CN119198677A - A SERS sensing platform for simultaneous joint detection of exosome proteins and its preparation method - Google Patents
A SERS sensing platform for simultaneous joint detection of exosome proteins and its preparation method Download PDFInfo
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- CN119198677A CN119198677A CN202411383138.4A CN202411383138A CN119198677A CN 119198677 A CN119198677 A CN 119198677A CN 202411383138 A CN202411383138 A CN 202411383138A CN 119198677 A CN119198677 A CN 119198677A
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- exosome
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Abstract
本发明属外泌体蛋白检测的技术领域，提供一种同步联合检测外泌体蛋白的SERS传感平台及其制备方法，包括以下步骤：差速离心法对初始样品进行预处理；制备磁珠‑外泌体复合物，用不同SERS标签对外泌体表面的三种蛋白分别进行标记；在免疫磁珠‑外泌体复合物原位沉积银纳米颗粒；制备金膜阵列基底进行SERS检测，利用多级信号放大的SERS协同增强效应，得到外泌体上各蛋白对应的SERS信号。构建了一种集分离、富集和检测为一体的SERS传感平台用于外泌体蛋白的高灵敏检测。显著提高了外泌体与贵金属拉曼探针的结合效率，为同一样本中多重蛋白的同步联合检测提高了可行性。该方法检灵敏度高、特异性好，可实现外泌体蛋白的高通量检测。
The present invention belongs to the technical field of exosome protein detection, and provides a SERS sensing platform for synchronous joint detection of exosome proteins and a preparation method thereof, comprising the following steps: pre-treating the initial sample by differential centrifugation; preparing a magnetic bead-exosome complex, and labeling three proteins on the surface of the exosomes with different SERS labels; in situ depositing silver nanoparticles on the immunomagnetic bead-exosome complex; preparing a gold film array substrate for SERS detection, and using the SERS synergistic enhancement effect of multi-stage signal amplification to obtain the SERS signals corresponding to each protein on the exosome. A SERS sensing platform integrating separation, enrichment and detection is constructed for high-sensitivity detection of exosome proteins. The binding efficiency of exosomes and precious metal Raman probes is significantly improved, and the feasibility of synchronous joint detection of multiple proteins in the same sample is improved. The method has high detection sensitivity and good specificity, and can realize high-throughput detection of exosome proteins.
Description
Technical Field
The invention belongs to the technical field of exosome protein detection, and particularly relates to a SERS sensing platform for synchronous combined detection of exosome proteins and a preparation method thereof.
Background
Exosomes (exosomes) are nanolipid vesicles secreted by cells, with a particle size of about 30-150 nm, which are secreted by almost all types of cells, especially in tumor cells, with high exosome production. The exosomes contain various bioactive molecules, including nucleic acid, lipid, protein and the like, perform information transmission among cells, play a significant role in intercellular communication, and particularly provide favorable conditions for proliferation and metastasis of tumors in aspects of regulating and controlling tumor microenvironment, promoting angiogenesis, assisting tumor cells to escape immune monitoring, enhancing tumor drug resistance and the like. Among them, exosome proteins are closely related to tumor growth and invasion and metastasis. For example, high-level expression of exosome protein CD97 in gastric cancer can promote angiogenesis, promote enhancement of proliferation capacity of gastric cancer cells, exosome membrane proteins, lectin α6β4 and α6β1, play a key role in lung metastasis, lectin αvβ5 plays a key role in liver metastasis, and SMAD3 protein in liver cancer serum exosomes can promote proliferation and lung metastasis of cancer cells. The results of these studies indicate that exosome proteins play an important role in regulating tumor proliferation and metastasis. Compared with the traditional tissue biopsy, the exosome detection has the advantages of convenient and quick sampling and small injury, so that exosome protein is hopefully a novel tumor liquid biopsy object and provides a novel marker for early diagnosis and metastasis monitoring of cancers.
At present, detection methods of exosome proteins comprise western immunoblotting analysis, an immune colloidal gold method, an enzyme-linked immunosorbent assay, a flow cytometry and the like, and the methods not only have large required sample size and lower sensitivity, but also further need to carry out complex sample pretreatment and other works on samples, so that the methods are difficult to implement and popularize in clinical application. Therefore, development of a detection method for detecting exosome proteins with high sensitivity, good specificity and high accuracy without pretreatment of the sample is needed.
