol-mercapto (DSPE-PEG-SH, 2k) used were purchased from Huateng Pharmaceutical, catalog number LP096003-2K. The polydimethylsiloxane reagent used was from Dow Corning, catalog number DC184.

[0046] like Figure 1As shown, a method for preparing a SERS sensing platform based on simultaneous joint detection of exosomal proteins includes the following steps: First, sample pretreatment using differential centrifugation; second, exosomal capture using immunomagnetic beads with gold shells; third, multiplex exosomal proteins labeled using Raman tags; fourth, in-situ deposition of silver nanoparticles on the immunomagnetic bead-exosomal complex; fifth, preparation of a gold film SERS array; and sixth, detection of exosomal proteins based on the synergistic enhancement effect of SERS. The specific method is as follows:

[0047] (1) Sample pretreatment was performed using differential centrifugation: Cell supernatant was collected and centrifuged at 300g for 10 min, 2000g for 10 min, and 10000g for 30 min to remove residual dead cells, cell debris, large vesicles, and other impurities. The liquid sample was then filtered through a 0.22 μm filter membrane, and the filtrate was collected for later use. All centrifugation steps were performed at 4℃.

[0048] (2) Immunosensitive gold-shelled magnetic beads for exosome capture: 1 mL of CD63 antibody-modified gold-shelled magnetic beads (10 mg / mL) was incubated with 50 mL of pretreated sample. The CD63 antibody specifically binds to the abundant CD63 protein on the surface of exosomes. After incubation at 4°C for 2 h, the magnetic bead-exosome complex (MB-Au@Exo) was obtained by magnetic separation and dispersed in 2.5 mL of 10 mM PBS. The immunosensitive gold-shelled magnetic beads MB-Au have a core-shell structure, including an inner core layer and an outer shell layer. The inner core layer is composed of iron oxide nanoparticles, and the outer shell layer is composed of gold nanoshells. CD63 antibody is modified on the surface of the gold nanoshells as a capture probe.

[0049] (3) Simultaneous labeling of multiple exosomal proteins with different Raman tags: 1 mM of different Raman reporter molecules were coupled with 0.1 mg / mL of the corresponding antibody molecules as Raman tags, and then simultaneously incubated with captured exosomes. The Raman reporter molecule has a thiol group at the end and a benzene ring conjugated structure. N-succinimide-4-(maleimide-methyl)cyclohexane carboxylate (SMCC) was used as a cross-linking agent. The maleimide at one end of SMCC was coupled with the thiol group of the Raman reporter molecule through a click chemical reaction, and the succinimide group at the other end of SMCC was coupled with the amino group in the antibody to obtain the Raman reporter molecule-antibody tag. The Raman tag includes 4-mercaptobenzonitrile-EGFR antibody, 4-nitrophenylthiol-GPC-3 antibody, and 4-ethynylphenylthiol-PD-L1 antibody. The three tags at the same concentration (250 μM) were simultaneously incubated with 1 mL of magnetic bead-exosome complex at 4 °C for 1 h. After incubation, the mixture was separated by a magnet for 1 min, and the product (MB-Au@Exo@Raman tag) was obtained by magnetic separation.

[0050] (4) In-situ deposition of silver nanoparticles from immunomagnetic bead-exosome complex: 100 μM and 10 μL of distearate-PEG-SH (2k) were added to 1 mL of exosomes obtained in step (3), and incubated together at 4 °C for 30 min. The DSPE-PEG-SH was inserted into the exosome lipid membrane to obtain thiol-coated exosomes, which were then washed three times with 10 mM PBS and redispersed in 1 mL of PBS. Then, 10 mM dilute ammonia was added to adjust the pH of the system to 10, and 10 μL of 100 mM silver nitrate solution and 10 μL of 250 mM ascorbic acid reducing agent were added, mixed evenly, and incubated at 37 °C for 1 h. Among them, the thiol group is an electron-donating group with a good affinity for metal ions, which can effectively enrich silver nitrate molecules and generate silver nanoparticles in situ on the exosomes as a SERS substrate. Simultaneously, silver nanoparticles were also generated on the surface of the gold-shelled magnetic beads, forming a gold-silver composite substrate. This doped composite structure can interact with the in-situ silver substrate of exosomes to generate a large number of SERS "hot spots," exhibiting a strong synergistic Raman enhancement effect. Finally, silver nanoparticles were simultaneously deposited in situ on both the exosomes and the gold-shelled magnetic beads to obtain MB-Au@Exo@Ag.

