CN114277106A - Method for counting multiple subpopulations of extracellular vesicles through single vesicle membrane protein expression profiling analysis and application of method - Google Patents
Method for counting multiple subpopulations of extracellular vesicles through single vesicle membrane protein expression profiling analysis and application of method Download PDFInfo
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Abstract
The invention discloses a method for counting multiple subpopulations of extracellular vesicles by analyzing expression profiles of single vesicle membrane proteins and application of the method, wherein the method comprises the following steps: incubating Extracellular Vesicles (EVs) with an antibody-conjugated nucleic acid complex, said antibody-conjugated nucleic acid complex being expressed universally by EVs and/or being conjugated with specific antibodies and nucleic acids; and then preparing a reaction system by the mixture of the EVs and the antibody coupled nucleic acid compound together with a primer and a probe, generating micro droplets by a droplet generator to realize the wrapping of a single EV, carrying out thermal cycle amplification reaction on the droplets by a PCR (polymerase chain reaction) instrument, and identifying and reading the droplets by the droplet generator so as to separate and analyze the EVs subgroup. The invention can realize the digital absolute quantification of EVs, and can realize the accurate detection and analysis of multiple EV subgroups only by replacing the antibody; the technology is simple, stable and mature, can realize clinical disease diagnosis by using a common PCR instrument, and is beneficial to clinical popularization and transformation.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a method for counting multiple subpopulations of extracellular vesicles by analyzing a single vesicle membrane protein expression profile and application thereof.
Background
Extracellular Vesicles (EVs) are membrane structures with lipid bilayer membranes of 30-2000nm diameter that are released from cells into the extracellular environment. Cells encapsulate specific bioactive molecules such as microRNAs, proteins, etc. into or bind to the surface of EVs, and are stably present in almost all biological fluids, such as serum, urine, amniotic fluid, cerebrospinal fluid, saliva, etc., and can be obtained in a non-invasive manner. From the first discovery of EVs, studies on EVs have been carried out with a rapid pace. Particularly, in recent years, with the intensive research on the action mechanism of EVs and the relationship between EVs and diseases, EVs is proved to be a novel biomarker with wide application prospect. However, EVs can be secreted by any nucleated cell, and indeed, those subpopulations of EVs that have disease-specific proteins or nucleic acids are truly diagnostic for disease. Therefore, accurate detection of specific subsets of EVs is currently an area of research that is of increasing interest for the diagnosis of diseases by EVs, and research on subsets of EVs can advance accurate diagnosis and treatment of diseases in human individuals.
Because the traditional EVs detection technology, such as Nanoparticle Tracking Analysis (NTA) determination of EVs concentration, Enzyme-linked immunosorbent assay (ELISA) or immuno-blot assay (Western blot) evaluation of EVs protein content, Northern blotting, microarray hybridization, RT-PCR and the like, needs a large sample amount, has complicated operation steps, and is not suitable for clinical detection. In recent years, the EVs detection technology is mature, a batch of EVs detection platforms such as optical, electrical (electrochemistry and electrodynamics) and small microfluidic technology platforms are developed, the sensitivity and specificity of EVs detection are obviously improved, the detection time is shortened, and the EVs detection platform is used for diagnosis and prognosis monitoring of diseases such as prostate cancer, liver cancer and colorectal cancer. However, these detection techniques all stay at the EVs batch analysis level, neglecting individual differences of EVs, neglecting, masking or losing molecular information carried by a single EV, and limiting accurate analysis of EVs.
The single-vesicle analysis technology platform can realize the digital ultra-sensitive detection of EVs and realize the accurate analysis of specific EVs subgroups. Therefore, a large number of scholars are attracted to study the problem. Currently, single vesicle analysis platforms for EVs specific protein subsets have been reported in the literature. Such as: kabe Y et al reported an EVs counting platform (Exocouter) which can count protein expression double positive EVs, and currently, the technology has been converted into a commercial instrument by the company Hesimecon, however, the detection cost of the technology is expensive (400-; he D and the like have developed a single-molecule imaging technology based on Total Internal Reflection Fluorescence (TIRF) technology, which can also realize the detection of a single EVs membrane protein, but the technology has higher requirements on fluorescence imaging equipment and currently only stays in scientific research. The research team of the applicant also focuses on the research of single EVs detection in disease diagnosis for a long time, and inspires the digital detection of rare nucleic acid single molecules by droplet digital PCR (droplet digital PCR) in the early work, so that a novel method for the digital detection of EVs is developed, the digital detection of single EVs membrane protein is realized, and the method is successfully used for the diagnosis of breast cancer (Liu C)#,Xu X#,Li B,Situ B,Pan W,Hu Y,An T,Yao S*,Zheng L*Single-Exosome-Counting Immunoassays for Cancer diagnostics Nano Letters,2018,18(7): 4226-. However, this technique is only an analysis of one specific subset of membrane proteins EVs, which greatly limits the classification analysis of multiple subsets of EVs. At present, the research of the single vesicle protein expression profiling for the multi-subgroup EVs digital detection new method and the disease early diagnosis based on the droplet microfluidic technology is still a hotspot and difficulty in the EVs research field.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for single vesicle membrane protein expression profiling for counting multiple subpopulations of extracellular vesicles and uses thereof.