Surface enhanced raman scattering spectroscopy (SERS), a phenomenon of light scattering that uses a roughened metal Surface to enhance the raman signal of an object under test. The technology has the unique advantages of high sensitivity, abundant spectral information, no light bleaching, no need of sample pretreatment, capability of realizing in-situ and nondestructive detection of samples, abundant sources of Raman reporter molecules, narrow spectral peaks, capability of simultaneously realizing the marking or encoding of various objects to be detected, and suitability for multi-component detection. In addition, some raman reporter molecules contain specific functional groups, including cyano, alkynyl, deuterated groups, etc., which can generate strong SERS signals in the raman silencing region 1800-2800 cm -1. The molecules are used as SERS labels, so that the background interference of complex biological endogenous substances can be avoided, a background-free SERS sensing technology is constructed, and the molecules are widely applied to biomedical fields such as tumor cell identification, cell metabolite detection, tissue imaging, nucleic acid or protein detection and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a SERS sensing platform for synchronously and jointly detecting exosome proteins and a preparation method thereof, wherein the SERS sensing platform is a SERS sensing platform with integrated separation, enrichment and detection and multi-stage signal amplification, the tumor exosome proteins are subjected to high-sensitivity and high-flux joint detection, and the method is used for synchronously and jointly detecting and analyzing tumor-related proteins (EGFR, GPC-3 and PD-L1) in liver cancer cell-derived exosome.
In order to achieve the aim, the technical scheme adopted by the invention is that the preparation method of the SERS sensing platform for synchronously and jointly detecting the exosome protein comprises the following steps:
(1) Pretreating a sample by a differential centrifugation method, namely collecting cell supernatant, carrying out differential centrifugation, centrifuging for 10min at 300g, centrifuging for 10min at 2000g, centrifuging for 30min at 10000g, removing residual dead cells, cell fragments, large vesicles and other impurities in the sample, carrying out all differential centrifugation steps at 4 ℃, filtering the supernatant obtained in the last step by adopting a filter membrane of 0.22 mu m, and collecting filtrate for later use;
(2) The immune Jin Keci bead captures exosomes, namely 1mL of CD63 antibody modified immune gold shell magnetic beads with the concentration of 10mg/mL are mixed with 50mL of the pretreated sample obtained in the step (1), incubated for 2 hours at 4 ℃, the CD63 antibody is specifically combined with CD63 protein on the surface of the exosomes, the obtained mixture is separated by a magnet for 2 minutes, and a magnetic bead-exosome compound MB-Au@exo is obtained and dispersed in 2.5mL of 10mM PBS;
(3) Synchronously labeling multiple exosome proteins with different Raman labels, namely coupling 1mM different Raman reporter molecules with corresponding antibody molecules of 0.1mg/mL, wherein the Raman reporter molecules are 4-mercaptobenzonitrile, 4-nitrobenzenethiol and 4-ethynylbenzenethiol as Raman labels, and the antibody molecules are EGFR antibodies, GPC-3 antibodies and PD-L1 antibodies;
The Raman labels are 4-mercaptobenzonitrile-EGFR antibody, 4-nitrobenzenethiol-GPC-3 antibody and 4-ethynylbenzenethiol-PD-L1 antibody, then three labels with the same concentration (250 mu M) are incubated with 1mL of the magnetic bead-exosome compound obtained in the step (2) simultaneously, after incubation for 1h at 4 ℃, the obtained crude product is separated by a magnet for 1min, and the obtained crude product is washed three times by 10mM PBS and dispersed in 1mL of 10mM PBS;
(4) Adding 100 mu M and 10 mu L distearoyl phosphatidylethanolamine-polyethylene glycol-sulfhydryl (DSPE-PEG-SH, 2 k) into 1mL of exosome obtained in the step (3), incubating for 30min at 4 ℃, inserting the DSPE-PEG-SH into a lipid membrane of the exosome to obtain a sulfhydryl-coated exosome, washing for three times by adopting 10mM PBS, redispersing in 1mL of PBS, then dropwise adding 10mM dilute ammonia water to adjust the pH=10, adding 10 mu L of 100mM silver nitrate solution and 10 mu L of 250mM ascorbic acid reducer, uniformly mixing, incubating for 1h at 37 ℃, and depositing silver nanoparticles on the exosome and gold shell magnetic beads in situ to obtain MB-Au@Exo@Ag;
(5) Preparing a gold film SERS array substrate, namely placing a positively charged anti-drop glass slide with the size of 2cm multiplied by 2.5cm into 10mL and 3mM chloroauric acid solution for incubation for 5min, adding concentrated ammonia water with the mass concentration of 28% for oscillation reaction for 1min, then cleaning for 3 times by deionized water, wherein the volume ratio of the chloroauric acid solution to the concentrated ammonia water is 50:1, placing the glass slide into 1mM sodium borohydride solution for incubation for 1min to generate light red gold nano seeds, placing the glass slide into 1mL,0.75mM chloroauric acid and 1mL,0.75mM hydroxylamine hydrochloride in an equal volume mixed growth solution, carrying out oscillation reaction for 10min at room temperature to further form a large-area rough plasma gold film distributed in an island shape, and finally assembling the glass slide with flexible PDMS array holes to obtain the gold film SERS array substrate;
(6) And (3) detecting exosome protein based on SERS synergistic enhancement effect, namely dripping 10 mu L of MB-Au@Exo@Ag obtained in the step (4) into the gold film SERS array obtained in the step (5), and detecting by using a laser confocal Raman spectrometer after a sample is dried to obtain a Raman signal corresponding to the exosome protein.