[0051] (5) Preparation of gold film SERS array substrate: A positively charged anti-detachment glass slide was placed in 10 mL of 3 mM chloroauric acid solution and incubated for 5 min. Then, 28% concentrated ammonia solution was added and the reaction was shaken for 1 min. The slide was then washed with deionized water. The volume ratio of chloroauric acid solution to concentrated ammonia solution was 50:1. The slide was then placed in 1 mM sodium borohydride solution and incubated for 1 min to generate light red gold nanoseeds. The slide was then washed with deionized water. The slide was then placed in 1 mL of 0.75 mM sodium borohydride solution and incubated for 1 min to generate light red gold nanoseeds. The slide was then washed with deionized water. A large-area, rough, island-shaped plasma gold film was prepared by mixing chloroauric acid with an equal volume of 1 mL of 0.75 mM hydroxylamine hydrochloride in a growth solution and reacting with shaking at room temperature for 10 min. This film was then assembled with a flexible PDMS array to obtain a gold film SERS array. The flexible PDMS array was prepared by adding a curing agent to polydimethylsiloxane at a mass ratio of 10:1, removing air bubbles from the mixture, pouring it into a mold, and heating it in an oven at 100°C for 2 h. The prepared PDMS film was then perforated to a pore size of 3 mm.

[0052] (6) Detection of exosomal proteins based on SERS synergistic enhancement effect: 10 μL of MB-Au@Exo@Ag obtained in step (4) was added to the gold film SERS array obtained in step (5). After the sample dried, it was detected by laser confocal Raman spectroscopy to obtain the Raman signal corresponding to the exosomal protein. A large number of SERS "hot spots" can be constructed between the silver substrate on the exosome, the gold-silver composite substrate on the magnetic beads, and the plasma gold film. This synergistic effect is conducive to forming a multi-level signal amplification SERS sensing platform, which significantly improves the detection sensitivity of the method. Detection was performed using laser confocal Raman spectroscopy. The test conditions were: 785nm laser, integration time of 10s, and laser power of 100%.

[0053] Figure 2 This is a schematic diagram illustrating the use of immunografted magnetic beads to capture exosomes in this invention. First, CD63 antibody is modified onto the gold-coated magnetic beads, which are then blocked with bovine serum albumin to reduce non-specific adsorption. Then, this immunograft probe is added to the pretreated sample, allowing it to specifically bind to the CD63 protein on the surface of the exosomes, enabling rapid separation of the exosomes using a magnet.

[0054] Figure 3 This is a structural characterization diagram of the gold-shelled magnetic bead-exosome complex in this invention. The exosomes captured by the immunomagnetic beads are shown below. Figure 3 As shown in Figure a, exosomes attached to the surface of magnetic beads can be observed under a transmission electron microscope. The captured exosomes were then analyzed using Western blotting, with the parental cell line serving as a control. Figure 3 The exosomes shown in c are rich in CD63 protein and also contain Alix protein, but do not express Calnexin protein, while cells do not express Alix protein but contain Calnexin protein. Figure 3 b shows the exosome capture process characterized by dynamic light scattering. The average particle size of the bare magnetic beads is approximately 210 nm, and the particle size of the immunomagnetic beads is approximately 240 nm. Compared to exosomes and magnetic beads alone, the average particle size of the exosome-magnetic bead complex increases to 606 nm. These experimental results further demonstrate that exosomes are successfully captured by immunomagnetic beads.

[0055] Figure 4 This is a structural characterization of the magnetic bead-exosome complex coated silver nanoparticles in this invention. Figure 4 a is the UV-Vis spectrum. The absorption peak at 414 nm in the figure originates from the silver nanoparticles, and the absorption peak at 515 nm originates from the gold shell on the surface of the magnetic beads. Figure 4 b is a transmission electron microscope image of silver nanoparticles deposited on the surface of exosomes.

