CN107741416B - SERS (surface enhanced Raman scattering) probe and substrate marked by multiple antibodies as well as preparation method and application of SERS probe and substrate - Google Patents
SERS (surface enhanced Raman scattering) probe and substrate marked by multiple antibodies as well as preparation method and application of SERS probe and substrate Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and particularly relates to SERS probes and substrates marked by various antibodies, and a preparation method and application thereof. The invention adopts dopamine to embed and combine a plurality of antibodies under the self-polymerization condition to obtain an SERS substrate and an SERS marker probe modified by polydopamine and the antibodies, realizes the high-sensitivity detection and diagnosis of exosomes extracted from pancreatic cancer cells and serum samples of clinical pancreatic cancer patients by utilizing a Raman spectroscopy technology through immunological combination acting force, and discovers that the tumor stage judgment of pancreatic cancer can be realized by an SERS platform based on an MIF antibody through statistical data analysis. The SERS probe marking method is simple and convenient, is used for detecting pancreatic cancer exosomes, is quick and sensitive, has high specificity, requires a small amount of samples, does not need a high-speed centrifugal separation process, and can be suitable for clinical diagnosis and staged tumor metastasis judgment.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to SERS probes and substrates marked by various antibodies, and a preparation method and application thereof.
Background
Pancreatic cancer is one of the worst gastrointestinal malignant tumors, the national tumor center analyzes and estimates 2012-year malignant tumor registration data reported by national registers collected by 2015-year national tumor registration center,the number of pancreatic cancer cases is 8.6 ten thousand, and the incidence rate is 6.35/105The 10 th position in the incidence of all malignant tumors has 7.7 ten thousand deaths and the mortality rate is 5.72/105And the bit is at the 6 th bit. Pancreatic cancer lacks specific clinical manifestations and a sensitive early diagnostic marker, the common serum marker for diagnosis and follow-up is CA19-9, but the diagnostic specificity is poor. The current diagnosis and staging of pancreatic cancer depends mainly on the performance of imaging including spiral CT enhanced scanning and Magnetic Resonance Imaging (MRI), and the staging (TNM) judgment of primary tumor size (T), N-regional lymph node (N) and M-distant metastasis (M) is carried out. CT scanning time is short, imaging speed is fast, price is cheap, but radiation dose is large, and the CT scanning method cannot be used as a conventional examination method. MRI soft tissue resolution is high, detail can be displayed, radiation is not generated, but examination time is long, the limitation of scanning range is difficult to evaluate the distant metastasis, and finally pathological examination is still needed for diagnosis. Therefore, there is an urgent need to develop new methods and techniques for early diagnosis and staging judgment of pancreatic cancer with high speed, sensitivity, specificity, non-invasiveness and appropriate price.
With the ongoing progress in tumor research, researchers have found that there are small amounts of Circulating Tumor Cells (CTCs) in the blood of tumor patients, as well as small amounts of circulating tumor dna (ctdna) released by necrotic cancer cells. The method of diagnosis and monitoring of patient tumors by detecting CTCs and ctDNA in blood is known as fluid biopsy (liquid biopsy). Compared with the traditional surgical biopsy and needle biopsy methods, the liquid biopsy method can solve the difficulty of clinical sampling, meet the requirement of high-frequency detection on patients, and has the advantages of small side effect, simple operation, repeated sampling and low cost compared with the needle biopsy. Therefore, the liquid biopsy is expected to play more and more important roles in the accurate medical aspects such as early screening of tumors, dynamic monitoring of tumor patients, individualized medication and the like.
The exosomes (exosomes) are also attracting increasing attention as a class of important vesicles in human bodies, since tumor-derived exosomes contain tumor-specific proteins and miRNAs and play an important role in the growth and metastasis of tumors, and can serve as potential tumor diagnosis markers, early detection of tumors is easier to achieve by exosomes extracted from body fluids such as plasma, serum and urine, which can contain tumor-related information, and the exosomes isolated from the serum of pancreatic cancer patients have found a phosphatidylinositol glycan-1 (GPC 1) glycoprotein, but have a very low content of GPC1 in exosomes isolated from other benign pancreatic diseases and normal human blood, which is more reliable than the detection of the carbohydrate antigen CA19-9 by the conventional E L ISA, the noninvasive pancreatic cancer diagnosis method established based on the exosome markers can very accurately diagnose cancerous lesions from good pancreatic diseases, and further has a role in predicting early metastasis of tumor-promoting expression of tumor cells after pancreatic cancer biopsy, and also has a significant effect on the prognosis of tumor metastasis of tumor-promoting expression of the tumor-associated with the tumor-associated protein and miRNA.