To achieve the above and other related objects, a first aspect of the present invention provides a method for single vesicle membrane protein expression profiling for counting multiple subpopulations of extracellular vesicles, comprising the steps of:
incubating Extracellular Vesicles (EVs) with antibody-conjugated nucleic acid complexes that are universally expressed by Extracellular Vesicles (EVs) and/or are conjugated with specific antibodies and nucleic acids; and then preparing a reaction system by a mixture of Extracellular Vesicles (EVs) and antibody-coupled nucleic acid complexes, a primer and a probe, generating micro droplets by a droplet generator to realize the wrapping of single EV, performing thermal cycle amplification reaction on the droplets by a PCR (polymerase chain reaction) instrument, and identifying and reading the droplets by the droplet generator so as to separate and analyze the EVs subgroup.
Further, the Extracellular Vesicles (EVs) are extracted from cell supernatants or derived from blood/plasma samples.
Further, the cell supernatant is derived from breast cancer cells; preferably, the breast cancer cells are selected from at least one of MCF7 cells, MDA-MB-231, SKBR-3.
Further, the blood/plasma sample is derived from a breast cancer patient.
Further, the method for extracting Extracellular Vesicles (EVs) from the cell supernatant includes the steps of: centrifuging the cell suspension, removing supernatant, adding a cell culture medium containing fetal calf serum, blowing, uniformly mixing, transferring to a cell culture dish, performing constant-temperature culture, performing cell passage when the cell growth density reaches 70-80%, adding a serum-free cell culture medium when the cell growth density reaches 60-70% for cell starvation treatment, adding the cell culture medium without the exosome fetal calf serum for continuous culture, collecting cell supernatant, performing high-speed centrifugation, sucking the supernatant, performing ultracentrifugation, removing the supernatant, adding PBS (phosphate buffer solution) for resuspension, and performing ultracentrifugation, namely extracting Extracellular Vesicles (EVs) from the cell supernatant.
Further, in the method for extracting Extracellular Vesicles (EVs) from a cell supernatant, the cell culture medium containing fetal calf serum is a cell culture medium containing 10% fetal calf serum, the cell culture medium containing exosome fetal calf serum is a cell culture medium containing 1-3% exosome fetal calf serum, and the cell culture medium is at least one selected from DMEM medium, MEM medium and 1640 medium.
Further, in the method for extracting Extracellular Vesicles (EVs) from the cell supernatant, after collecting the cell supernatant, centrifuging at 3000g for 20min, after sucking the supernatant, centrifuging at 16000g for 30min, and sucking the supernatant; then 135000g was ultracentrifuged for 70min, the supernatant was discarded, and resuspended with PBS, 135000g was ultracentrifuged for 70min, and Extracellular Vesicles (EVs) from which the cell supernatant was derived were extracted.
Further, the method for extracting Extracellular Vesicles (EVs) from a blood/plasma sample comprises the following steps: and (3) centrifuging the blood sample, sucking plasma to obtain a plasma sample, diluting the plasma sample with PBS, centrifuging, sucking supernatant, adding PBS, ultracentrifuging, discarding supernatant, and then resuspending with PBS to obtain plasma EVs.
Further, the method for extracting Extracellular Vesicles (EVs) from a blood/plasma sample comprises the following steps: centrifuging the blood sample after anticoagulation treatment, sucking plasma to obtain a plasma sample, and mixing the plasma sample according to the ratio of 1: 1 in PBS, centrifuging for 20min at 3000g, sucking the supernatant, centrifuging for 30min at 16000g, sucking the supernatant, adding PBS, ultracentrifuging for 40min at 120000g, discarding the supernatant, and then resuspending with PBS to obtain plasma EVs.
Further, the antibody-conjugated nucleic acid complex is formed by coupling Extracellular Vesicles (EVs) with universal expression and/or specific avidin antibodies and biotinylated nucleic acid chains.
Further, the antibody universally expressed by the Extracellular Vesicles (EVs) is selected from at least one of CD9, CD63 and CD81, the antibody specific to the Extracellular Vesicles (EVs) is selected from at least one of HER2 and EpCAM, the nucleic acid is selected from at least one of T1, T2 and T3, and the nucleotide sequences of the T1, the T2 and the T3 are respectively shown as SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO. 11.
Further, the primer is selected from at least one combination of Forward primer1 and Reverse primer1, Forward primer2 and Reverse primer2, Forward primer3 and Reverse primer3, and the nucleotide sequences of the Forward primer1, the Forward primer1, the Forward primer2, the Reverse primer2, the Forward primer3 and the Reverse primer3 are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12 and SEQ ID NO. 13.
Further, the Probe is selected from at least one of Probe1, Probe2 and Probe3, and the nucleotide sequences of Probe1, Probe2 and Probe3 are shown as SEQ ID No.4, SEQ ID No.8 and SEQ ID No.14 respectively.
