SERS (Surface Enhanced Raman Scattering) sensing platform for synchronously and jointly detecting exosome protein and preparation method of SERS sensing platform

Abstract

The invention belongs to the technical field of exosome protein detection, and provides an SERS (Surface Enhanced Raman Scattering) sensing platform for synchronously and jointly detecting exosome protein and a preparation method of the SERS sensing platform. Preparing a magnetic bead-exosome compound, and respectively marking the three proteins on the surface of the exosome by using different SERS tags; depositing silver nanoparticles in situ on the immunomagnetic bead-exosome compound; preparing a gold film array substrate for SERS (Surface Enhanced Raman Scattering) detection, and obtaining SERS signals corresponding to each protein on the exosome by utilizing an SERS synergistic enhancement effect of multistage signal amplification. A SERS sensing platform integrating separation, enrichment and detection is constructed and is used for high-sensitivity detection of exosome protein. The binding efficiency of the exosome and the precious metal Raman probe is remarkably improved, and the feasibility of synchronous combined detection of multiple proteins in the same sample is improved. The method is high in detection sensitivity and good in specificity, and high-throughput detection of exosome protein can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome protein detection, and specifically relates to a SERS sensing platform for synchronously and jointly detecting exosome proteins and a preparation method thereof. Background Art

[0002] Exosomes are nano-lipid vesicles secreted by cells, with a particle size of about 30-150 nm. They can be secreted by almost all types of cells, especially in tumor cells, where the production of exosomes is very high. Exosomes contain a variety of bioactive molecules, including nucleic acids, lipids, proteins, etc., which transmit information between cells and play a major role in intercellular communication. In particular, tumor-derived exosomes provide favorable conditions for tumor proliferation and metastasis in regulating the tumor microenvironment, promoting angiogenesis, assisting tumor cells to escape immune surveillance, and enhancing tumor drug resistance. Among them, exosome proteins are closely related to tumor growth, invasion and metastasis. For example, the high level of expression of exosomal protein CD97 in gastric cancer can promote angiogenesis and increase the proliferation ability of gastric cancer cells; exosomal membrane proteins lectins α6β4 and α6β1 play a key role in lung metastasis, and lectin αvβ5 plays a key role in liver metastasis; SMAD3 protein in liver cancer serum exosomes can promote cancer cell proliferation and lung metastasis. These research results all indicate that exosome proteins play an important role in regulating tumor proliferation and metastasis. Compared with traditional tissue biopsy, exosome detection has the advantages of convenient and rapid sampling and less damage. Therefore, exosome proteins are expected to become a new type of tumor liquid biopsy object, providing a new marker for early diagnosis of cancer and metastasis monitoring.

[0003] At present, the detection methods of exosome proteins include protein immunoblotting analysis, immunocolloidal gold method, enzyme-linked immunosorbent assay, flow cytometry, etc. These methods not only require a large sample volume and low sensitivity, but also require further complex sample pretreatment, making it difficult to promote them in clinical applications. Therefore, it is urgent to develop a detection method with high sensitivity, good specificity, high accuracy, and no sample pretreatment required for exosome protein detection.

