CN114878418A - Method and system for separating and detecting exosome based on micro-fluidic chip - Google Patents

Method and system for separating and detecting exosome based on micro-fluidic chip Download PDF

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CN114878418A
CN114878418A CN202210431876.6A CN202210431876A CN114878418A CN 114878418 A CN114878418 A CN 114878418A CN 202210431876 A CN202210431876 A CN 202210431876A CN 114878418 A CN114878418 A CN 114878418A
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exosomes
exosome
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徐震宇
吴钍荣
陈瑛娜
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Guangzhou Zhaorui Medical Biotechnology Co ltd
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Abstract

The application relates to the technical field of exosome separation, and particularly discloses a method and a system for separating and detecting exosomes based on a microfluidic chip. The method comprises the following specific steps: fluorescent labeling of exosomes: carrying out fluorescence labeling on the sample by using exosome-labeled fluorescent molecules to obtain a labeled sample; separation of exosomes: adding the marked sample into a microfluidic chip, and separating exosomes in the marked sample by using alternating current to provide dielectrophoresis; cleaning the microfluidic chip by using a cleaning solution under the same condition to obtain an exosome; detection of exosomes: and observing the appearance of the exosome by using a transmission scanning electron microscope, and determining the concentration and particle size distribution of the exosome by using NTA. This application can effectively shorten the separation time of exosome, and accomplishes quantitative determination when realizing exosome separation.

Description

Method and system for separating and detecting exosome based on micro-fluidic chip
Technical Field
The application relates to the technical field of exosome separation, in particular to a method and a system for separating and detecting exosomes based on a microfluidic chip.
Background
Exosomes are nanoscale (30-150nm) membrane vesicles of cellular origin, playing a key role in intercellular communication. In the past years, exosomes have been identified as diagnostic biomarkers, as they are widely present in body fluids and are closely associated with disease progression. Exosomes must be isolated from a blood (plasma or serum) sample before they are analyzed for their associated RNA and protein biomarkers. Ultracentrifugation is the main technology for separating exosome at present, but the method does not meet the standard required by clinical application because the purity, yield and integrity of the exosome obtained by separation are poor, and the processing time is too long.
In recent years, other methods such as polyethylene glycol (PEG) based precipitation, phosphatidylserine affinity capture, size exclusion chromatography, and membrane affinity have emerged in particular applications with limited success rates. Recently, asymmetric flow field flow fractionation methods have been used to sort EV (extracellular vesicles) subpopulations from cell and tumor culture media at high resolution, but their throughput is affected by cumbersome sample preparation procedures. The increased requirements for input EV concentrations and the time-consuming process of a single assay also limit their widespread use.
Due to the small size and low buoyant density of exosomes, the separation of exosomes from other components in blood in the related art requires a lot of time and effort, e.g., multiple processing steps are required, including a lot of ultracentrifugation and incubation steps from a few hours to overnight or plasma proteins in blood cannot be eliminated, etc., and time consuming multi-step processes and methods may damage exosomes and reduce overall collection efficiency. In addition, most of the detection of the secretion detection amount in the related art is performed after separation, and partial functions of a sample after detection and quantification are affected, so that the sample cannot be applied later.
Therefore, there is a need to develop new techniques to improve the drawbacks of exosome isolation and purification.
Disclosure of Invention
In order to shorten the separation time of exosomes and complete quantitative detection while realizing exosome separation, the application provides a method and a system for separating and detecting exosomes based on a microfluidic chip.
In a first aspect, the present application provides a method for separating and detecting exosomes based on a microfluidic chip, which adopts the following technical scheme:
a method for separating and detecting exosomes based on a microfluidic chip specifically comprises the following steps:
(1) fluorescent labeling of exosomes: carrying out fluorescence labeling on the sample by using exosome labeled fluorescent molecules to obtain a labeled sample;
(2) separation of exosomes: adding the marked sample obtained in the step (1) into a microfluidic chip, and separating exosomes in the marked sample by using alternating current to provide dielectrophoresis; cleaning the microfluidic chip by using a cleaning solution under the same condition to obtain an exosome;
(3) detection of exosomes: and (3) observing the appearance of the exosomes obtained in the step (2) by using a transmission scanning electron microscope, and determining the concentration and particle size distribution of the exosomes obtained in the step (2) by using NTA.