Further, the antibody-conjugated nucleic acid complex is combined with a primer and a probe, and the primer and the probe are selected from at least one of the following combination modes:
(1) CD9-T1 complex, Forward primer1, Reverse primer1 and Probe 1;
(2) CD63-T1 complex, Forward primer1, Reverse primer1 and Probe 1;
(3) CD81-T1 complex, Forward primer1, Reverse primer1 and Probe 1;
(4) EpCAM-T2 complex, Forward primer2, Reverse primer2 and Probe 2;
(5) HER2-T3 complex, Forward primer3, Reverse primer3 and Probe 3.
Further, the antibody-conjugated nucleic acid complex is purified by avidin magnetic beads.
Further, the 5 'end and the 3' end of the probe respectively carry a fluorescent group (fluorophore) and a fluorescence quenching group (quencher), the fluorescent group includes but is not limited to HEX, FAM, CY5, TAMRA and the like, and the quenching group includes but is not limited to MGB, BHQ1, BHQ3, IOWA and the like.
Further, the preparation method of the antibody-conjugated nucleic acid complex comprises the following steps:
a1, preparation of avidin antibody: treating the antibody by using a streptavidin coupling kit to obtain an avidin antibody;
a2, preparation of antibody-conjugated nucleic acid complex: and mixing the avidin antibody and biotinylated nucleic acid chain for reaction to obtain the antibody coupled nucleic acid compound.
Further, in the step A1, the antibody is at least one selected from the group consisting of CD9, CD63, CD81, HER2 and EpCAM.
Further, in the step A1, the Streptavidin coupling Kit is Streptavidin coupling Kit-
And (2) the kit, wherein the antibody is mixed with the LL-modifier reagent in the kit and then reacts for 3-16h at room temperature in a dark place, after the reaction is finished, the LL-querher reagent is added into the obtained mixed solution and reacts for 30-60min at room temperature to obtain the avidin antibody.
Further, in the step A2, the nucleic acid strand is selected from at least one of T1, T2 and T3, and the nucleotide sequences of the T1, the T2 and the T3 are respectively shown as SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO. 10.
Further, in the step A2, CD9 and T1, CD63 and T1, CD81 and T1, EpCAM and T1, and HER2 and T3 are reacted to obtain antibody-conjugated nucleic acid complexes.
Further, in the step A2, the mixing reaction time is 30-60min, preferably 60 min.
Further, the amount of biotinylated nucleic acid used in step a2 was higher than that used for the avidinated antibody, since a purification step was followed.
Further, the preparation method of the antibody-conjugated nucleic acid complex further comprises the steps of A3, purification of the antibody-conjugated nucleic acid complex: and D, diluting the antibody-coupled nucleic acid compound prepared in the step A2 with PBS, adding avidin magnetic beads, uniformly mixing for reaction, standing after the reaction is finished, sucking the supernatant, adding the avidin magnetic beads, repeating the operation, purifying for a plurality of times, and sucking the supernatant.
Further, in the step a3, the number of purification times is 5 to 12, preferably 8 to 10.
Further, in the step A3, the reaction time is 30-60min after the avidin magnetic beads are added, and the standing time is 2-10 min after the reaction is finished.
Further, in the step A3, the antibody-conjugated nucleic acid complex is diluted 1000 times after being prepared, wherein 1. mu.L of the avidin magnetic beads is added per 100. mu.L of the system.
Further, incubating the purified antibody-conjugated nucleic acid compound with Extracellular Vesicles (EVs), and staining cell nuclei after the incubation is finished; preferably, the cell nuclei are stained with a nuclear dye; preferably, the nuclear dye is selected from one of Hoechst and DAPI.
Further, the thermocycling amplification reaction procedure is as follows: the temperature is 95 ℃: 5 min; ② 94 ℃ C: 30 s; ③ 60 ℃: 2 min; (ii) + (iii): 50 cycles; fourthly, 4 ℃: and f, infinity.
In a second aspect, the invention provides a multiplex detection system, which comprises a mixture of Extracellular Vesicles (EVs) and antibody-conjugated nucleic acid complexes, primers and probes, wherein the antibody-conjugated nucleic acid complexes are formed by universally expressing the Extracellular Vesicles (EVs) and/or coupling specific antibodies and nucleic acids.
In a third aspect, the present invention provides the use of a method according to the first aspect, a multiplex detection system according to the second aspect, in the preparation of an EVs detection kit.
In a fourth aspect, the present invention provides an EVs detection kit constructed according to the method of the first aspect, or comprising the multiple detection system of the second aspect.
As described above, the method for single vesicle membrane protein expression profiling for counting multiple subpopulations of extracellular vesicles and the application thereof according to the present invention have the following beneficial effects:
1. the technology provided by the invention can realize the digital absolute quantification of EVs and can be used for the accurate classification analysis of EVs subgroups;
2. the technical platform provided by the invention is a universal detection method, and detection and analysis of multiple EV subgroups can be realized only by replacing antibodies;
3. the technical system provided by the invention is simple, stable and mature, can realize clinical disease diagnosis by using a common PCR instrument, and is beneficial to clinical popularization and transformation.