[0056] Figure 5The images show the SERS spectra of the three Raman reporter molecules screened in this invention. By detecting a series of conjugated organic molecules with terminal thiol groups, 4-mercaptobenzonitrile (4-MBN, characteristic peak: 2220 cm⁻¹) was finally selected. -1 ), 4-nitrobenzenethiol (4-NTP, characteristic peak: 1330 cm⁻¹) -1 ), 4-ethynylbenzenethiol (SH-alkyne, characteristic peak: 2100 cm⁻¹) -1 As a Raman reporter molecule, the three Raman characteristic peaks do not overlap and are used for labeling three corresponding antibodies.

[0057] Figure 6 This is a structural characterization of the rough gold film substrate in this invention. For example... Figure 6 As shown in the scanning electron microscope image in (a), the surface of the plasma-encapsulated gold film exhibits a uniform and dense distribution of nano-island structures, with a strong and broad UV-Vis absorption peak at 655 nm. Figure 6 b.

[0058] Figure 7 This is a working curve of the SERS sensing platform based on simultaneous joint detection used in this invention to analyze exosomal proteins derived from liver cancer cells. Figure 7 a represents exosomes (5.11 × 10⁻⁶) 9 Representative SERS spectra obtained from particles / mL. At 5.11 × 10⁻⁶. 3 ~5.11×10 9 Within the particle / mL range Figure 7 b is the choice of 1330 cm -1 The Raman signal at the location was used as the target signal for the GPC-3 protein, and the resulting working curve was Y = 1397.3X - 3989.6, with a linear correlation coefficient R. 2 =0.991, and the detection limit is 83 particles / mL. Figure 7 c represents the selection of 2220 cm. -1 The Raman signal at the target location was used as the target signal for the EGFR protein, resulting in the working curve Y = 347.2X - 925.5, with a linear correlation coefficient R. 2 =0.985, detection limit is 96 particles / mL. Figure 7 d represents the selection of 2100 cm -1 The Raman signal at the location was used as the target signal for the PD-L1 protein, and the resulting working curve was Y = 315.7X - 851.3, with a linear correlation coefficient R. 2 =0.981, and the detection limit is 116 particles / mL.

[0059] Figure 8This invention presents an interference experiment for detecting exosomal proteins based on the SERS sensing platform. Taking the detection of GPC-3 protein in HepG2 exosomes as an example, this study evaluates the interference of commonly present interfering substances in body fluids, including proteins, carbohydrates, and amino acids, on SERS detection results. As shown in the figure, the average SERS signal intensities corresponding to each group (human serum albumin (HSA, 100 μg / mL), immunoglobulin (IgG, 100 μg / mL), lysozyme (50 μg / mL), glucose (10 mM), glycine (10 mM), and cysteine ​​(10 mM)) ranged from 251 to 574, similar to the SERS signal of the blank group, but significantly different from the signal intensity of HepG2-derived exosomes (t-test, P < 0.01). This indicates that the method has good selectivity for exosomal protein detection and is not easily interfered with by other substances.

[0060] Figure 9 This invention utilizes the SERS sensing platform to detect three exosomal proteins (GPC-3, EGFR, and PD-L1) from different cell lines. As shown in the figure, all three proteins were negatively expressed in the blank control group and LO2 exosomes derived from normal hepatocytes. In exosomes derived from the HepG2 hepatocellular carcinoma line, the HeLa cervical cancer line, and non-small cell lung cancer (NSCLC), EGFR and PD-L1 were positively expressed, while GPC-3 was specifically expressed in HepG2 exosomes. This preliminary demonstration shows that this method can be used to differentiate between exosomes from normal cell lines and tumor cell lines, and can improve the selectivity of HepG2 exosome detection.

[0061] Further employing the standard addition method, exosomes in actual simulated samples were detected using a SERS sensing platform for simultaneous joint detection of exosomal proteins. The medium for the simulated samples was human serum albumin dissolved in 10 mM PBS solution. Taking the detection of GPC-3 protein in exosomes as an example, exosome standards obtained by ultracentrifugation were added to the same volume (50 mL) and concentration (10 mg / mL) of human serum albumin solution in each group, resulting in spiked samples with different concentration gradients (5 × 10⁻⁶ mM PBS). 4 ~5×10 7 The particles / mL were detected using the SERS sensing platform described in this invention. Each experiment was repeated three times, and the corresponding recovery rate and relative standard deviation for each group were calculated.