The immuno Jin Keci bead in the step (2) is used as a capture probe, and comprises a ferroferric oxide magnetic core with the particle size of 200nm and a gold shell outer layer modified by a CD63 antibody;
The preparation method of the immune gold shell magnetic beads comprises the steps of adding 1mL of 20mg/mL of magnetic beads into 50mL of aqueous solution, respectively adding 10mg/mL of chloroauric acid solution, 60mg/mL of sodium citrate solution and 5mL of sodium citrate solution, carrying out reflux reaction at 100 ℃ for 30min by adopting a solvothermal method to obtain gold shell coated magnetic beads, washing with ultrapure water for three times for preservation, then adopting 2mL of 0.2mg/mL of CD63 antibody to modify 20mL of 1mg/mL of gold shell magnetic beads in 10mM PBS medium, incubating for 1h at 37 ℃, separating by a magnet to obtain the immune gold shell magnetic beads, washing with 10mM PBS for three times, and then sealing by adopting bovine serum albumin with the mass fraction of 1%;
the different Raman labels in the step (3) comprise Raman reporter molecules and corresponding antibodies, wherein the tail end of the structure of each Raman reporter molecule contains sulfhydryl groups and has a benzene ring conjugated structure, N-succinimidyl 4- (maleimidomethyl) cyclohexane carboxylate SMCC is used as a cross-linking agent, maleimide at one end of the SMCC is coupled with sulfhydryl groups of the Raman reporter molecules through click chemical reaction, and the succinimide groups at the other end of the SMCC are coupled with amino groups in the antibodies to obtain the Raman reporter molecules-antibody labels;
the preparation method comprises the steps of mixing 1mM of Raman reporter molecule with 1.1mM of cross-linking agent N-succinimidyl 4- (maleimidomethyl) cyclohexane carboxylate SMCC, stirring at room temperature for reaction for 1h, incubating the obtained intermediate product with 0.1mg/mL of corresponding antibody in PBS medium at 37 ℃ for 1h, and removing residual Raman reporter molecule, SMCC and intermediate product by adopting a 30KD ultrafiltration tube.
The flexible PDMS array holes in the step (5) are obtained by uniformly mixing polydimethylsiloxane PDMS and a matched curing agent according to a mass ratio of 10:1, removing bubbles in a mixed system, pouring the mixture into a glass mold, placing the mold with the size of 80cm multiplied by 80cm in an oven at the temperature of 100 ℃, incubating for 2 hours, and punching the prepared PDMS film, wherein the aperture is 3mm.
The specific test condition for detection by adopting a laser confocal Raman spectrometer in the step (6) is 785nm laser, the integration time is 10s, and the laser power is 100%.
The invention also provides a SERS sensing platform for synchronously and jointly detecting the exosome protein, which is obtained by the preparation method.
The invention also provides application of the SERS sensing platform in synchronous joint analysis detection of tumor-associated proteins EGFR, GPC-3 and PD-L1.
In the detection process, the sulfhydryl is an electron donating group, has good affinity to metal ions, can effectively enrich silver nitrate molecules, and generates silver nano particles in situ in exosomes as SERS substrates. Meanwhile, silver nano particles are also generated on the surfaces of the gold-shell magnetic beads, so that a gold-silver composite substrate is formed. The doped composite structure can co-act with the nano silver substrate on the surface of the exosome, can generate a large number of SERS 'hot spots', and has a strong synergistic Raman enhancement effect.
A large number of hot spots can be generated between the silver substrate on the exosome, the gold-silver composite substrate on the magnetic bead and the solid-phase plasma gold film, and the synergistic effect is beneficial to forming a SERS sensing platform with multistage signal amplification, so that the detection sensitivity of the method is remarkably improved.
The invention provides an exosome protein analysis platform integrating separation, enrichment and detection by combining a magnetic separation technology and a multi-stage signal amplification SERS sensing technology. The method is different from the conventional SERS technology for marking noble metal colloid particles, and the method adopts silver nano particles deposited on an exosome-magnetic bead compound in situ as a SERS substrate, so that the problems that the exosome size is small, the noble metal colloid particles marking efficiency is low due to steric effect, and multiple protein synchronous joint detection is difficult to realize are remarkably improved. In addition, a rough gold film substrate is further combined, a sensing platform with a synergistic SERS enhancement effect is constructed, and the feasibility of detecting low-abundance target objects is further improved. The method has high sensitivity and good specificity, and can realize multichannel and high-flux detection of exosome membrane proteins.
Compared with the existing detection technology, the invention has the following advantages and beneficial effects:
The detection sensitivity is high, the exosomes have high heterogeneity, the expression level difference of each protein on the exosome membrane is large, and the method constructs a multi-stage signal amplification SERS sensing platform, can generate strong synergistic SERS enhancement effect, and realizes simultaneous detection of multiple proteins. Compared with the conventional SERS technology for labeling noble metal colloid particles, the sensitivity of the detection method is remarkably improved.