The method provided by the application realizes the separation of exosomes in the sample and other substances in the sample under the action of dielectrophoresis provided by alternating current, can strictly limit mechanical damage to exosomes under the condition of not diluting the sample (serum, plasma or blood sample), can effectively reduce the processing steps of separating the sample and exosomes, and shortens the separation time of exosomes. In addition, the method provided by the application further comprises a detection part of the exosome, the exosome can be detected in time after separation, and the application of the exosome is not influenced in the detection process.
In addition, in the case of immunoaffinity separation methods in the related art, exosome separation may exclude potentially important populations, and the process becomes dependent on the selectivity, specificity, and affinity (binding constant) of the antibody. It is clear that the methods provided herein are independent of antibody affinity binding and are capable of maximizing the viability of exosome RNA and protein biomarkers for subsequent detection, identification and analysis.
The wash solution may be PBS buffer or other solution.
Preferably, the cleaning solution comprises mannitol, tris hydrochloride and fatty acid diethanolamide.
Preferably, the cleaning solution comprises the following components in parts by weight, calculated by 1000 parts by weight: 12-18.4 parts of mannitol, 4-8.8 parts of tris (hydroxymethyl) aminomethane hydrochloride and 3.5-6.0 parts of fatty acid diethanolamide.
Preferably, the cleaning solution comprises the following components in parts by weight, calculated by 1000 parts by weight: 14.5-16.2 parts of mannitol, 5.6-8.2 parts of tris (hydroxymethyl) aminomethane hydrochloride and 4.4-5.2 parts of fatty acid diethanolamide.
In a specific embodiment, the mannitol in the cleaning solution may be present in an amount of 12 parts, 14.5 parts, 15.6 parts, 16.2 parts, 18.4 parts by weight.
In some embodiments, the mannitol in the cleaning solution may be present in an amount of 12-14.5 parts, 12-15.6 parts, 12-16.2 parts, 14.5-15.6 parts, 14.5-18.4 parts, 15.6-16.2 parts, 15.6-18.4 parts, 16.2-18.4 parts by weight.
In a specific embodiment, the parts by weight of tris hydrochloride in the cleaning solution may be 4 parts, 5.6 parts, 6.8 parts, 8.2 parts, 8.8 parts.
In some embodiments, the tris hydrochloride in the cleaning solution may be present in an amount of 4 to 5.6 parts, 4 to 6.8 parts, 4 to 8.2 parts, 5.6 to 6.8 parts, 5.6 to 8.8 parts, 6.8 to 8.2 parts, 6.8 to 8.8 parts, 8.2 to 8.8 parts by weight.
In a specific embodiment, the parts by weight of the fatty acid diethanolamide in the cleaning solution may be 3.5 parts, 4.4 parts, 4.7 parts, 5.2 parts, 6.2 parts.
In some embodiments, the weight parts of the fatty acid diethanolamide in the cleaning solution may be 3.5-4.4 parts, 3.5-4.7 parts, 3.5-5.2 parts, 4.4-4.7 parts, 4.4-6.2 parts, 4.7-5.2 parts, 4.7-6.2 parts, 5.2-6.2 parts.
Mannitol is a sugar alcohol, an isomer of sorbitol. Is easily soluble in water, is white crystalline powder, and has sweet taste similar to sucrose. Tris-HCl is commonly used in biological buffers. The fatty acid diethanolamide belongs to a nonionic surfactant and is easily soluble in water.
The method further optimizes the components of the cleaning solution, and utilizes the cleaning solution consisting of mannitol, tris hydrochloride and fatty acid diethanolamide to clean the microfluidic chip to obtain the exosome. Through experimental analysis, the addition amounts of mannitol, tris (hydroxymethyl) aminomethane hydrochloride and fatty acid diethanolamide in the cleaning solution are controlled within the range, and the content of exosome obtained through separation can be further effectively improved.
Preferably, the time for cleaning the microfluidic chip by the cleaning solution is 0.5-3.5 min.
Preferably, the time for cleaning the microfluidic chip by the cleaning solution is 1-3 min.