Drawings
FIG. 1 shows a schematic diagram of an experimental system of the present invention. Wherein, A is a flow chart of removing the unbound antibody nucleic acid compound by ultrafiltration after incubation of the EV and the antibody nucleic acid compound, and B is a flow chart of droplet wrapping and data analysis of a single EV.
FIG. 2 is a graph showing the results of the feasibility and activity verification experiment of the antibody-conjugated nucleic acid complex in the example of the present invention.
FIG. 3 is a graph showing the results of the concentration optimization experiment of the probe in the example of the present invention. The method comprises the following steps of A, B, C, D, E, F and F, wherein A is a FAM fluorescence channel 1D liquid drop scatter diagram before optimization, B is a FAM fluorescence channel 1D liquid drop scatter diagram after optimization, C is a CY5 fluorescence channel 1D liquid drop scatter diagram before optimization, D is a CY5 fluorescence channel 1D liquid drop scatter diagram after optimization, E is a 2D liquid drop scatter diagram before optimization, and F is a 2D liquid drop scatter diagram after optimization.
FIG. 4 is a graph showing the results of the EV ultrafiltration number optimization experiment in the example of the present invention. Wherein, A is a FAM fluorescence channel 1D droplet scattergram under different ultrafiltration times, B is a CY5 fluorescence channel 1D droplet scattergram under different ultrafiltration times, and C is a copy number histogram of targets under different ultrafiltration times.
FIG. 5 is a graph showing the results of a linear analysis of single vesicle membrane protein expression profiling for multiple subpopulations of extracellular vesicle counts in an example of the present invention. Wherein A is a fluorescence diagram of the droplet under different EV concentrations, and B is a detection linear range of the EV.
FIG. 6 is a graph showing the results of a diagnosis and analysis of breast cancer using clinical plasma samples according to the detection technique of the present invention.
Wherein A is different EV subgroups in the plasma of 1 healthy person and 1 cancer patient, B is a heat map showing the distribution of different EV subgroups in the plasma of 33 healthy, 24 tis-I, 25 II and 18 III-IV breast cancer patients, C is a bar chart showing the number of particles of 7 EV subgroups in healthy persons and different cancer stage patients, and D is a Weinn chart showing the number of particles of different EV subgroups in healthy persons and tis-I, II and III-IV stage patients.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The invention provides a method for counting multiple subpopulations of extracellular vesicles by analyzing expression profiles of single vesicle membrane proteins.
Specifically, the breast cancer is taken as a disease model, CD9/63/81 expressed universally by Extracellular Vesicles (EVs) and HER2 and EpCAM specific to diseases in the breast cancer EVs are selected as research objects, droplet microfluidics based is taken as a technical platform, and the droplet microfluidics technology is utilized to carry out single dispersed wrapping on the EVs, so that multiple detection of proteins with single EV precision is realized.
The implementation of the invention provides a new powerful tool for the molecular diagnosis and research of diseases, is helpful for further disclosing the disease development mechanism, and provides powerful technical support for promoting the wide application of EVs markers in clinical diagnosis and treatment and for early diagnosis, staging, prognosis and curative effect evaluation of diseases.
The following specific exemplary embodiments are provided to illustrate the practice of the present invention in detail. It should also be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention, and that numerous insubstantial modifications and adaptations of the invention described above will occur to those skilled in the art. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.
Example 1
1. Principle of
The experimental design principle is shown in fig. 1. Firstly, incubating EVs extracted by ultracentrifugation and an antibody coupling nucleic acid compound, then washing by an ultrafiltration tube to remove the antibody coupling nucleic acid compound which is not specifically combined, then generating micro-droplets on a microfluidic chip by a droplet generator by using reaction systems such as pretreated EVs, primer probes and the like to realize the wrapping of a single EV, then moving an EP tube filled with the droplets to a PCR instrument to perform thermal cycle reaction, then transferring the droplets to a droplet analysis chip, and realizing the separation and analysis of specific EVs subsets through the identification and reading of the droplet analyzer.
2. Materials and methods
2.1 materials
2.1.1 cell lines
Human breast cancer cell lines: MCF7, purchased from cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences, and stored in liquid nitrogen.
2.1.2 the main reagent consumables, instruments and DNA sequence tables required by the experiment are shown in Table 1, Table 2 and Table 3 respectively:
TABLE 1 Main reagent consumables table
TABLE 2 Instrument name and manufacturer summary sheet
| Drop generating instrument | Zhejiang Dapu Biotechnology Co.,Ltd. |
| Liquid drop analysis instrument | Zhejiang Dapu Biotechnology Co.,Ltd. |
| Gene amplification instrument | Hangzhou Bori Technology Co., Ltd. |
TABLE 3 DNA sequence Listing required for the experiment
The above DNA sequences were synthesized by Biotechnology engineering (Shanghai) Co., Ltd.