The separation speed is high, the target product is collected through magnetic separation in the whole experimental flow of the method, and the residual impurities of each step are removed, so that the detection efficiency is greatly improved.
The method has high specificity, adopts differential centrifugation and a filter membrane to remove impurities, captures exosomes through an antibody modified immunomagnetic bead, and adopts an antibody modified Raman reporter molecule label to label exosome proteins, so that the method has good specificity.
The method is suitable for synchronous detection of multiple membrane proteins in exosomes derived from different cells or body fluids, and only specific antibodies corresponding to the proteins need to be replaced.
Synchronous joint detection of exosome multiple proteins is realized: the exosomes are marked with various specific Raman labels in advance, and then the silver nano substrate is directly deposited in situ, so that compared with the traditional mode that after different noble metal Raman probes are mixed, the exosomes are incubated with the exosomes, the steric effect can be obviously reduced, and the multichannel simultaneous detection of proteins is facilitated.
Drawings
FIG. 1 is a schematic diagram of a SERS sensing platform based on synchronous joint detection of exosome proteins in the present invention;
FIG. 2 is a schematic diagram of capturing exosomes using immunogold-shelled magnetic beads in the present invention;
FIG. 3 is a diagram showing structural characterization of the gold-shell bead-exosome complex of the present invention;
FIG. 4 is a diagram showing the structural characterization of the magnetic bead-exosome complex coated silver nanoparticle according to the present invention;
FIG. 5 is a SERS spectrum of three Raman reporter molecules screened in the present invention;
FIG. 6 is a structural representation of a rough substrate of a plasma gold film in accordance with the present invention;
FIG. 7 is a graph showing the analysis of liver cancer cell-derived exosome proteins based on a synchronous joint detection SERS sensing platform;
FIG. 8 is an interference experiment for detecting exosome proteins based on SERS sensing platform in the present invention;
FIG. 9 shows the results of the detection of three exosome proteins (GPC-3, EGFR, PD-L1) from different cell lines based on SERS-sensing platforms according to the invention;
FIG. 10 shows the results of further validating SERS using dot immunoblotting in the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by persons of ordinary skill in the art without making creative efforts based on the embodiments of the present invention are all within the scope of protection of the present invention.
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, the disclosure of which is incorporated herein by reference as is commonly understood by reference.
Those skilled in the art will recognize that equivalents of the specific embodiments described, as well as those known by routine experimentation, are intended to be encompassed within the present application.
The experimental methods in the following examples are conventional methods unless otherwise specified. The apparatus used in the examples described below were all laboratory conventional apparatus unless otherwise specified, and the experimental materials used in the examples described below were all purchased from conventional biochemical reagent stores unless otherwise specified.
CD63 antibody (ab 134045), EGFR antibody (ab 40815), GPC-3 antibody (ab 216606), PD-L1 antibody (ab 243877) used in the present invention were purchased from Abcam, the positive charge anti-drop slide used was purchased from the chemical family, cat# HK2143-46052, the distearoyl phosphatidylethanolamine-polyethylene glycol-mercapto group (DSPE-PEG-SH, 2K) was purchased from Huateng, cat# LP096003-2K, the polydimethylsiloxane reagent (DOW CORNING/Takanin) used, and the cat# DC184.
As shown in FIG. 1, the preparation method of the SERS sensing platform based on synchronous joint detection of exosome proteins comprises the following steps of preprocessing a sample by adopting a differential centrifugation method, capturing exosome by using an immune Jin Keci bead, marking multiple exosome proteins by using a Raman label, depositing silver nano particles in situ on an immune magnetic bead-exosome complex, preparing a gold film SERS array, and detecting exosome proteins based on SERS synergistic enhancement effect, wherein the preparation method comprises the following steps:
(1) The sample is pretreated by adopting a differential centrifugation method, namely, cell supernatant is collected and subjected to differential centrifugation, and the cell supernatant is centrifuged for 10min at 300g, 10min at 2000g and 30min at 10000g in sequence, so that residual dead cells, cell fragments, large vesicles and other impurities are removed. The liquid sample was then filtered through a 0.22 μm filter and the filtrate was collected for further use. The centrifugation steps were all performed at 4 ℃.
(2) Immuno Jin Keci bead capture exosomes by incubating with gold-shell magnetic beads modified with CD63 antibodies (1 mL,10 mg/mL) with 50mL of pretreatment sample, CD63 antibodies can specifically bind to CD63 protein enriched on the exosome surface. After incubation for 2h at 4 ℃, the magnetic bead-exosome complex (MB-au@exo) was isolated by magnet and dispersed in 2.5mL of 10mM PBS. The immuno Jin Keci bead MB-Au is of a core-shell structure and comprises an inner core layer and an outer shell layer, wherein the inner core layer is ferroferric oxide nano particles, the outer shell layer is a gold nano shell, and a CD63 antibody is modified on the surface of the gold nano shell and is used as a capture probe.