In a specific embodiment, the time for the cleaning solution to clean the microfluidic chip may be 0.5min, 1min, 2min, 3min, 4min, 5 min.
In some specific embodiments, the time for the cleaning solution to clean the microfluidic chip may be 0.5-1min, 0.5-2min, 0.5-3min, 1-2min, 1-3.5min, 2-3min, 2-3.5 min.
Through experimental analysis, the time for cleaning the microfluidic chip by the cleaning solution is controlled within the range, and the content of the exosome obtained through separation can be further effectively improved.
Preferably, the sample injection flow rate after the marking is 3-5.5 muL/min.
Preferably, the sample injection flow rate after the marking is 4-5 muL/min.
In a specific embodiment, the sample injection flow rate after labeling can be 3 μ L/min, 4 μ L/min, 4.5 μ L/min, 5 μ L/min, 5.5 μ L/min.
In some embodiments, the sample flow rate of the labeled sample may be 3-4. mu.L/min, 3-4.5. mu.L/min, 3-5. mu.L/min, 4-4.5. mu.L/min, 4-5.5. mu.L/min, 4.5-5. mu.L/min, 4.5-5.5. mu.L/min, 5-5.5. mu.L/min.
Through experimental analysis, the sample introduction flow rate of the marked sample is controlled within the range, and the content of the exosome obtained by separation can be further effectively improved.
Preferably, the alternating current has a voltage of 5-8.5V and a frequency of 15 kHz.
Preferably, the exosome-labeled fluorescent molecule is PKH 26.
In a second aspect, the present application provides a system for exosome separation and detection using the above method, and the following technical scheme is adopted:
the system for separating and detecting the exosome by using the method specifically comprises a sample introduction part, a separation and optical imaging part and a detection part which are sequentially communicated.
Preferably, the separation and optical imaging part comprises a separation part of exosomes and an optical imaging part of an exosome separation process.
Preferably, the detection moiety comprises a transmission scanning electron microscope and NTA for analytical detection of exosomes.
The application provides an exosome separation and detecting system includes exosome's separation part and detection part, can in time carry out exosome's detection after exosome separation accomplishes, and the application of exosome is not influenced in the testing process. The system provided by the application comprises a separation part of exosomes and an optical imaging part in an exosome separation process, so that the separation condition of exosomes can be monitored in time. Meanwhile, the detection part comprises a transmission scanning electron microscope and NTA (nano particle tracking analysis), wherein the transmission scanning electron microscope is used for analyzing and detecting the exosomes, the transmission scanning electron microscope is used for observing the appearance of the exosomes, and the NTA is used for analyzing the concentration and the particle size of the exosomes. Therefore, the system can be used for completing the separation and detection of the exosome and obtaining the morphology, concentration and particle size parameters of the exosome.
In summary, the present application has the following beneficial effects:
(1) the method for separating and detecting the exosomes can effectively shorten the separation time of the exosomes, and achieves quantitative detection while the exosomes are separated, so that the exosomes are separated and quantified from body fluid in one step.
(2) The method provided by the application realizes the separation of exosomes in the sample and other substances in the sample under the action of dielectrophoresis provided by alternating current, can strictly limit mechanical damage to exosomes under the condition of not diluting the sample (serum, plasma or blood sample), can effectively reduce the processing steps of separating the sample and exosomes, and shortens the separation time of exosomes.
(3) The method provided by the application further comprises a detection part of the exosome, the exosome can be detected in time after separation, and the later application of the exosome is not influenced in the detection process.
(4) The methods provided herein are independent of antibody affinity binding and are capable of maximizing the viability of exosome RNA and protein biomarkers for subsequent detection, identification and analysis.
Drawings
Fig. 1 is a graph of the morphology, concentration and particle size distribution of exosomes obtained by the method provided in example 3 of the present application.
Fig. 2 shows the fluorescence distribution of the microfluidic chip before and after the application of the alternating current in the method provided in example 3 of the present application.
FIG. 3 shows the quantitative results of exosomes isolated from rabbit plasma at different concentrations using the method provided in example 3 of the present application.
Detailed Description
The application provides a method for separating and detecting exosomes based on a microfluidic chip, which specifically comprises the following steps:
(1) fluorescent labeling of exosomes: and carrying out fluorescence labeling on the sample by using exosome labeled fluorescent molecules to obtain a labeled sample.