The existing quantitative characterization methods for the exosomes mainly comprise Nanoparticle Tracking Analysis (NTA), electron microscope morphology observation, flow cytometry, and an E L ISA method or Western Blot method based on exosome surface proteins, and the methods have the advantages of large required sample amount, low sensitivity and specificity, complex and time-consuming sample treatment, small treatment flux, no clinical implementation, expensive instruments and unsuitability for clinical popularization.
The SERS immune method based on the anti-MIF has the highest sensitivity, the system is used for diagnosing a clinical pancreatic cancer serum sample, the statistical data result can obviously distinguish blood samples of a pancreatic cancer patient and a healthy person, and can classify tumor stages of different TNMs of the pancreatic cancer (whether P1,2 and P3 are transferred or not), the result coincidence degree with a pathological section is high, the method only needs a 2u L serum sample, high-speed centrifugation is not needed, and the detection time of a single sample only takes 15 minutes.
Disclosure of Invention
The invention aims to provide a SERS probe and a substrate marked by multiple antibodies (anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9 and anti-CD 63) for high-sensitivity detection of pancreatic cancer exosomes, a preparation method of the SERS probe and the substrate, and application of the SERS probe and the substrate in high-sensitivity detection of pancreatic cancer exosomes.
The invention applies the SERS immunization method of anti-MIF to the diagnosis of clinical pancreatic cancer serum samples, has the highest sensitivity and specificity, can remarkably distinguish blood samples of pancreatic cancer patients and healthy people according to the result of statistical data, can classify the tumor stages of different TNM of pancreatic cancer (P1, 2 and P3, whether metastasis occurs or not), and has high result matching degree with pathological sections.
The invention provides a preparation method of SERS probes and substrates marked by various antibodies, which comprises the following steps:
(1) adding six antibodies of anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9 and anti-CD63 into a gold-silver nanoparticle solution with a 2-20 mM core-shell structure, wherein the concentration of each antibody is 5-40 mu g/m L, the adding amount of each antibody is 30-150 mu L, adding a dopamine solution with the concentration of 1-20 mg/m L, reacting at room temperature for 0.5-4 hours, centrifuging the reacted solution, and dispersing with deionized water to obtain SERS probes marked by various antibodies;
(2) soaking a common glass slide in 8-14M NaOH for 4-7 h, adding 6-10 ml of dopamine solution with the concentration of 10-100 mg/ml into the solution, placing the culture dish on a special shaking table, shaking for 0.5-2 h, and then washing with deionized water;
placing the modified and cleaned glass slide into BSA (bovine serum albumin) containing six antibodies, namely anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9 and anti-CD63, with the volume concentration of 0.2-2%, wherein the concentration of each antibody is 1-10 mg/m L, reacting for 0.5-3 h, washing with deionized water for 1-3 times, and naturally airing to obtain a substrate.
The SERS probes and the substrates marked by the antibodies prepared by the invention can be used for high-sensitivity detection of pancreatic cancer exosomes, and the specific steps are as follows.
(1) Cell culture and exosome extraction
Culturing a pancreatic cancer cell line PANC-01 in an RPMI 1640 culture medium, adding 2-20% of fetal bovine serum, 50-150 mu g/m L benzylpenicillin and 50-150 mu g/m L streptomycin before using the culture medium, and placing the cells in a medium containing 2-10% of CO2Culturing in an incubator at 25-45 ℃ until the culture amount is enough.