[0004] Surface-enhanced Raman scattering (SERS) is a light scattering phenomenon that uses a rough metal surface to enhance the Raman signal of the object to be detected. This technology has the unique advantages of high sensitivity, rich spectral information, no photobleaching, and no need for sample pretreatment. It can realize in-situ and non-destructive detection of samples. In addition, the source of Raman reporter molecules is abundant, the spectral peak is narrow, and multiple objects to be detected can be labeled or encoded at the same time, which is suitable for multi-component detection. In addition, some Raman reporter molecules contain specific functional groups, including cyano, alkynyl, deuterated groups, etc., in the Raman silent zone of 1800-2800 cm -1 The internal energy can generate a strong SERS signal. Using this type of molecule as a SERS tag can avoid the background interference of complex biological endogenous substances and construct a background-free SERS sensing technology. It has been widely used in biomedical fields such as tumor cell identification, cell metabolite detection, tissue imaging, nucleic acid or protein detection, etc. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a SERS sensing platform for synchronous joint detection of exosome proteins and a preparation method thereof. The SERS detection method is a SERS sensing platform with multi-stage signal amplification integrating separation, enrichment and detection, and performs high-sensitivity and high-throughput joint detection of tumor exosome proteins. The method is used for the synchronous joint detection and analysis of tumor-related proteins (EGFR, GPC-3, PD-L1) in exosomes derived from liver cancer cells.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for preparing a SERS sensing platform for simultaneous joint detection of exosome proteins, comprising the following steps: (1) Pre-treat the sample by differential centrifugation: collect the cell supernatant and perform differential centrifugation at 300g for 10 min, 2000g for 10 min, and 10000g for 30 min to remove impurities such as dead cells, cell debris, and large vesicles remaining in the sample; all differential centrifugation steps were performed at 4°C; the supernatant obtained in the last step was filtered using a 0.22 μm filter membrane, and the filtrate was collected for later use; (2) Capturing exosomes with immunogold-shell magnetic beads: 1 mL of 10 mg / mL CD63 antibody-modified immunogold-shell magnetic beads 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. (3) Simultaneous labeling of multiple exosomal proteins with different Raman labels: 1 mM of different Raman reporter molecules were coupled with 0.1 mg / mL of corresponding antibody molecules as Raman labels. 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 labels are: 4-mercaptobenzonitrile-EGFR antibody, 4-nitrobenzenethiol-GPC-3 antibody, 4-ethynylbenzenethiol-PD-L1 antibody; then the three labels of the same concentration (250 μM) are co-incubated with 1 mL of the magnetic bead-exosome complex obtained in step (2), incubated at 4° C. for 1 hour, separated by a magnet for 1 minute, and the obtained crude product is washed three times with 10 mM PBS and dispersed in 1 mL, 10 mM PBS; (4) In situ deposition of silver nanoparticles on the immunomagnetic bead-exosome complex: Add 100 μM, 10 μL of distearoylphosphatidylethanolamine-polyethylene glycol-thiol (DSPE-PEG-SH, 2k) to 1 mL of exosomes obtained in step (3), and incubate at 4°C for 30 min. DSPE-PEG-SH is inserted into the exosome lipid membrane to obtain thiol-coated exosomes; then wash three times with 10 mM PBS and redisperse in 1 mL PBS; then add 10 mM dilute ammonia water to adjust the pH of the system to 10; add 10 μL of 100 mM silver nitrate solution and 10 μL of 250 mM ascorbic acid reducing agent, mix well, and incubate at 37°C for 1 h. Silver nanoparticles are simultaneously deposited on the exosomes and gold shell magnetic beads in situ to obtain MB-Au@Exo@Ag; (5) Preparation of gold film SERS array substrate: Place a 2 cm × 2.5 cm positively charged anti-shedding glass slide in 10 mL, 3 mM chloroauric acid solution and incubate for 5 min, then add 28% concentrated ammonia water for oscillation reaction for 1 min, and then wash with deionized water for 3 times, wherein the volume ratio of chloroauric acid solution to concentrated ammonia water is 50:1; then place the glass slide in 1 mM sodium borohydride solution for 1 min to generate light red gold nanoseeds; then place it in an equal volume mixed growth solution of 1 mL, 0.75 mM chloroauric acid and 1 mL, 0.75 mM hydroxylamine hydrochloride, and oscillate for 10 min at room temperature to further form a large area of ​​rough, island-shaped plasma gold film; finally, assemble it with the flexible PDMS array holes to obtain a gold film SERS array substrate; (6) Detection of exosome 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 using a laser confocal Raman spectrometer to obtain the Raman signal corresponding to the exosome protein.