Wherein, the exosome-labeled fluorescent molecule is PKH 26.
(2) Separation of exosomes: adding the marked sample obtained in the step (1) into a microfluidic chip, and separating exosomes in the marked sample by using alternating current to provide dielectrophoresis; and cleaning the microfluidic chip by using a cleaning solution under the same condition to obtain the exosome.
The cleaning solution comprises mannitol, tris hydrochloride and fatty acid diethanolamide. The cleaning solution comprises the following components in parts by weight, wherein the cleaning solution comprises the following components in parts by weight, based on 1000 parts by weight: 12-18.4 parts of mannitol, 4-8.8 parts of tris (hydroxymethyl) aminomethane hydrochloride and 3.5-6.0 parts of fatty acid diethanolamide. Further, the cleaning solution comprises the following components in parts by weight in 1000 parts by weight: 14.5 to 16.2 portions of mannitol, 5.6 to 8.2 portions of tris-hydroxymethyl aminomethane hydrochloride and 4.4 to 5.2 portions of fatty acid diethanolamide.
Wherein the voltage of the alternating current is 5-8.5V, and the frequency is 15 kHz. The sample injection flow rate after marking is 3-5.5 muL/min. The time for cleaning the microfluidic chip by the cleaning solution is 0.5-3.5 min.
(3) Detection of exosomes: and (3) observing the appearance of the exosomes obtained in the step (2) by using a transmission scanning electron microscope, and determining the concentration and particle size distribution of the exosomes obtained in the step (2) by using NTA.
The application also provides a system for separating and detecting exosomes by using the method, which specifically comprises a sample introduction part, a separation and optical imaging part and a detection part which are sequentially communicated. Wherein the separation and optical imaging part comprises an exosome separation part and an exosome separation process optical imaging part. The detection part comprises a transmission scanning electron microscope and NTA for analyzing and detecting the exosome.
Specifically, the system for separating and detecting the exosome by using the method comprises a sample introduction part, a separation and optical imaging part and a detection part which are sequentially communicated. The marked sample enters the separation and optical imaging part from the sample feeding part. The separation and optical imaging part comprises a separation part of exosomes and an optical imaging part of an exosome separation process, and the separation part comprises a microfluidic chip. And (3) the marked sample enters the microfluidic chip, and the microfluidic chip is cleaned by using a cleaning solution under the same condition under the action of dielectrophoresis provided by alternating current to obtain the exosome. The displacement change of the exosomes in the labeled sample can be observed by the optical imaging section. The obtained exosomes enter the detection section. The detection part comprises a transmission scanning electron microscope and NTA for analyzing and detecting the exosome. And observing the morphology of the obtained exosomes by using a transmission scanning electron microscope, and determining the concentration and particle size distribution of the obtained exosomes by using NTA.
The present application will be described in further detail below with reference to preparation examples 1 to 16, examples 1 to 24, and comparative examples 1 to 8.
Preparation example
Preparation examples 1 to 13
Preparation examples 1 to 13 each provide a cleaning solution.
The difference of the preparation examples is that: the amounts of the components added in the cleaning solution were varied, and are specifically shown in table 1.
The preparation method of the cleaning solution comprises the following steps: the components are dissolved in 1000ml of ddH according to the addition amount of the components 2 And (4) adding water.
TABLE 1 addition of the Components of preparation examples 1 to 13
Figure BDA0003611459440000061
Preparation examples 14 to 16
Preparation examples 14 to 16 each provide a cleaning solution.
The difference between the above preparation examples is that: the types of addition of the components in the cleaning solution were different, as shown in table 2.
TABLE 2 addition of the components in preparation example 3 and preparation examples 14 to 16
Figure BDA0003611459440000062
Figure BDA0003611459440000071
Examples
Examples 1 to 13
Examples 1-13 provide a method for the separation and detection of exosomes, respectively, based on microfluidic chips. Wherein, the sample is 1ml rabbit plasma.
The above embodiments differ in that: the types of cleaning solutions used in the process varied, as shown in table 3.