(3) Multiple exosome proteins were synchronously labeled with different raman tags by coupling 1mM of different raman reporter molecules to 0.1mg/mL of the corresponding antibody molecules as raman tags and then co-incubating with the captured exosomes simultaneously. The terminal of the Raman reporter molecule structure contains sulfhydryl and has a benzene ring conjugated structure, N-succinimidyl 4- (maleimidomethyl) cyclohexane carboxylate (SMCC) is used as a cross-linking agent, the maleimide at one end of the SMCC is coupled with the sulfhydryl of the Raman reporter molecule through click chemistry reaction, and the succinimidyl at the other end of the SMCC is coupled with amino in the antibody to obtain the Raman reporter molecule-antibody label. The Raman tag comprises a 4-mercaptobenzonitrile-EGFR antibody, a 4-nitrobenzenethiol-GPC-3 antibody, a 4-ethynylbenzenethiol-PD-L1 antibody, three tags with the same concentration (250 mu M) are simultaneously incubated with 1mL of a magnetic bead-exosome complex, after incubation for 1h at 4 ℃, the tags are separated by a magnet for 1min, and a product (MB-Au@Exo@Raman tag) is obtained through magnet separation.
(4) Immunomagnetic bead-exosome complex in-situ deposition of silver nano particles, namely adding 100 mu M and 10 mu L of distearoyl phosphatidylethanolamine-polyethylene glycol-sulfhydryl (DSPE-PEG-SH, 2 k) into 1mL of exosome obtained in the step (3), incubating for 30min at 4 ℃, inserting the DSPE-PEG-SH into an exosome lipid membrane to obtain a sulfhydryl-coated exosome, washing three times by adopting 10mM PBS, redispersing in 1mL of PBS, then dropwise adding 10mM of diluted ammonia water to adjust the pH=10 of the system, adding 10 mu L of 100mM silver nitrate solution and 10 mu L of 250mM ascorbic acid reducer, mixing uniformly, and incubating for 1h at 37 ℃. Wherein, sulfhydryl is electron donating group, has good affinity to metal ion, can effectively enrich silver nitrate molecule, and can generate silver nano particles in situ in exosomes as SERS substrate. Meanwhile, silver nano particles are also generated on the surfaces of the gold-shell magnetic beads, so that a gold-silver composite substrate is formed. The doped composite structure can co-act with an exosome in-situ silver substrate to generate a large number of SERS 'hot spots', and has a strong synergistic Raman enhancement effect. And finally, simultaneously depositing silver nano particles on the exosomes and the gold-shell magnetic beads in situ to obtain MB-Au@Exo@Ag.
(5) The preparation method of the gold film SERS array substrate comprises the steps of placing a positively charged anti-drop glass slide in 10mL,3mM chloroauric acid solution for 5min, adding concentrated ammonia water with the mass concentration of 28% for oscillation reaction for 1min, washing with deionized water, wherein the volume ratio of the chloroauric acid solution to the concentrated ammonia water is 50:1, placing the glass slide in 1mM sodium borohydride solution for incubation for 1min to generate light red gold nano seeds, washing with deionized water, placing the glass slide in 1mL,0.75mM chloroauric acid and 1mL,0.75mM hydroxylamine hydrochloride in an equal volume mixed growth solution, carrying out oscillation reaction at room temperature for 10min to prepare a large-area rough island-shaped plasma gold film, and finally assembling the large-area rough island-shaped plasma gold film with flexible PDMS array holes to obtain the gold film SERS array, wherein the flexible PDMS array holes are uniformly mixed with a curing agent, wherein the mass ratio of the two is 10:1, removing bubbles in a mixed system, pouring the glass slide into a mold, placing the mold in a baking oven, and heating for 2h. The prepared PDMS film was perforated to a pore size of 3mm.
(6) And (3) detecting exosome protein based on SERS synergistic enhancement effect, namely dripping 10 mu L of MB-Au@Exo@Ag obtained in the step (4) into the gold film SERS array obtained in the step (5), and detecting by using a laser confocal Raman spectrometer after a sample is dried to obtain a Raman signal corresponding to the exosome protein. A large number of SERS 'hot spots' can be constructed between a silver substrate on an exosome, a gold-silver composite substrate on a magnetic bead and a plasma gold film, and the synergistic effect is beneficial to forming a SERS sensing platform with multistage signal amplification, so that the detection sensitivity of the method is remarkably improved. The detection is carried out by adopting a laser confocal Raman spectrometer, the test condition is 785nm laser, the integration time is 10s, and the laser power is 100%.