[0007] The immunogold-shell magnetic beads described in step (2) are used as capture probes, comprising a magnetic core of ferroferric oxide with a particle size of 200 nm and a gold-shell outer layer modified with CD63 antibodies; The specific preparation method of the immune gold shell magnetic beads is as follows: 1 mL of 20 mg / mL magnetic beads is added to 50 mL of aqueous solution, and then 10 mg / mL and 5 mL of chloroauric acid solution and 60 mg / mL and 5 mL of sodium citrate solution are added respectively, and a solvent thermal method is used to reflux for 30 minutes at 100° C. to obtain gold shell-coated magnetic beads, and the gold shell-coated magnetic beads are washed three times with ultrapure water for storage; then 2 mL and 0.2 mg / mL of CD63 antibody are used to modify 20 mL and 1 mg / mL of gold shell magnetic beads in a 10 mM PBS medium, and incubated at 37° C. for 1 hour; then the immune gold shell magnetic beads are separated by a magnet, and washed three times with 10 mM PBS, and then blocked with 1% by mass bovine serum albumin; The different Raman tags in step (3) include a Raman reporter molecule and a corresponding antibody, wherein the Raman reporter molecule structure contains a thiol group at the end and has a benzene ring conjugated structure, and N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate SMCC is used as a cross-linking agent, and the maleimide at one end of SMCC is coupled with the thiol group of the Raman reporter molecule through a click chemistry reaction, and the succinimide group at the other end of SMCC is coupled with the amino group in the antibody to obtain a Raman reporter molecule-antibody tag; The specific preparation method is as follows: 1 mM Raman reporter molecule is mixed with 1.1 mM cross-linker N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate SMCC, and the reaction is stirred at room temperature for 1 hour. The obtained intermediate product is incubated with 0.1 mg / mL of the corresponding antibody in PBS medium at 37°C for 1 hour; then a 30KD ultrafiltration tube is used to remove the residual Raman reporter molecule, SMCC and intermediate product.

[0008] The flexible PDMS array holes in step (5) are as follows: polydimethylsiloxane PDMS and a matching curing agent are mixed at a mass ratio of 10:1, and after removing bubbles in the mixed system, the mixture is poured into a glass mold with a size of 80 cm×80 cm, placed in an oven at 100°C, and incubated for 2 hours; the prepared PDMS membrane is punched with holes with a hole diameter of 3 mm.

[0009] The specific test conditions for the detection using the laser confocal Raman spectrometer in step (6) are: 785 nm laser, integration time of 10 s, and laser power of 100%.

[0010] The present invention also provides a SERS sensing platform for synchronously and jointly detecting exosome proteins obtained by the preparation method.

[0011] The present invention also provides an application of the SERS sensing platform in the simultaneous combined analysis and detection of tumor-related proteins EGFR, GPC-3, and PD-L1.

[0012] In the detection process of the present invention, the thiol group is an electron-donating group with good affinity for metal ions, which can effectively enrich silver nitrate molecules and generate silver nanoparticles in situ in the exosomes as SERS substrates. At the same time, silver nanoparticles are also generated on the surface of the gold-shell magnetic beads to form a gold-silver composite substrate. This doped composite structure can work together with the nanosilver substrate on the surface of the exosomes to generate a large number of SERS "hot spots", which has a strong synergistic Raman enhancement effect.

[0013] A large number of "hot spots" can be generated between the silver substrate on the exosomes, the gold-silver composite substrate on the magnetic beads, and the solid-phase plasma gold film. This synergistic effect is conducive to the formation of a SERS sensing platform with multi-level signal amplification, which significantly improves the detection sensitivity of this method.

[0014] The present invention combines magnetic separation technology with SERS sensing technology with multi-stage signal amplification, and proposes an exosome protein analysis platform that integrates separation, enrichment and detection. The inventive method is different from the traditional SERS technology of labeling precious metal colloidal particles. It uses silver nanoparticles deposited in situ on the exosome-magnetic bead complex as the SERS substrate, which significantly improves the problem of low labeling efficiency of precious metal colloidal particles due to the small size of exosomes and the existence of steric hindrance, making it difficult to achieve simultaneous joint detection of multiple proteins. In addition, further combined with a rough gold film substrate, a sensing platform with a synergistic SERS enhancement effect is constructed, which further improves the feasibility of detecting low-abundance targets. The method has high sensitivity and good specificity, and can achieve multi-channel and high-throughput detection of exosome membrane proteins.

[0015] Compared with the existing detection technology, the present invention has the following advantages and beneficial effects: High detection sensitivity: Exosomes are highly heterogeneous, and the expression levels of various proteins on the exosome membrane vary greatly. This method constructs a SERS sensing platform with multi-level signal amplification, which can produce a strong synergistic SERS enhancement effect and achieve simultaneous detection of multiple proteins. Compared with the traditional SERS technology of labeling precious metal colloidal particles, the sensitivity of the detection method is significantly improved.

[0016] Fast separation speed: This method collects the target product through magnetic separation throughout the entire experimental process and removes residual impurities at each step, which greatly improves the detection efficiency.