The method specifically comprises the following steps:
(1) fluorescent labeling of exosomes: and carrying out fluorescent labeling on the sample by using an exosome-labeled fluorescent molecule PKH26 to obtain a labeled sample.
(2) Separation of exosomes: and (2) adding the marked sample obtained in the step (1) into a microfluidic chip (ACE chip), and separating exosomes in the marked sample by using alternating current to provide dielectrophoresis. Wherein, the voltage of the alternating current is 5V, and the frequency is 15 kHz. The flow rate of the sample after labeling was 4.5. mu.L/min.
And cleaning the microfluidic chip by using a cleaning solution under the same condition to obtain the captured exosomes. Wherein, the time for cleaning the microfluidic chip by the cleaning solution is 2 min.
(3) Detection of exosomes: eluting the exosomes captured in the step (2) by using PBS buffer solution at the flow rate of 5 mu L/min, and eluting for 2min to obtain an exosome solution. And observing the appearance of the exosome by using a transmission scanning electron microscope, and determining the concentration and particle size distribution of the exosome by using NTA (nanoparticle tracking analysis).
The system for separating and detecting the exosome by using the method specifically comprises a sample introduction part, a separation and optical imaging part and a detection part which are sequentially communicated. And (2) enabling the marked sample obtained in the step (1) to enter a separation and optical imaging part from the sample introduction part. The separation and optical imaging part comprises a separation part of exosomes and an optical imaging part of an exosome separation process, and the separation part comprises a microfluidic chip. And (3) the marked sample enters the microfluidic chip, and the microfluidic chip is cleaned by using a cleaning solution under the same condition under the action of dielectrophoresis provided by alternating current to obtain the exosome. The displacement change of the exosomes in the labeled sample can be observed by the optical imaging section. The obtained exosomes enter the detection section. The detection part comprises a transmission scanning electron microscope and NTA for analyzing and detecting the exosome. And observing the appearance of the obtained exosomes by using a transmission scanning electron microscope, and determining the concentration and particle size distribution of the obtained exosomes by using NTA.
Table 3 types of cleaning solutions used in the processes provided in examples 1-13
Figure BDA0003611459440000081
Examples 14 to 17
Examples 14-17 provide a method for the separation and detection of exosomes, respectively, based on microfluidic chips.
The above embodiments differ in that: the time for cleaning the microfluidic chip by the cleaning solution in the method is different, and is specifically shown in table 4.
Table 4 time for washing microfluidic chip with washing solution in the methods provided in example 3 and examples 14 to 17
Example number Cleaning time (min)
3 2
14 0.5
15 1
16 3
17 3.5
18 4
19 5
Examples 20 to 23
Examples 20-23 provide a method for the separation and detection of exosomes, respectively, based on microfluidic chips.
The above embodiments differ in that: the sample injection flow rates of the marked samples in the method are different, and are specifically shown in table 5.
TABLE 5 sample feed flow rates in the methods provided in example 3 and examples 20-23
Example number Sample injection flow rate (μ L/min)
3 4.5
20 3
21 4
22 5
23 5.5
Example 24
The embodiment provides a method for separating and detecting exosomes based on a microfluidic chip.
This embodiment differs from embodiment 3 in that: the cleaning solution in the method is PBS buffer solution.
Comparative example
Comparative examples 1 to 6
Comparative examples 1 to 6 provide a method for separating and detecting exosomes based on a microfluidic chip, respectively.
The above comparative examples differ in that: the types of cleaning solutions used in the method were varied, as shown in table 6.
Table 6 types of cleaning solutions used in the processes provided by comparative examples 1-6
Comparative example No. Type of cleaning liquid
1 Preparation example 14
2 Preparation example 15
3 Preparation example 16
4 Preparation example 17
5 Preparation example 18
6 Preparation example 19
Comparative example 7
This comparative example provides a method for the isolation of exosomes using ultracentrifugation.