2.2 methods
2.2.1 preparation and validation of antibody-conjugated nucleic acid complexes
(1) Preparation of antibody-conjugated nucleic acid complexes:
preparation of avidinated antibody: according to the Streptavidin Conjugation Kit-
The kit was prepared by mixing 10. mu.L of each antibody (CD9, CD63, CD81, HER2, EpCAM) at a concentration of 1mg/mL with 1. mu.L of LL-modifier in the kit, shaking gently, mixing well, adding each
In a mix bottle, reacting for 3 hours at room temperature without light, after the reaction is finished, adding 1 mu L of LL-querher into the mixed solution, and reacting for 30 minutes at room temperature;
preparing an antibody-coupled nucleic acid complex: respectively taking the prepared avidin antibodies CD9, CD63, CD81, HER2 and EpCAM 2 mu L into an EP tube, wherein 2 mu L of biotinylated T1 is respectively added into the EP tube added with avidin CD9, CD63 and CD81, 2 mu L of biotinylated T2 is added into the avidin EpCAM and 2 mu L of biotinylated T3 is added into avidin HER2, uniformly mixing, and reacting at room temperature for 1 h;
(2) purification of antibody-conjugated nucleic acid complexes: diluting the prepared antibody-coupled nucleic acid compound by 1000 times by PBS, wherein 1 mu L of avidin magnetic beads with the concentration of 10mg/mL (washed once by PBS and resuspended) are added into each 100 mu L of system, the mixture is placed on a sample mixer to react for 30min at room temperature at the rotating speed of 15rpm, then the mixture is placed on a magnetic frame and kept stand for 2min, supernatant is sucked into a new EP tube, 1 mu L of magnetic beads are added, the operation is repeated, the purification is carried out for 8-10 times totally, and finally the supernatant is sucked into the new EP tube for standby.
2.2.2 EVs specimen pretreatment
(1) Extraction of cell supernatant EV:
cell culture: taking out a freezing tube containing the breast cancer MCF7 cell strain frozen in a liquid nitrogen tank, quickly transferring the tube into a 37 ℃ constant-temperature water bath box to quickly dissolve the cell, adding the cell suspension in the freezing tube into a new 15mL centrifuge tube in a biological safety cabinet after the cell suspension is completely dissolved, centrifuging the tube at 800rpm for 3min, discarding the supernatant, adding 10mL of complete culture medium (90% DMEM culture medium, 10% fetal calf serum and 1% double antibody), lightly blowing and uniformly mixing the cell suspension, transferring the cell suspension into a cell culture dish (d is 10mm), culturing the cell suspension in a constant-temperature incubator at 37 ℃ and 5% CO2 for 2-3 days, observing the cell state and density condition, and carrying out passage when the cell growth density reaches 70-80%;
② cell passage: discarding old culture medium in the culture dish, washing the culture dish for 2-3 times by PBS, then adding 1mL of pancreatin, slightly shaking the culture dish to uniformly distribute the pancreatin, observing under an inverted microscope after 2min, if the cell gap is obviously increased and the cell morphology begins to become round, adding 200 microliter of fetal calf serum to stop digestion, adding 1mLPBS to lightly blow and beat the cells, transferring the cell suspension to a 15mL centrifuge tube, centrifuging at 800rpm for 3min, then discarding the supernatant, adding DMEM culture medium containing 10% of fetal calf serum to resuspend, and carrying out 1: culturing in a humidified constant-temperature incubator with 5% CO2 at 37 ℃ after 4 passages;
③ treating the cells by hungry treatment and collecting cell supernatant: discarding old culture medium in the dish when cell growth density reaches 60-70%, washing with PBS for 2-3 times, adding serum-free DMEM culture medium and 1% double antibody, culturing in incubator for 12 hr, discarding old culture medium in the dish, washing the dish with PBS for 2-3 times, adding 1-2% Exo-FBSTM(exosome-removed fetal calf serum) and a 1% double-antibody DMEM medium are continuously cultured for 48 hours, and finally MCF7 cell supernatant is collected;
pretreatment of cell supernatant: centrifuging at 3000g for 20min, sucking the supernatant, centrifuging at 16000g for 30min, and sucking the supernatant;
ultracentrifugation of cell supernatant: the supernatant is added into an ultracentrifuge tube (cat # 326823, Beckman), 135000g of ultracentrifuge is carried out for 70min, the supernatant is discarded, PBS is added for resuspension, 135000g of ultracentrifuge is carried out for 70min, and EV from MCF7 cell supernatant can be extracted.
(2) Extraction of plasma EV
Firstly, taking an EDTA anticoagulated whole blood sample, centrifuging for 15min twice at 2500g, and sucking plasma into a new EP tube;
collecting 100 mu L of plasma specimen, and mixing the sample according to the proportion of 1: diluting with PBS at a ratio of 1, centrifuging at 3000g for 20min, sucking supernatant, centrifuging at 16000g for 30min, and sucking supernatant;
③ the supernatant was put into a 4mL ultracentrifuge tube (cat # 355645, Beckman), the volume was replenished to 4mL with PBS, ultracentrifugation was carried out at 120000g for 40min, the supernatant was discarded, and it was resuspended in 50. mu.L of PBS to obtain plasma exosomes.