[0017] High specificity: This method uses differential centrifugation and filtration membrane to remove impurities, captures exosomes through antibody-modified immunomagnetic beads, and labels exosomal proteins with antibody-modified Raman reporter molecule tags, making this method highly specific.

[0018] Strong scalability: This method is suitable for the simultaneous detection of multiple membrane proteins in exosomes from different cells or body fluids, and only requires replacing the specific antibodies corresponding to each protein.

[0019] The simultaneous joint detection of multiple proteins in exosomes was achieved: a variety of specific Raman tags were pre-labeled on the exosomes, and then the silver nanosubstrate was directly deposited in situ. Compared with the traditional method of mixing different types of precious metal Raman probes and then incubating them with exosomes, this can significantly reduce the steric effect and is conducive to the multi-channel simultaneous detection of proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of a SERS sensing platform based on simultaneous joint detection of exosome proteins in the present invention; Figure 2 Schematic diagram of using immune gold shell magnetic beads to capture exosomes in the present invention; Figure 3 This is a structural representation diagram of the gold shell magnetic bead-exosome complex in the present invention; Figure 4 This is a structural characterization diagram of the magnetic bead-exosome complex coated with silver nanoparticles in the present invention; Figure 5 The SERS spectra of three Raman reporter molecules screened in the present invention; Figure 6 It is a structural characterization of the rough substrate of the plasma gold film in the present invention; Figure 7 This is a working curve diagram of analyzing exosome proteins derived from liver cancer cells based on the synchronous combined detection SERS sensing platform in the present invention; Figure 8 This is an interference experiment for detecting exosome proteins based on the SERS sensing platform in the present invention; Fig. 9 The results of the present invention using the SERS sensing platform to detect three exosomal proteins (GPC-3, EGFR, and PD-L1) from different cell lines; Fig.10 The dot immunoblotting method was used in the present invention to further verify the SERS detection results. DETAILED DESCRIPTION

[0021] In order to make the purpose, 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 part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0022] Unless defined otherwise, 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 belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0023] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] The CD63 antibody (ab134045), EGFR antibody (ab40815), GPC-3 antibody (ab216606), and PD-L1 antibody (ab243877) used in the present invention were purchased from Abcam, the positively charged anti-shedding slide used was purchased from Chemical Science, item number HK2143-46052, the distearoylphosphatidylethanolamine-polyethylene glycol-thiol (DSPE-PEG-SH, 2k) used was purchased from Huateng Pharmaceutical, item number LP096003-2K, and the polydimethylsiloxane reagent used (DOW CORNING / Dow Corning) was item number DC184.

[0026] like Figure 1 As shown, a preparation method of a SERS sensing platform based on simultaneous joint detection of exosome proteins includes the following steps: the first step: pre-treating the sample by differential centrifugation; the second step: capturing exosomes by immunogold shell magnetic beads; the third step: labeling multiple exosome proteins by Raman tags; the fourth step: in situ deposition of silver nanoparticles on the immunomagnetic bead-exosome complex; the fifth step: preparing a gold film SERS array; the sixth step: detecting exosome proteins based on the SERS synergistic enhancement effect. The specific method is as follows: (1) Pre-treat the sample by differential centrifugation: collect the cell supernatant and perform differential centrifugation, 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. Then filter the liquid sample with a 0.22 μm filter membrane and collect the filtrate for later use. The centrifugation steps are all performed at 4°C.

[0027] (2) Immunogold-shell magnetic beads to capture exosomes: Gold-shell magnetic beads (1 mL, 10 mg / mL) modified with CD63 antibodies were incubated with 50 mL of pretreated samples. CD63 antibodies can specifically bind to the abundant CD63 protein on the surface of exosomes. After incubation at 4°C for 2 hours, the magnetic bead-exosome complex (MB-Au@Exo) was separated by magnet and dispersed in 2.5 mL of 10 mM PBS. The immune gold-shell magnetic beads MB-Au are core-shell structures, including an inner core layer and an outer shell layer. The inner core layer is ferroferric oxide nanoparticles, and the outer shell layer is a gold nanoshell. CD63 antibodies are modified on the surface of the gold nanoshell as a capture probe.