FIG. 2 is a schematic representation of the capture of exosomes using immunogold-shelled magnetic beads in the present invention. First, CD63 antibody is modified on magnetic beads coated by gold shell, and blocking is carried out by bovine serum albumin so as to reduce nonspecific adsorption. And then adding the immunocapture probe into the pretreated sample, and carrying out specific binding with CD63 protein on the surface of the exosome, and carrying out rapid separation on the exosome by a magnet.
FIG. 3 is a diagram showing structural characterization of the gold-shell bead-exosome complex of the present invention. The exosomes captured by the immunomagnetic beads are shown in fig. 3a, and the exosomes are observed to be attached to the surfaces of the magnetic beads under a transmission electron microscope. The captured exosomes were then subjected to western blot analysis using the parental cell line as a control, the exosomes shown in fig. 3c were enriched for CD63 protein, also containing Alix protein, but not Calnexin protein, and cells were not expressing Alix protein, but containing Calnexin protein. Fig. 3b shows a capturing process of exosomes characterized by dynamic light scattering, wherein the average particle size of bare magnetic beads is about 210 nm, the particle size of immunogold-coated magnetic beads is about 240: 240 nm, and the average particle size of exosome-magnetic bead complex is increased to 606: 606 nm compared to exosomes and magnetic beads alone. These experimental results further demonstrate that exosomes are successfully captured by immunomagnetic beads.
FIG. 4 is a structural representation of a magnetic bead-exosome complex coated silver nanoparticle according to the present invention. Fig. 4a is an ultraviolet-visible spectrum, in which the absorption peak at 414 nm comes from the silver nanoparticle, the absorption peak at 515 nm comes from the gold shell on the surface of the magnetic bead, and fig. 4b is a transmission electron microscope of the deposition of silver nanoparticle on the surface of the exosome.
FIG. 5 is a SERS spectrum of three Raman reporter molecules screened in the present invention. By detecting a series of conjugated organic molecules with mercapto groups at the tail end, 4-mercaptobenzonitrile (4-MBN, characteristic peak: 2220 cm -1), 4-nitrobenzenethiol (4-NTP, characteristic peak: 1330 cm -1) and 4-ethynyl benzenethiol (SH-alkyne, characteristic peak: 2100 cm -1) are obtained through screening and used as Raman reporter molecules, and three Raman characteristic peaks are not overlapped with each other and are used for labeling three corresponding antibodies.
FIG. 6 is a structural representation of a roughened substrate of gold film in accordance with the present invention. As shown in the scanning electron microscope image in fig. 6a, the surface of the plasma gold film is distributed with uniform and compact nano island structural units, and has a stronger and wider ultraviolet-visible absorption peak at 655 nm, as shown in fig. 6b.
Fig. 7 is a working graph of the SERS sensing platform for analyzing liver cancer cell-derived exosome proteins based on synchronous joint detection in the present invention. FIG. 7a is a representative SERS spectrum detected in exosomes (5.11X10 9 parts/mL). In the range of 5.11X10 3～5.11×109 parts/mL, FIG. 7b shows the resulting working curve Y= 1397.3X-3989.6 with linear correlation coefficient R 2 =0.991 and detection limit of 83 particles/mL, where the Raman signal at 1330 cm -1 is chosen as the target signal for GPC-3 protein. Fig. 7c shows the working curve y=347.2X-925.5, the linear correlation coefficient R 2 =0.985, and the detection limit of 96 particles/mL, when the raman signal at 2220 cm -1 is selected as the target signal for the EGFR protein. Fig. 7d shows the result of selecting the raman signal at 2100 cm -1 as the target signal for PD-L1 protein, resulting in an operating curve y=315.7x-851.3 with a linear correlation coefficient R 2 =0.981, and a detection limit of 116 particles/mL.
FIG. 8 is an experimental set of the interferents for detecting exosome proteins based on SERS sensing platforms in accordance with the present invention. Taking GPC-3 protein detection in HepG2 exosomes as an example, interference substances commonly present in body fluids, including interference experiments of proteins, saccharides, amino acids on SERS detection results, are evaluated. As shown in the figure, the average SERS signal intensity corresponding to each group of human serum albumin (HSA, 100 mug/mL), immunoglobulin (IgG, 100 mug/mL), lysozyme (lysozyme, 50 mug/mL), glucose (glucose, 10 mM), glycine (glycine, 10 mM) and cysteine (10 mM) is between 251 and 574, which is similar to that of a blank group, and the signal intensity of the serum from HepG2 source is greatly different, so that the method has good selectivity for detecting the exosome protein and is not easy to be interfered by other substances (t test, P < 0.01).
FIG. 9 shows the detection of three exosome proteins (GPC-3, EGFR, PD-L1) from different cell lines based on SERS-sensing platforms according to the invention. As shown, in the LO2 exosomes derived from the blank and normal liver cell lines, all three proteins were expressed negatively. EGFR and PD-L1 are positively expressed in exosomes derived from liver cancer cell lines HepG2, cervical cancer cell lines HeLa and non-small cell cancer NSCLC, and GPC-3 is specifically expressed in HepG2 exosomes. Preliminary demonstration shows that the method can be used for distinguishing normal cell line exosomes from tumor cell line exosomes, and can improve the detection selectivity of HepG2 exosomes.