And (3) extracting the exosome by adopting a high-speed centrifugation method. Firstly, placing cell culture solution in a centrifuge for 2-10 min at 500-2000 g (g is centrifugal force, sometimes expressed by rotating speed, but high-speed centrifugation is expressed by centrifugal force g, the same is applied below) and then centrifuging for 2-10 min at 1000-3000 g, removing cell debris, taking supernatant, filtering through a 0.22 mu m filter membrane, placing filtrate in an ultracentrifuge for 1-5 h at 4 ℃ and 120000-200000 g, taking lower-layer sediment, adding PBS (phosphate buffer solution) for dissolution, placing in an ultracentrifuge for 1-5 h at 4 ℃ and 120000-200000 g, abandoning supernatant, obtaining sediment, namely enriched exosome, adding PBS for dissolution, and placing in a-20 ℃ refrigerator for storage.
The characterization of the exosome adopts Nanosight NS300 of Malvern company to analyze the particle size distribution of the exosome, 2-20 mu L exosomes are taken and diluted by PBS by 10-200 times, an sCMOS is selected by a Nanosight NS300 lens, Blue488 is selected by laser, the experimental temperature is 22.4-22.6 ℃, each sample is measured for 3-6 times, the appearance of the exosome is observed by a field emission Transmission Electron Microscope (TEM), 5-20 mu L exosomes are taken and placed on a 400-mesh carbon film pattern grid, redundant liquid is wiped from the edge of the grid by a filter paper sheet, the grid is dried at room temperature, 2-20 mu L3% phosphotungstic acid solution is added, the negative dyeing is carried out at room temperature for 1.5 min, and the filter paper sheet is heated and dried under a 65 ℃ incandescent lamp for 2-5 min after absorbing the dyeing liquid.
(2) Detection of pancreatic cancer exosomes with multiple antibody-labeled SERS probes
Soaking a polydopamine-modified glass sheet (namely a substrate) in 0.01-0.1% BSA solution at 25-40 ℃ for 20-80 minutes, and then washing the glass sheet with PBS for 3-5 times; exosome solutions were diluted with PBS to different concentrations: 5.44 beta 102--2.72Χ1010Exosomes/ml; dripping 2-10 ul of exosome solution onto a polydopamine-modified glass sheet coated with a specific antibody, reacting for 20-80 minutes at 25-40 ℃, and then washing for 3-5 times by PBS; dripping 1-10 ul of SERS probe marked with a specific antibody on a corresponding glass sheet, reacting for 30-90 minutes at 25-40 ℃, and then washing with deionized water; the prepared glass sheet is subjected to spectral scanning on a Raman spectrometer (e.g., Horiba Jobinyvon, Germany) at an excitation wavelength of 785 nm.
(3) And (6) analyzing results. The SERS probe marked by the multiple antibodies obtained in the way has surface enhanced Raman signal at 1073.51cm-1Has the strongest and narrow Raman signal moleculepSpectral signal peak of ATP.
For convenience of data display, 1073.51cm in SERS spectrogram is adopted in the invention-1The logarithmic value of the raman intensity at (a) is analyzed. For different concentrations (5.44 beta 10)2to 2.72Χ1010The result shows that anti-MIF has the maximum sensitivity, anti-GPC1 has the minimum sensitivity, and the detection limits (L OD) of the four antibody platforms, namely anti-MIF, anti-GPC1, anti-EGFR and anti-EpCAM, reach 9X10-19mol/L。2uL 5.44Χ102A sample with a single/ml concentration containing only 1 exosome indicates that the SERS system of the invention is capable of single exosome detection, whereas commercial MIF E L ISA kits are minimally capable of detecting 2.72 beta 108And 2, each exosome sample with L concentration per m, and the required amount of the sample reaches 100u L. therefore, compared with the pancreatic cancer exosome detection of the commercial E L ISA kit, the sensitivity of the SERS probe immune detection of the pancreatic cancer exosomes marked by the multiple antibodies of the invention is improved by 6 orders of magnitude.
The SERS probe and the substrate marked by the multiple antibodies can be used for analyzing a serum sample of a pancreatic cancer patient and judging the tumor stage and metastasis of the pancreatic cancer.
(1) The specific steps of the centrifugation-free multi-antibody detection of the serum sample are as follows:
the method comprises the steps of diluting a serum sample by 2-5 times with PBS (phosphate buffer solution) before use, filtering with a 0.22-micrometer filter membrane, finally dropping 1-10 u L diluted sample on a polydopamine and antibody-modified glass sheet, reacting the glass sheet for 20-80 minutes at 25-40 ℃, then washing with PBS for 3-5 times, dropping 1-10 ul SERS (surface enhanced Raman scattering) probes respectively marked with anti-MIF (anti-MIF), anti-GPC1 and anti-EGFR (epidermal growth factor receptor) antibodies on the corresponding glass sheet, reacting for 30-90 minutes at 25-40 ℃, then washing with deionized water, and performing spectral scanning on the prepared glass sheet on a Raman spectrometer, wherein the wavelength of excitation light is 785 nm.