[0028] (3) Simultaneous labeling of multiple exosome proteins with different Raman tags: 1mM different Raman reporter molecules were coupled with 0.1mg / mL corresponding antibody molecules as Raman tags, and then incubated with the captured exosomes at the same time. The Raman reporter molecule structure contains a thiol group at the end and has a benzene ring conjugated structure. N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC) is used as a cross-linker. The maleimide at one end of SMCC is coupled with the thiol group of the Raman reporter molecule through a click chemistry reaction, and the succinimide group at the other end of SMCC is coupled with the amino group in the antibody to obtain a Raman reporter molecule-antibody tag. The Raman tags include 4-mercaptobenzonitrile-EGFR antibody, 4-nitrobenzenethiol-GPC-3 antibody, and 4-ethynylbenzenethiol-PD-L1 antibody. The three tags of the same concentration (250 μM) were incubated with 1 mL of magnetic beads-exosome complex at the same time. After incubation at 4°C for 1 hour, they were separated by a magnet for 1 minute, and the product (MB-Au@Exo@Raman tag) was obtained by magnetic separation.

[0029] (4) In situ deposition of silver nanoparticles on immunomagnetic beads-exosome complex: Add 100μM, 10μL of distearoylphosphatidylethanolamine-polyethylene glycol-thiol (DSPE-PEG-SH, 2k) to 1mL of exosomes obtained in step (3), and incubate at 4°C for 30min; DSPE-PEG-SH is inserted into the exosome lipid membrane to obtain thiol-coated exosomes, which are then washed three times with 10mM PBS and redispersed in 1mL PBS; then 10mM dilute ammonia water is added dropwise to adjust the system pH to 10, and then 10μL, 100mM silver nitrate solution and 10μL, 250mM ascorbic acid reducing agent are added, mixed evenly, and incubated at 37°C for 1h. Among them, thiol is an electron-donating group with good affinity for metal ions, which can effectively enrich silver nitrate molecules and generate silver nanoparticles in situ in exosomes as SERS substrates. At the same time, silver nanoparticles were also generated on the surface of the gold-shell magnetic beads, forming a gold-silver composite substrate. This doped composite structure can work together with the in-situ silver substrate of the exosomes to produce a large number of SERS "hot spots", with a strong synergistic Raman enhancement effect. Finally, silver nanoparticles were deposited in-situ on the exosomes and gold-shell magnetic beads to obtain MB-Au@Exo@Ag.

[0030] (5) Preparation of gold film SERS array substrate: The positively charged anti-shedding glass slide was placed in 10 mL, 3 mM chloroauric acid solution and incubated for 5 min. Then, concentrated ammonia water with a mass concentration of 28% was added for oscillation reaction for 1 min. The glass slide was then washed with deionized water. The volume ratio of chloroauric acid solution to concentrated ammonia water was 50:1. The glass slide was then placed in 1 mM sodium borohydride solution and incubated for 1 min to generate light red gold nanoseeds. The glass slide was then washed with deionized water. The glass slide was then placed in 1 mL, 0.75 mM sodium borohydride solution and incubated for 1 min to generate light red gold nanoseeds. Chloroauric acid and 1mL, 0.75mM hydroxylamine hydrochloride in an equal volume mixed growth solution, oscillate and react at room temperature for 10 minutes to prepare a large area of ​​rough, island-shaped plasma gold film; finally, it is assembled with a flexible PDMS array hole to obtain a gold film SERS array; the flexible PDMS array hole is made by adding a curing agent to polydimethylsiloxane and mixing them, wherein the mass ratio of the two is 10:1, and after removing the bubbles in the mixed system, pour it into a mold, place it in an oven at 100°C, and heat it for 2 hours. The prepared PDMS membrane is punched with a hole diameter of 3mm.

[0031] (6) Detection of exosome 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 spectrometry to obtain the Raman signal corresponding to the exosome protein. A large number of SERS "hot spots" can be constructed between the silver substrate on the exosomes, the gold-silver composite substrate on the magnetic beads, and the plasma gold film. This synergistic effect is conducive to the formation of a multi-level signal amplification SERS sensing platform, which significantly improves the detection sensitivity of this method. The detection was carried out by laser confocal Raman spectrometry, and the test conditions were: 785 nm laser, integration time of 10 s, and laser power of 100%.