And detecting the exosomes in the actual simulation sample by further adopting a standard addition method based on a SERS sensing platform for synchronously and jointly detecting the exosome proteins. The medium in which the simulated sample was placed was human serum albumin dissolved in 10mM PBS solution. Taking GPC-3 protein detection as an example, exosome standard substances obtained by an ultracentrifugation method are respectively added into human serum albumin solutions with the same volume (50 mL) and the same concentration (10 mg/mL) of each group to obtain labeled samples (5×10 4～5×107 parts/mL) with different concentration gradients, the SERS sensing platform is adopted for detection, each group of experiments is repeated three times, and the corresponding recovery rate and relative standard deviation of each group are calculated.
The results are shown in Table 1. Table 1 shows the detection effect of the method on the actual sample by detecting exosomes in the actual simulated sample based on the SERS sensing platform for simultaneous combined detection of exosome proteins in the present invention.
TABLE 1 detection of exosomes in a simulated sample based on SERS sensing platforms in the present invention.
Table 1 shows the detection of exosomes in a simulated sample based on a SERS sensing platform for simultaneous, joint detection of exosome proteins in accordance with the present invention. When the concentration of the exosome standard substance is 5.11 multiplied by 10 4～5.11×107 parts per mL, the recovery rate of the method is 95.6% -108.7%, the method meets the allowable range of the standard recovery rate, and the relative standard deviation is 2.89% -4.53%, and belongs to an acceptable range, so that the method has potential application value in actual sample detection.
FIG. 10 shows the results of further validating SERS using dot immunoblotting in the present invention. Dot blot analysis is performed on the magnetic bead-exosome compound coated by silver nano particles in situ, and the fact that the surface of the magnetic bead-exosome compound is provided with blotting points of EGFR, GPC-3 and PD-L1, the exosome is used as a positive control, the blotting points of the three proteins are also provided, and gold shell magnetic beads are used as a negative control, so that no western blotting points are provided. This result further demonstrates that on HepG2 exosomes, EGFR, GPC-3, PD-L1 proteins were all expressed positively, consistent with the SERS assay results.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.
Claims (7)
1. A preparation method of a SERS sensing platform for synchronously and jointly detecting exosome proteins is characterized by comprising the following steps of
(1) Pretreating a sample by a differential centrifugation method, namely collecting cell supernatant, carrying out differential centrifugation, centrifuging for 10min at 300g, centrifuging for 10min at 2000g, centrifuging for 30min at 10000g, removing residual dead cells, cell fragments, large vesicles and other impurities in the sample, carrying out all differential centrifugation steps at 4 ℃, filtering the supernatant obtained in the last step by adopting a filter membrane of 0.22 mu m, and collecting filtrate for later use;
(2) The immune Jin Keci bead captures exosomes, 1mL,10mg/mL of CD63 antibody modified immune gold shell magnetic beads are mixed with 50mL of the pretreated sample obtained in the step (1), incubated for 2h at 4 ℃, CD63 antibody is specifically combined with CD63 protein on the surface of the exosomes, the obtained mixture is separated by a magnet for 2min, and a magnetic bead-exosome complex MB-Au@Exo is obtained and dispersed in 2.5mL of 10mM PBS;
(3) Synchronously labeling multiple exosome proteins with different Raman labels, namely coupling 1mM different Raman reporter molecules with corresponding antibody molecules of 0.1mg/mL, wherein the Raman reporter molecules are 4-mercaptobenzonitrile, 4-nitrobenzenethiol and 4-ethynylbenzenethiol as Raman labels, and the antibody molecules are EGFR antibodies, GPC-3 antibodies and PD-L1 antibodies;
the Raman labels are a 4-mercaptobenzonitrile-EGFR antibody, a 4-nitrobenzenethiol-GPC-3 antibody and a 4-ethynylbenzenethiol-PD-L1 antibody, then three labels with the same concentration of 250 mu M are simultaneously incubated with 1mL of the magnetic bead-exosome complex obtained in the step (2), after incubation for 1h at 4 ℃, the obtained mixture is separated by a magnet for 1min, and the obtained crude product is washed three times by 10mM PBS and dispersed in 1mL of 10mM PBS;
(4) Adding 100 mu M and 10 mu L distearoyl phosphatidylethanolamine-polyethylene glycol-sulfhydryl (DSPE-PEG-SH, 2 k) into 1mL of exosome obtained in the step (3), incubating for 30min at 4 ℃, inserting the DSPE-PEG-SH into a lipid membrane of the exosome to obtain a sulfhydryl-coated exosome, washing for three times by adopting 10mM PBS, redispersing in 1mL of PBS, then dropwise adding 10mM dilute ammonia water to adjust the pH=10, adding 10 mu L of 100mM silver nitrate solution and 10 mu L of 250mM ascorbic acid reducer, mixing uniformly, incubating for 1h at 37 ℃, and depositing silver nanoparticles on the exosome and gold shell magnetic beads in situ at the same time to obtain MB-Au@Exo@Ag;