The method specifically comprises the following steps:
(1) centrifuging the cell supernatant at 4 deg.C and 300g for 10min, removing cells and dead cells, centrifuging, and collecting supernatant;
(2) centrifuging the supernatant obtained in the step (1) for 10min at 4 ℃ under 2000g, removing cell debris, and centrifuging to remove the supernatant for later use;
(3) centrifuging the supernatant obtained in the step (2) for 30min at 4 ℃ under 10000g, removing large membrane bubbles, and centrifuging to remove the supernatant for later use;
(4) placing the supernatant obtained in the step (3) in an ultracentrifuge tube to a position with a tube opening of 2-3mm, and placing the ultracentrifuge tube in an ultracentrifuge with the temperature of 4 ℃ and the weight of 120000g for centrifugation for 70 min; after centrifugation, removing the supernatant as much as possible (care is taken to operate, pellet enriched with the exosome is easily lost), and then resuspending the precipitate at the bottom of the ultracentrifuge tube by using DPBS (double-stranded phosphate buffer solution), thus obtaining an exosome primary sample;
(5) and (4) re-suspending the primary exosome sample obtained in the step (4) with DPBS for ultracentrifugation, removing the supernatant as much as possible, and re-suspending the precipitate at the bottom of the ultracentrifuge tube with DPBS to obtain an exosome solution.
Comparative example 8
This comparative example provides a method for the isolation of exosomes using immunoaffinity. In particular to a method for separating CD9 in serum by using immunomagnetic beads + A method of exosomes.
The method specifically comprises the following steps:
(1) collecting fresh serum, placing in a centrifuge, centrifuging at room temperature of 2000g for 30min, discarding cell debris precipitate, and collecting centrifugation supernatant.
(2) Vortex oscillation streptavidin modification
Figure BDA0003611459440000101
MyOne TM Streptavidine T1 magnetic beads, after the magnetic bead suspension is uniform, 5 mul of magnetic bead suspension is added into 500 mul of separation buffer solution for suspension cleaning, a magnetic frame is used for separating the magnetic beads, and the cleaning is carried out for three times. After completion of washing, 500. mu.lSeparating buffer solution suspension magnetic beads;
(3) mu.l of mouse anti-human biotinylated CD9 monoclonal antibody produced by Ancell Co., Ltd was added to the magnetic bead suspension obtained in the above method (2), and the mixture was mixed by inversion and placed in
Figure BDA0003611459440000102
Incubate on sampleMixer incubator for 30min (parameters for 90 ° rotation upside down, 5s tilt, 5 ° shake for 1 s); and (3) finishing incubation, placing the incubation solution on a magnetic frame for 5min, discarding clear solution, suspending the magnetic beads by using 500 mu l of separation buffer solution, carrying out suspension cleaning, separating the magnetic beads by using the magnetic frame, and cleaning for three times. After washing, 500. mu.l of separation buffer suspended the beads.
(4) Adding 10 μ l of the serum obtained in the method (1) into the magnetic bead suspension obtained in the method (3), mixing the mixture evenly by inversion, and placing the mixture in a container
Figure BDA0003611459440000103
Incubate on the SampleMixer incubator for 2h (parameters of 90 ° rotation upside down, 5s tilt, 5 ° shake for 1 s); after the incubation is finished, placing the incubation liquid on a magnetic frame, discarding clear liquid, suspending the magnetic beads by using 500 mu l of separation buffer solution, performing suspension cleaning, separating the magnetic beads by using the magnetic frame, and performing cleaning for three times; after the washing is finished, the compound separated with the CD9+ exosome, namely the magnetic bead-antibody-CD 9 is obtained + An exosome.
Performance test
Test for detection
The following tests were carried out on the exosome solutions obtained by the methods provided in examples 1 to 24 and comparative examples 1 to 8, respectively, and the test results are shown in table 7.
The detection method specifically comprises the following steps: the exosome solution prepared above was each subjected to RNA extraction using miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was then reverse-transcribed into cDNA, and 8. mu.L of the reverse-transcribed cDNA, 1. mu. L B2M (a housekeeping gene) primer, probe mixture, 1. mu.L of enzyme-free water, and 10. mu.L of ddPCR were added to each ddPCR well TM Supermix for Probes (NodUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200 TM The housekeeping gene B2M quantification in exosomes was performed. According to the housekeeping gene B2The amount of M reflects the amount of exosomes.