2.2.3 analysis of Single vesicle Membrane protein expression profiles for feasibility analysis of multiple subpopulation extracellular vesicle counting
A negative control group and an experimental group are respectively arranged, and the activity of the core 'antibody-coupled nucleic acid complex' of the method is verified through confocal microscope observation at a cell level, so that feasibility analysis is carried out:
(1) cell culture: culturing breast cancer MCF7 cells in a confocal dish, discarding the culture medium when the density reaches 80-90%, and washing with PBS for 2 times;
(2) cell fixation:
adding 1mL of 4% paraformaldehyde fixing solution into a dish, and incubating for 30min at room temperature;
discarding cell fixing solution, washing 3 times on a shaking table by using 1mL of PBS;
③ adding 1mL of 0.5 percent TritonX-100 into the dish, and processing for 10min on a shaking table;
fourthly, removing the penetrating agent, and washing the mixture for 3 times on a shaking table by using 1mL of PBS;
(3) negative control group and experimental group treatment:
negative control: adding 2 mu L of biotinylated T1-FITC with the concentration of 10 mu M to a confocal dish of MCF7 cells, and incubating for 1 h;
experimental group: adding the 2mL of purified fluorophore-labeled antibody-coupled nucleic acid complex to a confocal dish of MCF7 cells, and incubating for 1 h;
(4) and (3) cell nucleus staining: discarding the supernatant, washing with PBS for 3 times, adding 2mL of basal medium and 2 μ L of Hoechst stock solution (10mg/mL) to a final concentration of 10 μ g/mL, placing in an incubator, and incubating for 10-15 min;
(5) PBS was washed to remove excess Hoechst, 1ml PBS was added to cover the cells, and finally the cells were observed under a confocal microscope, and the results are shown in FIG. 2. As can be seen from FIG. 2, the FITC fluorescent channel of the negative control group is non-fluorescent, while the FITC fluorescent channel of the antibody nucleic acid coupling experimental group is fluorescent, which proves that the antibody and the nucleic acid are successfully coupled.
2.2.4 analysis of Single vesicle Membrane protein expression profiles for optimization of Multi-subpopulation extracellular vesicle counting conditions
(1) And (3) optimizing the probe concentration combination of the multiple detection systems:
preparing Primer mix: taking 4.5 muL of Forward Primer1, Reverse Primer1, Forward Primer2, Reverse Primer2, Forward Primer3 and Reverse Primer3 with the concentration of 100 muM respectively, adding 23 muL of TE buffer solution to make up the volume to 50 muL, and preparing 10 Xprimer mix;
secondly, setting an experimental group with probes combined at different concentrations for ddPCR detection, wherein a specific sample adding system is shown in Table 4:
TABLE 4 sample adding table for different probe concentration combination systems
The results are shown in FIG. 3. As can be seen from FIG. 3, the 1D and 2D fluorescence droplet scattergrams of the respective subsets before optimization of the probe concentration were not sufficiently separated, whereas the 1D and 2D fluorescence droplet scattergrams of the respective subsets after optimization of the probe concentration were sufficiently separated
(2) Washing frequency optimization
Treatment of control group and each experimental group: respectively taking 2 microlitres of each of CD9/63/81-T1, EpCAM-T2 and HER2-T3 which are subjected to 300KD filtration treatment (800rpm, centrifugation for 2min and filtrate taking) in each group of EP tubes, wherein the compound dosage verification group does not need to be washed, and the compound is mixed for standby application; supplementing the volume of the mixed solution to 500 μ L with PBS for the rest tubes, adding into 300KD ultrafiltration tube, vortex oscillating at 150rpm for 5s, centrifuging at 13800g for 2min, filtering the liquid completely, discarding the filtrate, adding 500 μ LPBS, repeating the above washing steps, washing for 3, 5, 7, 8 and 9 times, discarding the filtrate, adding 45 μ LPBS, blowing gently to remove the filter membrane for resuspension, and collecting into new EP tube;
preparing a premix according to table 5:
TABLE 5 premix loading System-1
And then respectively adding 5 mu L of mixed solution of the control group and the experimental group into each EP tube filled with the premixed solution, uniformly mixing, generating liquid drops, and performing amplification reaction in a thermal cycler according to the following procedures:
①95℃：5min；
②94℃：30s；
③60℃：2min
(②+③：50cycles)
④4℃：∞；
finally, signal reading and analysis are performed on a droplet analysis instrument of dapu biotechnology ltd, zhejiang, with the results shown in fig. 4. As can be seen from FIG. 4, the unspecifically adsorbed antibody nucleic acid complexes can be efficiently removed by 8 times of ultrafiltration washing.