(2) Serum samples were collected from a hospital in the shanghai from 22 pancreatic cancer patients and 20 healthy people during the period from 12 months 2012 to 8 months 2016. The average age of pancreatic cancer patients was 60.45 ± 9.81 years, 9 women and 13 men, and the average age of healthy group was 57.75 ± 8.16 years, of which 7 women and 13 men. The study was conducted with approval from the ethical committee of the hospital medical research, and all patients had informed consent prior to collection of the samples. Serum samples were subjected to centrifugation-free multiple antibody detection. The statistical data was analyzed as follows: 1073.51cm is used in the obtained SERS spectrogram-1The log of the signal is analyzed. Data were plotted and analyzed for statistical data using Graph Prism 6.0, statistical resolution software SPSS21.0 and plotting software origin 7.5.
And (4) carrying out statistical analysis on the detection results of the serum samples of the pancreatic cancer patients and the healthy people. Shapiro-Wilk test showed pancreatic cancer patient groupW experiment =0.901,P=0.032<0.10 of healthy human control groupW control =0.913,P=0.072<0.10, which indicates that neither group of data exhibits a normal distribution. We used a non-parametric Mann-Whitney test on these two independent sets of samples,Z=-5.541,P<0.0001, which indicates that the two groups of samples have statistically significant differences. The average values of the pancreatic cancer patient group and the healthy human control group were 3.79. + -. 0.11 and 2.08. + -. 0.218, respectively. The results show that the method adopting the SERS probe marking based on the antibody MIF can distinguish pancreatic cancer patients from healthy people, provides a reference range for clinical diagnosis, and is expected to be applied to the field of actual clinical liquid biopsy diagnosis.
Further, the present invention provides TNM staging of 17 pancreatic cancer patient samples (excluding 5 pancreatic cancer samples without TNM staging) based on the value of SERS signal intensity into two groups, P1-2 and P3 according to histopathological reports, these patient samples have been divided into normal, non-invasive (invasive to parapancreatic tissues such as adipose tissue, nerves, duodenal mucosa, bile ducts), metastatic (metastatic to liver, spleen, adrenal gland, lymph nodes) based on SERS data of anti-MIF statistical results from Mann-Whitney tests found that there is a statistically significant difference between normal, P2-2, metastatic and invasive non-metastatic groups (P < 0.05) and in agreement with histopathological reports we surprisingly found that SERS data of anti-MIF simultaneously distinguished P3875 tumor staging from P64-2 staging, which means that the present invention can provide more accurate tumor diagnosis for further staging of tumors and achieve accurate tumor diagnosis, and further the present invention provides more accurate results in human serum samples than ISA 5 and ISA 3-869, and further shows that there is more accurate results in human serum samples.
In addition, the invention also adopts an anti-GPC1 and anti-EGFR modified SERS system to test and analyze the statistical data result of pancreatic cancer patients and healthy human serum samples. The result shows that the SERS system based on the two antibodies can also distinguish healthy people from pancreatic cancer, the healthy people and PT 1-2 stage, and the healthy people and the pancreatic cancer have no metastasis after infiltration, but cannot distinguish PT 1-2 stage and PT3 stage of the pancreatic cancer, and cannot distinguish metastatic blood samples from non-metastatic infiltration blood samples.
Compared with the existing reports, the method for labeling the exosome-associated Migration Inhibitory Factor (MIF) -labeled SERS probe is simple and convenient, is used for detecting pancreatic cancer exosomes, is rapid and sensitive, has high specificity and small required sample amount, does not need a high-speed centrifugal separation process, realizes the detection of serum samples of clinical pancreatic cancer patients and healthy people, and can be used for the metastasis and staged judgment of pancreatic cancer. The invention is also suitable for the development and application of other various antibody SERS platforms.