[0032] Figure 2 The schematic diagram of the use of immune gold-shell magnetic beads to capture exosomes in the present invention. First, CD63 antibodies are modified on the gold-shell-coated magnetic beads and blocked with bovine serum albumin to reduce nonspecific adsorption. Then, the immune capture probe is added to the pretreated sample, which can specifically bind to the CD63 protein on the surface of the exosomes and quickly separate the exosomes by a magnet.

[0033] Figure 3 Figure 2 is a structural representation diagram of the gold shell magnetic bead-exosome complex in the present invention. Figure 3 As shown in a, exosomes can be observed attached to the surface of magnetic beads under transmission electron microscopy. Then, the captured exosomes were subjected to protein immunoblotting analysis, with the parental cell line as a control. Figure 3 As shown in c, there is abundant CD63 protein on the exosomes, which also contain Alix protein but do not express Calnexin protein, while the cells do not express Alix protein but contain Calnexin protein. Figure 3 b is the capture process of exosomes characterized by dynamic light scattering. The average particle size of bare magnetic beads is about 210 nm, and the particle size of immunogold shell magnetic beads is about 240 nm. Compared with the individual exosomes and magnetic beads, the average particle size of the exosome-magnetic bead complex increases to 606 nm. These experimental results further prove that exosomes are successfully captured by immunomagnetic beads.

[0034] Figure 4 This is the structural characterization of the magnetic bead-exosome complex coated with silver nanoparticles in the present invention. Figure 4 a is a UV-visible spectrum. The absorption peak at 414 nm comes from the silver nanoparticles, and the absorption peak at 515 nm comes from the gold shell on the surface of the magnetic beads. Figure 4 b is a transmission electron microscopy image of silver nanoparticles deposited on the surface of exosomes.

[0035] Figure 5The SERS spectra of the three Raman reporter molecules screened in the present invention are shown in Figure 2. By detecting a series of conjugated organic molecules containing thiol groups at the end, 4-mercaptobenzonitrile (4-MBN, characteristic peak: 2220 cm -1 )、4-nitrobenzenethiol (4-NTP, characteristic peak: 1330 cm -1 ), 4-ethynylbenzenethiol (SH-alkyne, characteristic peak: 2100 cm -1 ) is used as a Raman reporter molecule, and the three Raman characteristic peaks do not overlap with each other and are used to label the three corresponding antibodies.

[0036] Figure 6 This is a structural characterization of the rough substrate of the gold film in the present invention. Figure 6 As shown in the scanning electron microscope image in a, the surface of the plasma gold film is distributed with uniform and dense nano-island structural units, and has a strong and broad UV-visible absorption peak at 655 nm, as shown in Figure 6 b.

[0037] Figure 7 This is a working curve diagram of analyzing exosome proteins derived from liver cancer cells using the SERS sensing platform based on synchronous combined detection in the present invention. Figure 7 a is exosomes (5.11×10 9 Representative SERS spectra obtained by detecting 5.11×10 3 ~5.11×10 9 particles / mL range, Figure 7 b is 1330 cm -1 The Raman signal at was used as the target signal of GPC-3 protein, and the obtained 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 is selected as 2220 cm -1 The Raman signal at 1000 nm was used as the target signal of EGFR protein, and the obtained working curve was Y=347.2X-925.5, with a linear correlation coefficient R 2 =0.985, and the detection limit is 96 particles / mL. Figure 7 d is selected as 2100 cm -1 The Raman signal at PD-L1 protein was used as the target signal, and the obtained 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.

[0038] Figure 8This is an interference experiment for detecting exosome proteins based on the SERS sensing platform in the present invention. Taking the detection of GPC-3 protein in HepG2 exosomes as an example, the interference experiment of interfering substances commonly present in body fluids, including proteins, sugars, and amino acids, on the SERS detection results was evaluated. As shown in the figure, the average SERS signal intensity corresponding to each group of human serum albumin (HSA, 100μg / mL), immunoglobulin (IgG, 100μg / mL), lysozyme (lysozyme, 50μg / mL), glucose (glucose, 10mM), glycine (glycine, 10mM), and cysteine ​​(cysteine, 10mM) is between 251 and 574, which is similar to the SERS signal of the blank group, but very different from the signal intensity of HepG2-derived exosomes, with a significant difference (t test, P<0.01), indicating that this method has good selectivity for exosome protein detection and is not easily interfered by other substances.