2.2.5 analysis of Single vesicle Membrane protein expression profiles for multiple subpopulations of extracellular vesicle counting sensitivity and Linear Range
(1) Dilution of exosomes: diluting exosomes of a healthy person mixed plasma source by 10 times, 100 times and 1000 times by using PBS respectively;
(2) taking 3 mu L of the purified CD9-T1 compound (the total dilution multiple is 1000 times), respectively and uniformly mixing with 10 mu L of exosomes from the mixed plasma of healthy people with different dilution times, wherein the blank control group is formed by mixing PBS and the CD9-T1 compound, and reacting for 1h at room temperature;
(3) after the reaction is finished, the volume of the mixed solution is supplemented to 500 mu L by PBS, the mixed solution is added into a 300KD ultrafiltration tube, vortex oscillation is carried out for 5s at 150rpm, centrifugation is carried out for 2min at 13800g, the liquid is completely filtered, the filtrate is discarded, then 500 mu LPBS is added into the filter tube, the washing operation is repeated for 7 times, the total washing is carried out for 8 times, the filtrate is discarded at last, 45 mu LPBS is added, the filter membrane is lightly blown and used for resuspension, and the mixture is collected into a new EP tube;
(4) premix was prepared as per table 6:
TABLE 6 premix addition System-2
| Component | 20ul reaction | |
| mix | ||
| 10 | | |
| Forward Primer | ||
| 1 | 1.8 | |
| Reverse Primer | ||
| 1 | 1.8 | 900nM |
| Probe 1(10μM) | 0.6 | 300nM |
| DNase Free dH2O | 0.8 |
And (3) respectively adding 5 mu L of the resuspended liquid in each group in the step (3) into each EP tube filled with the premix, uniformly mixing, performing droplet generation, and performing amplification reaction in a thermal cycler according to the following procedures:
①95℃：5min；
②94℃：30s；
③60℃：2min
(②+③：50cycles)
④4℃：∞；
finally, signal reading and analysis are performed on a droplet analysis instrument of dapu biotechnology ltd, zhejiang, with the results shown in fig. 5. As can be seen from FIG. 5, with the dilution of EV concentration, the number of fluorescent droplets under the fluorescent channel is gradually reduced, the technology has good linear relation to single-vesicle EV expression profiling analysis, and the linear detection range reaches 4 orders of magnitude.
2.2.5 assessment of detectability of clinical specimens
(1) Centrifuging the purified CD9/63/81-T1, EpCAM-T2 and HER2-T3 complex (the total dilution multiple is 1000 times) for 2min by a 300KD ultrafiltration tube 8000rmp, taking filtrate, respectively taking 3 mu L of the filtrate and pre-treated patient plasma EV (30 healthy people and 67 breast cancer patients, wherein 24 patients with Tis-stage I, 25 patients with stage II and 18 patients with stage III-IV) to be uniformly mixed, and reacting at room temperature for 1 h;
(2) after the reaction is finished, the volume of the mixed solution is supplemented to 500 mu L by PBS, the mixed solution is added into a 300KD ultrafiltration tube, vortex oscillation is carried out for 5s at 150rpm, centrifugation is carried out for 2min at 13800g, the liquid is completely filtered, the filtrate is discarded, then 500 mu LPBS is added into the filter tube, the washing operation is repeated for 7 times, the total washing is carried out for 8 times, the filtrate is discarded at last, 45 mu LPBS is added, the filter membrane is lightly blown and used for resuspension, and the mixture is collected into a new EP tube;
(3) premix was prepared as described in Table 5 above
(4) Then, 5 μ L of the resuspended solution was added to each EP tube containing the premix, and after mixing, droplet generation and amplification reaction were performed in a thermal cycler according to the following procedures:
①95℃：5min；
②94℃：30s；
③60℃：2min
(②+③：50cycles)
④4℃：∞；
finally, signal reading and analysis are performed on a droplet analysis instrument of dapu biotechnology ltd, zhejiang, with the results shown in fig. 6. As can be seen from fig. 6, there is some variability in the EV subpopulations in the plasma of healthy persons and cancer patients, and counting the number of particles of different EV subpopulations has the potential for breast cancer diagnosis.
In conclusion, the invention establishes a new method capable of counting single vesicles, can be used for EV protein expression profile analysis, realizes absolute counting of multiple specific protein EV subgroups, and proves that the method has the potential for breast cancer diagnosis by collecting some clinical plasma samples and detecting and analyzing the plasma samples.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be accomplished by those skilled in the art without departing from the spirit and scope of the present invention as set forth in the appended claims.
SEQUENCE LISTING
<110> southern hospital of southern medical university
<120> method for counting multiple subpopulations of extracellular vesicles through single vesicle membrane protein expression profiling analysis and application thereof
<130> PCQNF2111590-HZ
<160> 12
<170> PatentIn version 3.5
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Claims (10)
1. A method for single vesicle membrane protein expression profiling for counting multiple subpopulations of extracellular vesicles, comprising the steps of:
incubating Extracellular Vesicles (EVs) with antibody-conjugated nucleic acid complexes that are universally expressed by Extracellular Vesicles (EVs) and/or are conjugated with specific antibodies and nucleic acids; and then preparing a reaction system by a mixture of Extracellular Vesicles (EVs) and antibody-coupled nucleic acid complexes, a primer and a probe, generating micro droplets by a droplet generator to realize the wrapping of single EV, performing thermal cycle amplification reaction on the droplets by a PCR (polymerase chain reaction) instrument, and identifying and reading the droplets by the droplet generator so as to separate and analyze the EVs subgroups.