(1) The method for synthesizing the SERS probe is simple and convenient: the glass sheet modification and SERS antibody labeling method is carried out by adopting a mild embedding method, complex instruments are not needed, harsh conditions such as high-temperature strong acid, strong base and the like are not needed, the reaction conditions are very mild, and complex special chemical reaction reagents are not needed. The glass sheet is uniformly modified, the SERS marker is high in stability, and aggregation can not occur after the glass sheet is placed for months.
(2) Fast and efficient: after the cell line or clinical serum sample is added, the reaction only needs 1 hour to achieve effective capture. Serum samples need only be filtered through a 0.22um membrane without the need for complex and time-consuming high-speed centrifugation processes. The SERS signal is collected quickly.
(3) The sensitivity is high: the minimum detected concentration of exosomes was 9X10-19The mol/L, 2u L sample only contains 1 exosome, so the detection of a single exosome can be realized.
(4) The specificity is high: the system is used for detecting clinical complex sample serum, and is only filtered by a 0.22um filter membrane, and other components do not interfere with detection. The detection of clinical blood samples can significantly distinguish blood samples of pancreatic cancer patients from those of healthy people, so the method can be used for early diagnosis of clinical pancreatic cancer.
(5) Can be used for auxiliary diagnosis: the SERS probe method of the MIF antibody can be used for distinguishing different stages of pancreatic cancer patients and can assist doctors in carrying out stage judgment. The serum detection has no wound to the patient, is a non-invasive liquid biopsy method, and is particularly suitable for the high-frequency postoperative monitoring and curative effect evaluation of the tumor.
(6) Strong expansibility: besides anti-MIF, other antibody-labeled SERS methods can be used for diagnosis and staging of pancreatic cancer, but detection performance varies due to differences in binding force and specificity of antibodies. However, the method can select an antibody with higher sensitivity and stronger specificity to establish a SERS labeling method suitable for other diseases, and can also combine a plurality of antibodies.
Drawings
Fig. 1 is a diagram of SERS marker, exosome. Wherein, (A) TEM picture of exosome extracted from pancreatic cancer cell PANC-01 cell line, and (B) NTA particle size distribution diagram of exosome extracted from pancreatic cancer cell PANC-01.
FIG. 2 is a Raman spectrum diagram of an exosome extracted from an anti-CD9/CD63/MIF/GPC1 SERS marker system used for detecting PANC-01 cell line.
Fig. 3 is a raman spectrum of the SERS marker. Wherein (A) is a Raman spectrogram of SERS markers constructed by anti-MIF under different exosome concentrations, and (B) is 1073.51cm-1The variation curve of Raman signal intensity along with the concentration of the exosome, (C) the linear standard working curve of the Raman intensity and the concentration of the exosome.
Fig. 4 is a histogram of the raman signal intensity distribution and analysis of test statistics for a set of SERS-labeled serum samples. The Raman signal intensity distribution histogram of the pancreatic cancer patient serum sample group based on the anti-MIF SERS marker is shown in the formula (A), the Raman signal intensity distribution histogram of the anti-MIF SERS marker healthy human serum sample group is shown in the formula (B), and the Raman signal intensity distribution histogram of the serum sample group is shown in the formula (C).
FIG. 5 is a diagram of the Shapiro-Wilk statistical analysis of staging and metastasis determinations for serum samples from pancreatic cancer patients. Wherein, the stage judgment of pancreatic cancer (A) distinguishes PT2-1 and PT3, and the infiltration or metastasis judgment of pancreatic cancer (B) is carried out.
Detailed Description
The invention is further described below by means of specific examples.
Example 1:
(1) preparation of SERS (surface enhanced Raman Scattering) probe and substrate marked by multiple antibodies
Adding 50 mu L10 mu g/M L six antibodies of anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9 and anti-CD63 into 5mM gold-silver nanoparticle solution with a core-shell structure, reacting with 60 mu L10 mg/M L dopamine solution at room temperature for 1 hour, centrifuging the reacted solution, dispersing with deionized water, soaking a common glass slide into 12M NaOH for 5 hours, adding 20mg/ml dopamine solution into the solution for 8ml, placing the culture dish on a special shaking bed for 1 hour, then washing with deionized water, placing the modified and washed slide into 1% BSA containing 5mg/M L anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9 and anti-CD63 for 2 hours, reacting with 1% deionized water, drying, and naturally washing with water for 3 hours.