[0062] The results are shown in Table 1. Table 1 shows the detection of exosomes in actual simulated samples by the SERS sensing platform based on simultaneous joint detection of exosome proteins in this invention, in order to evaluate the detection effect of this method on actual samples.

[0063] Table 1: Exosomes detected in simulated samples based on the SERS sensing platform in this invention.

[0064]

[0065] Table 1 shows the detection of exosomes in simulated samples using the SERS sensing platform based on simultaneous joint detection of exosome proteins in this invention. When the concentration of exosome standard added was 5.11 × 10⁻⁶... 4 ~5.11×10 7 When the particle count is 95.6% to 108.7%, the recovery rate of the method of the present invention is within the allowable range of spiked recovery rate, and the relative standard deviation is within the acceptable range of 2.89% to 4.53%, indicating that the method of the present invention has potential application value in actual sample detection.

[0066] Figure 10 To further validate the SERS detection results in this invention, dot immunoblotting was used. In-situ dot immunoblotting analysis was performed on the silver nanoparticle-coated magnetic bead-exosome complex, revealing the formation of EGFR, GPC-3, and PD-L1 blots on its surface. Exosomes alone, as a positive control, also produced blots for these three proteins. Gold-shelled magnetic beads, as a negative control, showed no protein blots. This result further demonstrates that EGFR, GPC-3, and PD-L1 are positively expressed on HepG2 exosomes, consistent with the SERS detection results.

[0067] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a SERS sensing platform for simultaneous and joint detection of exosomal proteins, characterized in that: Includes the following steps (1) Pretreatment of samples by differential centrifugation: Collect cell supernatant and centrifuge at 300g for 10 min, 2000g for 10 min, and 10000g for 30 min to remove residual dead cells, cell debris, and large vesicle impurities from the sample; all differential centrifugation steps are performed at 4℃; filter the supernatant obtained in the last step through a 0.22μm filter membrane and collect the filtrate for later use; (2) Immunoglobin magnetic beads capture exosomes: 1 mL of immunoglobin magnetic beads modified with 10 mg / mL CD63 antibody was mixed with 50 mL of the pretreated sample obtained in step (1) and incubated at 4 °C for 2 h. The CD63 antibody specifically binds to the CD63 protein on the surface of the exosomes. The mixture was separated by a magnet for 2 min to obtain the magnetic bead-exosome complex MB-Au@Exo, which was dispersed in 2.5 mL of 10 mM PBS. (3) Simultaneous labeling of multiple exosomal proteins with different Raman tags: 1 mM of different Raman reporter molecules were coupled with 0.1 mg / mL of the corresponding antibody molecules as Raman tags. The Raman reporter molecules were 4-mercaptobenzonitrile, 4-nitrobenzenethiol and 4-ethynylbenzenethiol; the antibody molecules were EGFR antibody, GPC-3 antibody and PD-L1 antibody. The Raman tags are: 4-mercaptobenzonitrile-EGFR antibody, 4-nitrobenzenethiol-GPC-3 antibody, and 4-ethynylbenzenethiol-PD-L1 antibody; then the three tags at the same concentration of 250 μM are simultaneously incubated with 1 mL of the magnetic bead-exosome complex obtained in step (2). After incubation at 4°C for 1 h, the mixture is separated by a magnet for 1 min. The crude product is washed three times with 10 mM PBS and dispersed in 1 mL of 10 mM PBS. (4) In-situ deposition of silver nanoparticles in immunomagnetic bead-exosome complex: 100 μM, 10 μL of distearate-PEG-SH, 2k, was added to 1 mL of exosomes obtained in step (3). The mixture was incubated at 4 °C for 30 min. DSPE-PEG-SH was inserted into the lipid membrane of the exosomes to obtain thiol-coated exosomes. The mixture was then washed three times with 10 mM PBS and redispersed in 1 mL of PBS. 10 mM dilute ammonia was added to adjust the pH of the system to 10. 10 μL of 100 mM silver nitrate solution and 10 μL of 250 mM ascorbic acid reducing agent were added and mixed evenly. The mixture was incubated at 37 °C for 1 h to deposit silver nanoparticles in situ on both the exosomes and the gold-shelled magnetic beads to obtain MB-Au@Exo@Ag. (5) Preparation of gold film SERS array substrate: A positively charged anti-detachment glass slide with a size of 2cm×2.5cm was placed in 10mL of 3mM chloroauric acid solution and incubated for 5min. Then, 28% concentrated ammonia solution was added and the reaction was shaken for 1min. The slide was then washed 3 times with deionized water. The volume ratio of chloroauric acid solution to concentrated ammonia solution was 50:

1. The slide was then placed in 1mM sodium borohydride solution and incubated for 1min to generate light red gold nanoseeds. The slide was then placed in an equal volume mixed growth solution of 1mL of 0.75mM chloroauric acid and 1mL of 0.75mM hydroxylamine hydrochloride and the reaction was shaken at room temperature for 10min to further form a large-area rough, island-shaped plasma gold film. Finally, the slide was assembled with the flexible PDMS array holes to obtain the gold film SERS array substrate. (6) Detection of exosomal proteins based on SERS synergistic enhancement effect: 10 μL of MB-Au@Exo@Ag obtained in step (4) was added to the gold film SERS array obtained in step (5). After the sample was dried, it was detected by laser confocal Raman spectroscopy to obtain the Raman signal corresponding to the exosomal protein.

2. The method for preparing a SERS sensing platform for simultaneous and joint detection of exosomal proteins according to claim 1, characterized in that: The immunogold-shelled magnetic beads described in step (2) serve as a capture probe, comprising a magnetic core of magnetite with a particle size of approximately 200 nm and a gold shell modified with CD63 antibody. The specific preparation method of the immunogold-shelled magnetic beads is as follows: 1 mL of 20 mg / mL magnetic beads is added to 50 mL of aqueous solution, followed by 10 mg / mL and 5 mL of chloroauric acid solution and 60 mg / mL and 5 mL of sodium citrate solution, respectively. The mixture is refluxed at 100 °C for 30 min using a solvothermal method to obtain gold-coated magnetic beads. The beads are washed three times with ultrapure water and stored. Then, 20 mL of 1 mg / mL gold-shelled magnetic beads are modified with 2 mL of 0.2 mg / mL CD63 antibody in 10 mM PBS medium and incubated at 37 °C for 1 h. The immunogold-shelled magnetic beads are then separated by magnetism and washed three times with 10 mM PBS, followed by blocking with 1% bovine serum albumin.

3. The method for preparing a SERS sensing platform for simultaneous joint detection of exosomal proteins according to claim 1, characterized in that: The different Raman tags mentioned in step (3) include Raman reporter molecules and corresponding antibodies. The Raman reporter molecule has a thiol group at the end of its structure and has a benzene ring conjugated structure. N-succinimide-4-(maleimide-methyl)cyclohexane carboxylate SMCC is used as a crosslinking agent. The maleimide at one end of SMCC is coupled to the thiol group of the Raman reporter molecule through a click chemical reaction. The succinimide group at the other end of SMCC is coupled to the amino group in the antibody to obtain the Raman reporter molecule-antibody tag. The specific preparation method is as follows: 1 mM Raman reporter molecule is mixed with 1.1 mM crosslinking agent N-succinimide-4-(maleimide-methyl)cyclohexanecarboxylate SMCC, and the mixture is stirred at room temperature for 1 h. The resulting intermediate product is incubated with 0.1 mg / mL of the corresponding antibody in PBS medium at 37 °C for 1 h. Then, the residual Raman reporter molecule, SMCC and intermediate product are removed using a 30 KD ultrafiltration tube.

4. The method for preparing a SERS sensing platform for simultaneous joint detection of exosomal proteins according to claim 1, characterized in that: The flexible PDMS array pores in step (5) are prepared by mixing polydimethylsiloxane PDMS with a matching curing agent at a mass ratio of 10:1, removing air bubbles from the mixture, pouring it into a glass mold with a mold size of 80cm×80cm, and heating it in an oven at 100℃ for 2 hours; and punching holes in the prepared PDMS film with a pore diameter of 3mm.

5. The method for preparing a SERS sensing platform for simultaneous joint detection of exosomal proteins according to claim 1, characterized in that: The specific test conditions for using a laser confocal Raman spectrometer in step (6) are: 785 nm laser, integration time of 10 s, and laser power of 100%.

6. A SERS sensing platform for simultaneous joint detection of exosomal proteins obtained by any of the preparation methods described in claims 1-5.

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