TABLE 7 test results of examples 1 to 24 and comparative examples 1 to 8
Figure BDA0003611459440000111
Compared with a method for separating and detecting exosomes based on a microfluidic chip and a method for separating exosomes based on ultracentrifugation and a method for separating exosomes based on immunoaffinity, the method provided by the application not only can effectively shorten the separation time of exosomes, but also can integrate the separation and detection of exosomes. As is apparent from Table 7, it is understood from the results of the tests of comparative examples 1 to 24 and comparative examples 7 to 8 that the content of B2M obtained by separation using the method provided herein is greater than the content of B2M obtained by separation using ultracentrifugation and is almost close to the content of B2M obtained by immunoaffinity separation. The B2M content may reflect the exosome content obtained from the treatment. Therefore, according to the method provided by the application, on the basis of effectively shortening the separation time of the exosomes and realizing the integration of the separation and detection of the exosomes, the content of the exosomes obtained by separation can be close to the content of the exosomes obtained by immunoaffinity separation of the exosomes.
By comparing the detection results of examples 1 to 23 and example 24, it can be seen that the content of B2M in the washing solution provided by the present application can be further increased, that is, the content of exosome obtained by separation can be effectively increased, compared with the content of PBS buffer.
As can be seen from the results of the tests of comparative examples 1 to 6 of comparative example 3, when mannitol, tris hydrochloride or fatty acid diethanolamide was used alone, or when the washing solutions prepared from any two of them were used in the method for separating and detecting exosomes, the B2M content obtained by the test was low and much lower than the B2M content obtained in the method for separating and detecting exosomes using the prepared washing solutions simultaneously for three. From this, it is found that mannitol, tris hydrochloride and fatty acid diethanolamide, used alone or in combination of any two of them, do not achieve effective separation of exosomes.
As can be seen from the results of comparative examples 1 to 5, the content of exosomes obtained by separation can be increased by controlling the addition amount of mannitol in the washing solution to 12 to 18.4 parts. Furthermore, the addition amount of mannitol in the cleaning solution is controlled to be 14.5-16.2 parts, so that the content of exosome obtained by separation can be further improved.
As is clear from the results of comparison between example 3 and examples 6 to 9, the content of exosomes obtained by separation can be increased by controlling the amount of tris hydrochloride added to the washing solution to 4 to 8.8 parts. Furthermore, the content of exosomes obtained by separation can be further improved by controlling the adding amount of tris hydrochloride in the cleaning solution to 5.6-8.2 parts.
From the results of comparison between examples 3 and 10 to 13, it is understood that the content of exosomes obtained by separation can be increased by controlling the amount of fatty acid diethanolamide added to the wash solution to 3.5 to 6.0 parts. Furthermore, the content of the exosome obtained by separation can be further increased by controlling the addition amount of the fatty acid diethanolamide in the cleaning solution to be 4.4-5.2 parts.
As is clear from the results of comparing examples 3 and 14 to 19, when the washing time of the washing solution is 4min and 5min, the content of the exosomes obtained by separation is substantially the same, and the content of the exosomes obtained by separation can be increased by controlling the washing time of the washing solution to 0.5 to 3.5 min. Further, the cleaning time of the cleaning fluid is controlled to be 1-3min, so that the content of the exosomes obtained by separation can be further improved.
As can be seen from the results of comparing examples 3 and 20 to 23, the amount of exosomes obtained by separation can be increased by controlling the sample injection flow rate of the sample to 3 to 5.5. mu.L/min. Furthermore, the sample injection flow rate is controlled to be 4-5 mu L/min, so that the content of exosomes obtained by separation can be further improved.
Test 2
The results observed by transmission scanning electron microscopy and NTA detection using the method provided in example 3 are shown in FIG. 1.
Fig. 1 is a graph of the morphology, concentration and particle size distribution of exosomes obtained by the method provided in example 3 of the present application.
As can be seen from FIG. 1, the particle size distribution of the exosomes obtained by the method provided in example 3 is in the range of 150-200nm, which meets the requirement of exosome distribution, and the obtained exosomes have high purity and no hybrid protein.
Test III
The change of the fluorescence distribution on the micro-fluidic chip before and after the application of the alternating current to the micro-fluidic chip in example 3 was observed by using a macro-zoom microscope.
Fig. 2 shows the fluorescence distribution of the microfluidic chip before and after the application of the alternating current in the method provided in example 3 of the present application.