(2) Cell culture and exosome extraction
Pancreatic cancer cell line PANC-01 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 60. mu.g/m L benzylpenicillin, and 80. mu.g/m L streptomycin before use, and cells were plated in 6% CO2Incubate at 35 ℃ until a sufficient number of cells are obtained.
The exosome is extracted by a high-speed centrifugation method. Firstly, placing the culture solution in a centrifuge, centrifuging for 5min at 1200 g, centrifuging for 6min at 2000g to remove cell debris, taking supernatant, filtering through a 0.22 mu m filter membrane, placing the filtrate in an ultracentrifuge, centrifuging for 2h at 140000 g at 4 ℃, taking lower-layer precipitate, adding PBS for dissolving, placing in the ultracentrifuge, centrifuging for 2h at 140000 g at 4 ℃, discarding supernatant, namely, precipitating to obtain enriched exosome, adding PBS for dissolving, and placing in a refrigerator at-20 ℃ for storage.
(3) Characterization of exosomes
The particle size distribution of exosomes is analyzed by adopting Nanosight NS300 of Malvern company, 5 mu L exosomes are taken and diluted by PBS for 20 times, the lens of the Nanosight NS300 selects sCMOS, laser selects Blue488, the experimental temperature is 22.4-22.6 ℃, each sample is measured for 4 times, the appearance of exosomes is observed by adopting a field emission Transmission Electron Microscope (TEM), 10 mu L exosomes are taken and arranged on a 400-mesh carbon film pattern layer grid, redundant liquid is wiped off from the edge of the grid by using a filter paper sheet, drying is carried out at room temperature, 10 mu L3% phosphotungstic acid solution is added, negative dyeing is carried out at room temperature for 2 min, the filter paper sheet is used for sucking the dye solution, and then heating and drying are carried out for 4min under a 65 ℃ incandescent lamp.
(4) SERS (surface enhanced Raman Scattering) immunoassay pancreatic cancer exosome based on multiple antibody markers
Polydopamine-modified glass slides were first soaked with 0.05% BSA solution for 50 minutes at 30 ℃ and then rinsed 4 times with PBS. The exosome solution was diluted to different concentrations with PBS, 3ul of exosome solution was dropped onto a polydopamine-modified glass slide coated with a specific antibody, reacted at 36 ℃ for 30 minutes, and then washed 3 times with PBS. And then 3ul of SERS probe marked with specific antibody is dropped on the corresponding glass sheet, and reacts for 60 minutes at 35 ℃, and then is washed by deionized water. The prepared glass sheet was subjected to spectral scanning on a Raman spectrum (horiba JobinYvon, Germany) with an excitation wavelength of 785 nm.
(5) Centrifugation-free multiple antibody detection of serum samples
Serum samples of 10 pancreatic cancer patients and 11 healthy people from 2012 to 2016 over 8 months were collected from Shanghai Changhai Hospital, the pancreatic cancer patients had an average age of 60.45 + -9.81 years, 4 women and 6 men, and the healthy group had an average age of 57.75 + -8.16 years, of which 4 women and 7 men, the study was performed under approval of the Changhai Hospital ethical Committee for medical research, all patients had informed consent before collecting the samples, the serum samples were diluted 3-fold with PBS before use, then filtered with 0.22um filter, and finally 2u L diluted samples were dropped onto polydopamine, antibody-modified glass slides, which were reacted for 50 minutes at 30 ℃ and then washed 3 times with PBS, 2ul probes, respectively labeled with anti-MIF, anti-SERS 1, anti-EGFR antibodies, were dropped onto corresponding glass slides, prepared for 50 minutes at 30 ℃, then ion-reacted for 3 minutes, and scanned with a spectrometer, excited by a spectrometer, and washed with 785nm water.
(6) The SERS spectrogram obtained by statistical data analysis adopts 1073.51cm-1The log of the signal is analyzed. Data were plotted and analyzed for statistical data using Graph Prism 6.0, statistical resolution software SPSS21.0 and plotting software origin 7.5.