(5) Preparing a gold film SERS array substrate, namely placing a positively charged anti-drop glass slide with the size of 2cm multiplied by 2.5cm into 10mL and 3mM chloroauric acid solution for incubation for 5min, adding concentrated ammonia water with the mass concentration of 28% for oscillation reaction for 1min, then cleaning for 3 times by deionized water, wherein the volume ratio of the chloroauric acid solution to the concentrated ammonia water is 50:1, placing the glass slide into 1mM sodium borohydride solution for incubation for 1min to generate light red gold nano seeds, placing the glass slide into 1mL,0.75mM chloroauric acid and 1mL,0.75mM hydroxylamine hydrochloride in an equal volume mixed growth solution, carrying out oscillation reaction for 10min at room temperature to further form a large-area rough plasma gold film distributed in an island shape, and finally assembling the glass slide with flexible PDMS array holes to obtain the gold film SERS array substrate;
(6) And (3) detecting exosome protein based on SERS synergistic enhancement effect, namely dripping 10 mu L of MB-Au@Exo@Ag obtained in the step (4) into the gold film SERS array obtained in the step (5), and detecting by using a laser confocal Raman spectrometer after a sample is dried to obtain a Raman signal corresponding to the exosome protein.
2. The method for preparing a SERS sensing platform for synchronously and jointly detecting exosome proteins according to claim 1, wherein the immuno Jin Keci beads in the step (2) are used as capture probes, and comprise a ferroferric oxide magnetic core with the particle size of about 200nm and a CD63 antibody modified gold shell outer layer;
The preparation method of the immune gold shell magnetic beads comprises the steps of adding 1mL of 20mg/mL of magnetic beads into 50mL of aqueous solution, adding 10mg/mL of chloroauric acid solution, 60mg/mL of sodium citrate solution and 5mL of sodium citrate solution respectively, carrying out reflux reaction at 100 ℃ for 30min by adopting a solvothermal method to obtain gold shell coated magnetic beads, washing with ultrapure water for three times for preservation, then modifying 20mL of 1mg/mL of gold shell magnetic beads by adopting 2mL of 0.2mg/mL of CD63 antibody in 10mM PBS medium, incubating for 1h at 37 ℃, separating by adopting a magnet to obtain the immune gold shell magnetic beads, washing with 10mM PBS for three times, and then blocking by adopting bovine serum albumin with the mass fraction of 1%.
3. The preparation method of the SERS sensing platform for synchronously and jointly detecting the exosome protein according to claim 1, wherein the different Raman labels in the step (3) comprise Raman reporter molecules and corresponding antibodies, wherein the tail end of the structure of each Raman reporter molecule contains sulfhydryl and has a benzene ring conjugated structure, N-succinimidyl 4- (maleimidomethyl) cyclohexane carboxylate SMCC is used as a cross-linking agent, maleimide at one end of the SMCC is coupled with the sulfhydryl of each Raman reporter molecule through click chemical reaction, and the succinimide group at the other end of the SMCC is coupled with amino in the antibodies to obtain the Raman reporter molecule-antibody label;
the preparation method comprises the steps of mixing 1mM of Raman reporter molecule with 1.1mM of cross-linking agent N-succinimidyl 4- (maleimidomethyl) cyclohexane carboxylate SMCC, stirring at room temperature for reaction for 1h, incubating the obtained intermediate product with 0.1mg/mL of corresponding antibody in PBS medium at 37 ℃ for 1h, and removing residual Raman reporter molecule, SMCC and intermediate product by adopting a 30KD ultrafiltration tube.
4. The preparation method of the SERS sensing platform for synchronously and jointly detecting the exosome protein, which is disclosed in claim 1, is characterized in that in the step (5), the flexible PDMS array holes are formed by uniformly mixing polydimethylsiloxane PDMS and a matched curing agent according to the mass ratio of 10:1, removing bubbles in a mixed system, pouring the mixture into a glass mold, heating the mold for 2 hours in an oven at 100 ℃, and punching the prepared PDMS film, wherein the aperture is 3mm.
5. The preparation method of the SERS sensing platform for synchronously and jointly detecting exosome multiple proteins, which is disclosed in claim 1, is characterized in that the specific test condition for detecting by adopting a laser confocal Raman spectrometer in the step (6) is 785 nm lasers, the integration time is 10s, and the laser power is 100%.
6. A SERS sensing platform for simultaneous combined detection of exosome proteins obtained according to the method of any one of claims 1-5.
7. Use of the SERS sensing platform of claim 6 in simultaneous joint analysis detection of tumor associated proteins EGFR, GPC-3, PD-L1.
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