As can be seen from FIG. 2, after the alternating current is applied, the red fluorescent exosomes are separated and concentrated around the edges of the strongest microelectrode of the DEP high-field area, which shows that the microfluidic chip can realize the separation of the exosomes.
Test four
The method provided in example 3 was used to separate and quantify exosomes from rabbit plasma at different concentrations, and the signal intensity of PKH26 fluorescent molecules on the microfluidic chip was monitored by an optical imaging system, with the results shown in fig. 3.
FIG. 3 shows the quantitative results of exosomes isolated from rabbit plasma at different concentrations using the method provided in example 3 of the present application.
As can be seen from FIG. 3, the exosome concentrations obtained by the above-described isolation method were 1.034X 10, respectively 4 particles/mL、1.034×10 5 particles/mL、1.034×10 6 particles/mL、1.034×10 7 particles/mL、1.034×10 8 particles/mL. And the fluorescence signal detected by the optical imaging system has good correlation with the exosome concentration obtained by separation. The detection limit of the method provided by the application can reach 1.034 multiplied by 10 4 particles/mL。
The method and the device have the advantages that the dielectric field generated by the alternating current is combined with the characteristic of the exosome to detect the fluorescently-labeled molecular exosome, an exosome separation and detection system integrating separation and detection is constructed, so that the nondestructive exosome is obtained by separating and quantifying in a sample completed in one step, and the exosome can be used for further biological purposes.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims (10)

1. A method for separating and detecting exosomes based on a microfluidic chip is characterized by specifically comprising the following steps:
(1) fluorescent labeling of exosomes: carrying out fluorescence labeling on the sample by using exosome-labeled fluorescent molecules to obtain a labeled sample;
(2) separation of exosomes: adding the marked sample obtained in the step (1) into a microfluidic chip, and separating exosomes in the marked sample by using alternating current to provide dielectrophoresis; cleaning the microfluidic chip by using a cleaning solution under the same condition to obtain an exosome;
(3) detection of exosomes: and (3) observing the appearance of the exosomes obtained in the step (2) by using a transmission scanning electron microscope, and determining the concentration and particle size distribution of the exosomes obtained in the step (2) by using NTA.
2. The microfluidic chip based method for separating and detecting exosomes according to claim 1, wherein the cleaning solution comprises mannitol, tris hydrochloride and fatty acid diethanolamide.
3. The microfluidic chip based exosome separation and detection method according to claim 2, wherein the cleaning solution comprises the following components in parts by weight, based on 1000 parts by weight: 12-18.4 parts of mannitol, 4-8.8 parts of tris (hydroxymethyl) aminomethane hydrochloride and 3.5-6.0 parts of fatty acid diethanolamide.
4. The microfluidic chip based exosome separation and detection method according to claim 2, wherein the cleaning solution comprises the following components in parts by weight, based on 1000 parts by weight: 14.5 to 16.2 portions of mannitol, 5.6 to 8.2 portions of tris-hydroxymethyl aminomethane hydrochloride and 4.4 to 5.2 portions of fatty acid diethanolamide.
5. The microfluidic chip based method for separating and detecting exosomes according to claim 1, wherein the time for the washing solution to wash the microfluidic chip is 0.5-3.5 min.
6. The microfluidic chip based exosome separation and detection method according to claim 1, wherein the sample injection flow rate after labeling is 3-5.5 μ L/min.
7. The microfluidic chip based method for separating and detecting exosomes according to claim 1, wherein the alternating current has a voltage of 5-8.5V and a frequency of 15 kHz.
8. System for exosome separation and detection using the method according to any one of claims 1-7, characterized in that it comprises in particular a sample introduction part, a separation and optical imaging part, a detection part, in sequential communication.
9. A system for exosome isolation and detection according to claim 8, characterised in that the isolation and optical imaging portion comprises an exosome isolation portion and an optical imaging portion of an exosome isolation process.
10. The microfluidic chip based method for separating and detecting exosomes according to claim 8, wherein the detection part comprises a transmission scanning electron microscope and NTA for analyzing and detecting exosomes.
CN202210431876.6A 2022-04-23 2022-04-23 Method and system for separating and detecting exosome based on micro-fluidic chip Pending CN114878418A (en)

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