Example 2:
(1) preparation of SERS (surface enhanced Raman Scattering) probe and substrate marked by multiple antibodies
Adding antibodies such as 80 mu L20 mu g/M L anti-i-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63 and the like into an 8mM gold-silver nanoparticle solution with a core-shell structure, reacting with a 10mg/M L dopamine solution of 60 mu L at room temperature for 1 hour, centrifuging the solution after reaction, dispersing with deionized water, soaking a common glass slide in 12M NaOH for 5 hours, adding 8ml of 20mg/ml dopamine solution into the solution, placing the culture dish on a special shaking bed for 1 hour, then washing with deionized water, placing the modified and washed slide in a 1% concentration BSA containing 10mg/M L anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9 and anti-CD63 for 2 hours, reacting with deionized water, and drying in air, and washing with water for 3 hours.
(2) Cell culture and exosome extraction
Pancreatic cancer cell line PANC-01 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 60. mu.g/m L benzylpenicillin, and 80. mu.g/m L streptomycin before use, and cells were plated in 6% CO2Incubate at 35 ℃ until a sufficient number of cells are obtained.
The exosome is extracted by a high-speed centrifugation method. Firstly, placing the culture solution in a centrifuge, centrifuging for 5min at 1200 g, centrifuging for 6min at 2000g to remove cell debris, taking supernatant, filtering through a 0.22 mu m filter membrane, placing the filtrate in an ultracentrifuge, centrifuging for 2h at 140000 g at 4 ℃, taking lower-layer precipitate, adding PBS for dissolving, placing in the ultracentrifuge, centrifuging for 2h at 140000 g at 4 ℃, discarding supernatant, namely, precipitating to obtain enriched exosome, adding PBS for dissolving, and placing in a refrigerator at-20 ℃ for storage.
(3) The characterization of the exosome adopts Nanosight NS300 of Malvern company to analyze the particle size distribution of the exosome, 5 mu L exosome is taken and diluted by PBS by 20 times, the lens of the Nanosight NS300 selects sCMOS, laser selects Blue488, the experimental temperature is 22.4-22.6 ℃, each sample is measured for 4 times, the appearance of the exosome is observed by a field emission Transmission Electron Microscope (TEM), 10 mu L exosome is taken and placed on a 400-mesh carbon film layer grid, redundant liquid is wiped off from the edge of the grid by a filter paper sheet, the filter paper sheet is dried at room temperature, 10 mu L3% phosphotungstic acid solution is added, the negative dyeing is carried out at room temperature for 2 min, the filter paper sheet is used for sucking the dyeing liquid, and then the heating and drying are carried out for 4min under a 65 ℃ incandescent lamp.
(4) SERS (surface enhanced Raman Scattering) immunoassay pancreatic cancer exosome based on multiple antibody markers
Polydopamine-modified glass slides were first soaked with 0.05% BSA solution for 50 minutes at 30 ℃ and then rinsed 4 times with PBS. The exosome solution was diluted to different concentrations with PBS, 3ul of exosome solution was dropped onto a polydopamine-modified glass slide coated with a specific antibody, reacted at 36 ℃ for 30 minutes, and then washed 3 times with PBS. And then 3ul of SERS probe marked with specific antibody is dropped on the corresponding glass sheet, and reacts for 60 minutes at 35 ℃, and then is washed by deionized water. The prepared glass sheet was subjected to spectral scanning on a Raman spectrum (horiba JobinYvon, Germany) with an excitation wavelength of 785 nm.
(5) Centrifugation-free multiple antibody detection of serum samples
Serum samples of 12 pancreatic cancer patients and 9 healthy people from 2012 to 2016 over 8 months were collected from Shanghai Changhai Hospital, the pancreatic cancer patients had an average age of 60.45 + -9.81 years, 5 women and 7 men, and the healthy group had an average age of 57.75 + -8.16 years, of which 3 women and 6 men, the study was performed under approval of the Changhai Hospital ethics Committee for medical research, all patients had informed consent before collecting the samples, the serum samples were diluted 5-fold with PBS before use, then filtered with a 0.22um filter, and finally 6u L diluted samples were dropped onto polydopamine, antibody-modified glass slides, reacted for 70 minutes at 37 ℃ and then washed 5 times with PBS, 5ul probes labeled with anti-MIF, anti-SERS 1, anti-EGFR antibodies, respectively, were dropped onto corresponding glass slides, prepared at 37 ℃ and reacted for 70 minutes, and then rinsed with deionized water at 785nm for excitation spectroscopy.