[0039] Fig. 9 The present invention detects three exosome proteins (GPC-3, EGFR, PD-L1) from different cell lines based on the SERS sensing platform. As shown in the figure, in the blank group and the LO2 exosomes derived from the normal liver cell line, the three proteins are negatively expressed. In the exosomes derived from the liver cancer cell line HepG2, the cervical cancer cell line HeLa, and the non-small cell carcinoma NSCLC, EGFR and PD-L1 are both positively expressed, while GPC-3 is specifically expressed in the HepG2 exosomes. It is preliminarily proved that this method can be used to distinguish between exosomes from normal cell lines and exosomes from tumor cell lines, and can improve the selectivity of HepG2 exosome detection.

[0040] The standard addition method was further used to detect exosomes in the actual simulated samples based on the SERS sensing platform for simultaneous detection of exosome proteins. The medium of the simulated sample was human serum albumin dissolved in 10mM PBS solution. Taking the detection of GPC-3 protein in exosomes as an example, the exosome standard obtained by ultracentrifugation was added to the human serum albumin solution of the same volume (50mL) and the same concentration (10mg / mL) in each group to obtain spiked samples with different concentration gradients (5×10 4 ~5×10 7 particles / mL), the SERS sensing platform of the present invention was used for detection, each group of experiments was repeated three times, and the corresponding recovery rate and relative standard deviation of each group were calculated.

[0041] 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 the simultaneous combined detection of exosome proteins in the present invention, so as to evaluate the detection effect of this method on actual samples.

[0042] Table 1: Detection of exosomes in simulated samples based on the SERS sensing platform in the present invention.

[0043] Table 1 shows the detection of exosomes in simulated samples by the SERS sensing platform based on simultaneous combined detection of exosome proteins in the present invention. 4 ~5.11×10 7 particles / mL, the recovery rate of the method of the present invention is between 95.6% and 108.7%, which is in line with the allowable range of spiked recovery rate, and the relative standard deviation is between 2.89% and 4.53%, which is within the acceptable range, indicating that the method of the present invention has potential application value in actual sample detection.

[0044] Fig.10 The dot immunoblotting method was used in the present invention to further verify the SERS detection results. The dot blotting analysis was performed in situ on the silver nanoparticle-coated magnetic bead-exosome complex, and it was found that EGFR, GPC-3, and PD-L1 were produced on the surface; the exosomes alone were used as positive controls, and the above three protein spots were also produced; while the gold shell magnetic beads were used as negative controls, and no protein spots appeared. This result further proves that the three proteins EGFR, GPC-3, and PD-L1 are all positively expressed on HepG2 exosomes, which is consistent with the SERS detection results.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 joint detection of exosome proteins, characterized in that: The following steps are included (1) Pre-treat the sample by differential centrifugation: collect the cell supernatant and perform differential centrifugation at 300g for 10 min, 2000g for 10 min, and 10000g for 30 min to remove impurities such as dead cells, cell debris, and large vesicles remaining in the sample; all differential centrifugation steps were performed at 4°C; the supernatant obtained in the last step was filtered using a 0.22 μm filter membrane, and the filtrate was collected for later use; (2) Capturing exosomes with immunogold-shell magnetic beads: 1 mL of 10 mg / mL CD63 antibody-modified immunogold-shell magnetic beads 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 obtained 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 labels: 1 mM of different Raman reporter molecules were coupled with 0.1 mg / mL of corresponding antibody molecules as Raman labels. 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 labels are: 4-mercaptobenzonitrile-EGFR antibody, 4-nitrobenzenethiol-GPC-3 antibody, 4-ethynylbenzenethiol-PD-L1 antibody; then the three labels at the same concentration of 250 μM are co-incubated with 1 mL of the magnetic bead-exosome complex obtained in step (2), incubated at 4°C for 1 hour, separated by a magnet for 1 minute, and the obtained crude product is washed three times with 10 mM PBS and dispersed in 1 mL, 10 mM PBS; (4) In situ deposition of silver nanoparticles on the immunomagnetic bead-exosome complex: Add 100 μM, 10 μL of distearoylphosphatidylethanolamine-polyethylene glycol-thiol (DSPE-PEG-SH, 2k) to 1 mL of the exosomes obtained in step (3), and incubate at 4°C for 30 min. DSPE-PEG-SH is inserted into the exosome lipid membrane to obtain thiol-coated exosomes; then wash three times with 10 mM PBS and redisperse in 1 mL PBS; then add 10 mM dilute ammonia water to adjust the pH of the system to 10; then add 10 μL of 100 mM silver nitrate solution and 10 μL of 250 mM ascorbic acid reducing agent, mix well, and incubate at 37°C for 1 h. Silver nanoparticles are simultaneously deposited on the exosomes and gold shell magnetic beads in situ to obtain MB-Au@Exo@Ag; (5) Preparation of gold film SERS array substrate: Place a 2 cm × 2.5 cm positively charged anti-shedding glass slide in 10 mL, 3 mM chloroauric acid solution and incubate for 5 min, then add 28% concentrated ammonia water for oscillation reaction for 1 min, and then wash with deionized water for 3 times, wherein the volume ratio of chloroauric acid solution to concentrated ammonia water is 50:1; then place the glass slide in 1 mM sodium borohydride solution for 1 min to generate light red gold nanoseeds; then place it in an equal volume mixed growth solution of 1 mL, 0.75 mM chloroauric acid and 1 mL, 0.75 mM hydroxylamine hydrochloride, and oscillate for 10 min at room temperature to further form a large area of ​​rough, island-shaped plasma gold film; finally, assemble it with the flexible PDMS array holes to obtain a gold film SERS array substrate; (6) Detection of exosome 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 using a laser confocal Raman spectrometer to obtain the Raman signal corresponding to the exosome protein.