2. The method of claim 1, wherein: the Extracellular Vesicles (EVs) are extracted from cell supernatants or derived from blood/plasma samples;
and/or the antibody-conjugated nucleic acid complex is formed by coupling Extracellular Vesicles (EVs) with universal expression and/or specific avidin antibodies with biotinylated nucleic acid chains;
and/or the antibody universally expressed by the Extracellular Vesicles (EVs) is selected from at least one of CD9, CD63 and CD81, the antibody specific to the Extracellular Vesicles (EVs) is selected from at least one of HER2 and EpCAM, the nucleic acid is selected from at least one of T1, T2 and T3, and the nucleotide sequences of the T1, the T2 and the T3 are respectively shown as SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO. 11;
and/or the primer is selected from at least one combination of Forward primer1 and Reverse primer1, Forward primer2 and Reverse primer2, Forward primer3 and Reverse primer3, and the nucleotide sequences of the Forward primer1, the Reverse primer1, the Forward primer2, the Reverse primer2, the Forward primer3 and the Reverse primer3 are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12 and SEQ ID NO. 13;
and/or the Probe is selected from at least one of Probe1, Probe2 and Probe3, and the nucleotide sequences of the Probe1, Probe2 and Probe3 are shown as SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO.14 respectively.
3. The method of claim 2, wherein: the cell supernatant is derived from breast cancer cells and the blood/plasma sample is derived from a breast cancer patient.
4. The method of claim 2, wherein: the antibody-conjugated nucleic acid complex, a primer and a probe are selected from at least one of the following combination modes:
(1) CD9-T1 complex, Forward primer1, Reverse primer1 and Probe 1;
(2) CD63-T1 complex, Forward primer1, Reverse primer1 and Probe 1;
(3) CD81-T1 complex, Forward primer1, Reverse primer1 and Probe 1;
(4) EpCAM-T2 complex, Forward primer2, Reverse primer2 and Probe 2;
(5) HER2-T3 complex, Forward primer3, Reverse primer3 and Probe 3.
5. The method of claim 1, wherein: the thermocycling amplification reaction procedure was as follows: the temperature is 95 ℃: 5 min; ② 94 ℃ C: 30 s; ③ 60 ℃: 2 min; (ii) + (iii): 50 cycles; fourthly, 4 ℃: and f, infinity.
6. A multiple detection system, comprising: the kit comprises a mixture of Extracellular Vesicles (EVs) and antibody-conjugated nucleic acid complexes, primers and probes, wherein the antibody-conjugated nucleic acid complexes are formed by universally expressing the Extracellular Vesicles (EVs) and/or coupling specific antibodies and nucleic acids.
7. The detection system according to claim 6, wherein: the Extracellular Vesicles (EVs) are extracted from cell supernatants or derived from blood/plasma samples;
and/or the antibody-conjugated nucleic acid complex is formed by coupling Extracellular Vesicles (EVs) with universal expression and/or specific avidin antibodies with biotinylated nucleic acid chains;
and/or the antibody universally expressed by the Extracellular Vesicles (EVs) is selected from at least one of CD9, CD63 and CD81, the antibody specific to the Extracellular Vesicles (EVs) is selected from at least one of HER2 and EpCAM, the nucleic acid is selected from at least one of T1, T2 and T3, and the nucleotide sequences of the T1, the T2 and the T3 are respectively shown as SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO. 11;
and/or the primer is selected from at least one combination of Forward primer1 and Reverse primer1, Forward primer2 and Reverse primer2, Forward primer3 and Reverse primer3, and the nucleotide sequences of the Forward primer1, the Reverse primer1, the Forward primer2, the Reverse primer2, the Forward primer3 and the Reverse primer3 are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12 and SEQ ID NO. 13;
and/or the Probe is selected from at least one of Probe1, Probe2 and Probe3, and the nucleotide sequences of the Probe1, Probe2 and Probe3 are shown as SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO.14 respectively.
8. The detection system according to claim 7, wherein: the antibody-conjugated nucleic acid complex, a primer and a probe are selected from at least one of the following combination modes:
(1) CD9-T1 complex, Forward primer1, Reverse primer1 and Probe 1;
(2) CD63-T1 complex, Forward primer1, Reverse primer1 and Probe 1;
(3) CD81-T1 complex, Forward primer1, Reverse primer1 and Probe 1;
(4) EpCAM-T2 complex, Forward primer2, Reverse primer2 and Probe 2;
(5) HER2-T3 complex, Forward primer3, Reverse primer3 and Probe 3.
9. Use of the method according to any one of claims 1 to 6, the detection system according to any one of claims 7 to 8 for the preparation of an EVs detection kit.
10. An EVs detection kit, characterized in that: constructed according to the method of any one of claims 1 to 6, or comprising the detection system of any one of claims 7 to 8.
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