(6) And (4) carrying out statistical data analysis, wherein Graph Prism 6.0, statistical analysis software SPSS21.0 and mapping software origin7.5 are adopted to carry out mapping and statistical data analysis on the obtained SERS data.
Claims (1)
1. A high-sensitivity detection method for pancreatic cancer cell exosomes is characterized by comprising the following steps:
(1) cell culture and exosome extraction
Culturing a pancreatic cancer cell line PANC-01 in an RPMI 1640 culture medium, adding 2-20% of fetal bovine serum, 50-150 g/m L benzylpenicillin and 50-150 g/m L streptomycin before using the culture medium, and placing the cells in a medium containing 2-10% of CO2Culturing the cells in the incubator at 25-45 ℃ until the cells have enough number;
extracting exosome by adopting a high-speed centrifugation method: firstly, placing cell culture solution in a centrifuge for centrifuging for 2-10 min at 500-2000 g, then centrifuging for 2-10 min at 1000-3000 g, removing cell fragments, filtering supernatant liquor by a 0.22 mu m filter membrane, placing filtrate in an ultracentrifuge for centrifuging for 1-5 h at 4 ℃ and 120000-200000 g, adding PBS (phosphate buffer solution) to dissolve sediment on a lower layer, then placing in an ultracentrifuge for centrifuging for 1-5 h at 4 ℃ and 120000-200000 g, discarding supernatant liquor, obtaining sediment which is enriched exosome, adding PBS to dissolve, and placing in a refrigerator at-20 ℃ for storage;
(2) method for detecting pancreatic cancer cell exosomes by using SERS (surface-enhanced Raman scattering) probe and SERS substrate labeled by multiple antibodies
The SERS probe marked by various antibodies is prepared by adding six antibodies of anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9 and anti-CD63 into a gold-silver nanoparticle solution with a 2-20 mM core-shell structure, wherein the concentration of each antibody is 5-40 mu g/m L, the adding amount of each antibody is 30-150 mu L, adding a dopamine solution with the concentration of 1-20 mg/m L to 40-150 mu L, reacting for 0.5-4 hours at room temperature, centrifuging the reacted solution, and dispersing with deionized water to obtain SERS probes marked by various antibodies;
the preparation method of the SERS substrate marked by the multiple antibodies comprises the following steps of putting a common glass slide into a culture dish containing 8-14M NaOH solution, soaking for 4-7 hours, adding 6-10 ml of dopamine solution with the concentration of 10-100 mg/ml into the solution, putting the culture dish on a special shaking table, shaking for 0.5-2 hours, and then washing with deionized water, putting the modified and washed glass slide into BSA containing six antibodies anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9 and anti-CD63 and having the volume concentration of 0.2-2%, reacting for 0.5-3 hours, wherein the concentration of each antibody is 1-10 mg/m L, washing with deionized water for 1-3 times after the reaction is finished, and naturally airing to obtain the SERS substrate marked by the multiple antibodies;
firstly, soaking a SERS substrate marked by various antibodies in 0.01-0.1% BSA solution for 20-80 minutes at 25-40 ℃, then washing the SERS substrate for 3-5 times by PBS, and diluting the exosome solution obtained in the step (1) by PBS to the concentration of 5.44 × 102~2.72×1010Exosomes/ml; dripping 2-10 ul of diluted exosome solution onto SERS substrates marked by various antibodies, reacting for 20-80 minutes at 25-40 ℃, and then washing for 3-5 times by PBS; dripping 1-10 ul of SERS probes marked by multiple antibodies onto the SERS substrates marked by the multiple antibodies, reacting for 30-90 minutes at 25-40 ℃, and then washing with deionized water; carrying out spectrum scanning on the SERS substrates marked by various antibodies on a Raman spectrometer, wherein the wavelength of excitation light is 785 nm;
(3) and (4) analyzing results: the obtained SERS spectrogram adopts 1073.51cm-1The logarithmic value of the signal was analyzed at 1073.51cm-1Has the strongest and narrow Raman signalSpectral signal peaks for molecule No. pATP.
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