2. The method for preparing a SERS sensing platform for simultaneous joint detection of exosome proteins according to claim 1, characterized in that: The immunogold-shell magnetic beads described in step (2) are used as capture probes, comprising a magnetic core of ferroferric oxide with a particle size of about 200 nm and a gold-shell outer layer modified with CD63 antibodies; The specific preparation method of the immune gold shell magnetic beads is as follows: 1 mL of 20 mg / mL magnetic beads is added to 50 mL of aqueous solution, and then 10 mg / mL and 5 mL of chloroauric acid solution and 60 mg / mL and 5 mL of sodium citrate solution are added respectively, and a solvent thermal method is used to reflux for 30 minutes at 100° C. to obtain gold shell-coated magnetic beads, which are then washed three times with ultrapure water for storage; then 2 mL and 0.2 mg / mL of CD63 antibodies are used to modify 20 mL and 1 mg / mL of gold shell magnetic beads in a 10 mM PBS medium, and the beads are incubated at 37° C. for 1 hour; then the immune gold shell magnetic beads are separated by a magnet, and the beads are washed three times with 10 mM PBS, and then blocked with 1% by mass bovine serum albumin.

3. The method for preparing a SERS sensing platform for simultaneous joint detection of exosome proteins according to claim 1, characterized in that: The different Raman tags in step (3) include a Raman reporter molecule and a corresponding antibody, wherein the Raman reporter molecule structure contains a thiol group at the end and has a benzene ring conjugated structure, and N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate SMCC is used as a cross-linking agent, and the maleimide at one end of SMCC is coupled with the thiol group of the Raman reporter molecule through a click chemistry reaction, and the succinimide group at the other end of SMCC is coupled with the amino group in the antibody to obtain a Raman reporter molecule-antibody tag; The specific preparation method is as follows: 1 mM Raman reporter molecule is mixed with 1.1 mM cross-linker N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate SMCC, and the reaction is stirred at room temperature for 1 hour. The obtained intermediate product is incubated with 0.1 mg / mL of the corresponding antibody in PBS medium at 37°C for 1 hour; then a 30KD ultrafiltration tube is used to remove the residual Raman reporter molecule, SMCC and intermediate product.

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

5. The method for preparing a SERS sensing platform for simultaneous joint detection of multiple proteins in exosomes according to claim 1, characterized in that: The specific test conditions for the detection using the 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 exosome proteins obtained according to any one of the preparation methods described in claims 1-5.

7. Application of the SERS sensing platform according to claim 6 in the simultaneous combined analysis and detection of tumor-related proteins EGFR, GPC-3, and PD-L1.

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