A method and system for separating and detecting exosomes based on a microfluidic chip

Abstract

This application relates to the technical field of exosome separation, and specifically discloses a method and system for separating and detecting exosomes based on a microfluidic chip. Specifically as follows: Fluorescent labeling of exosomes: A sample is fluorescently labeled using an exosome-labeling fluorescent molecule to obtain a labeled sample; Separation of exosomes: The labeled sample is added to the microfluidic chip, and an alternating current is used to provide dielectrophoresis to separate the exosomes in the labeled sample; and the microfluidic chip is washed with a washing solution under the same conditions to obtain exosomes; Detection of exosomes: The morphology of exosomes is observed using a transmission scanning electron microscope, and the concentration and particle size distribution of exosomes are determined using NTA. This application can effectively shorten the separation time of exosomes and achieve quantitative detection while separating exosomes.

Description

Technical Field

[0001] The present application relates to the technical field of exosome separation. More specifically, it relates to a method and system for separating and detecting exosomes based on a microfluidic chip. Background Art

[0002] Exosomes are cell-derived nanoscale (30 - 150 nm) membrane vesicles that play a key role in cell-to-cell communication. In the past few years, due to the widespread presence of exosomes in body fluids and their close association with disease development, exosomes have been identified as diagnostic biomarkers. Before analyzing exosome-related RNA and protein biomarkers, exosomes must be isolated from blood (plasma or serum) samples. Ultracentrifugation is currently the main technique for exosome separation, but due to the poor purity, yield, and integrity of the isolated exosomes, and the excessive processing time, this method does not meet the standards required for clinical applications.

[0003] In recent years, other methods such as polyethylene glycol (PEG)-based precipitation, phosphatidylserine affinity capture, size exclusion chromatography, and membrane affinity have emerged for specific applications, but the success rates are all limited. Recently, asymmetric flow field-flow fractionation has been used to sort EV (extracellular vesicle) subpopulations from cells and tumor culture media with high resolution, but its throughput is affected by cumbersome sample preparation procedures. The increasing requirements for input EV concentration and the time-consuming process for a single analysis also limit its wide application.

[0004] Due to the small size and low buoyant density of exosomes, separating exosomes from other components in blood in related technologies requires a large amount of time and effort. For example, it requires multiple processing steps, including a large number of ultracentrifugation and incubation steps from several hours to overnight, or plasma proteins in blood cannot be excluded, and the time-consuming multi-step processes and methods may damage exosomes and reduce the overall collection efficiency. In addition, in related technologies, most exosome detection and quantification are performed by separating first and then detecting, and some functions of the sample are affected after detection and quantification and cannot be used for subsequent applications.

[0005] Therefore, there is a current need to develop new technologies to improve the defects in exosome isolation and purification. Summary of the Invention

[0006] In order to shorten the exosome separation time and achieve quantitative detection while separating exosomes, the present application provides a method and system for separating and detecting exosomes based on a microfluidic chip.

[0007] In a first aspect, the present application provides a method for separating and detecting exosomes based on a microfluidic chip, adopting the following technical solution:

[0008] A method for separating and detecting exosomes based on a microfluidic chip, the method specifically comprising the following steps:

[0009] (1) Fluorescent labeling of exosomes: Using an exosome-labeling fluorescent molecule to perform fluorescent labeling on a sample to obtain a labeled sample;

[0010] (2) Separation of exosomes: Adding the labeled sample obtained in step (1) to a microfluidic chip, and using an alternating current to provide dielectrophoresis to separate the exosomes in the labeled sample; and using a cleaning solution to clean the microfluidic chip under the same conditions to obtain exosomes;

[0011] (3) Detection of exosomes: Using a transmission scanning electron microscope to observe the morphology of the exosomes obtained in step (2), and using NTA to measure the concentration and particle size distribution of the exosomes obtained in step (2).

[0012] The method provided by the present application realizes the separation of exosomes in a sample from other substances in the sample under the action of dielectrophoresis provided by an alternating current. Without the need to dilute the sample (serum, plasma or blood sample), it can strictly limit the mechanical damage to exosomes, and at the same time can effectively reduce the processing steps of sample and exosome separation, and shorten the separation time of exosomes. In addition, the method provided by the present application also includes a detection part for exosomes, which can detect exosomes in time after separation, and the detection process does not affect the application of exosomes.

[0013] In addition, in the case of an immunoaffinity separation method 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. Obviously, the method provided by the present application does not rely on antibody affinity binding, and can maximize the vitality of exosome RNA and protein biomarkers for subsequent detection, identification and analysis.

[0014] The cleaning solution can be PBS buffer or other solutions.

[0015] Preferably, the cleaning solution comprises mannitol, tris(hydroxymethyl)aminomethane hydrochloride and fatty acid diethanolamide.

[0016] Preferably, based on 1000 parts by weight, the cleaning solution comprises the following components in 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.

[0017] Preferably, based on 1000 parts by weight, the cleaning solution comprises the following components in 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.

[0018] In a specific embodiment, the weight parts of mannitol in the cleaning solution can be 12 parts, 14.5 parts, 15.6 parts, 16.2 parts, 18.4 parts.

[0019] In some specific embodiments, the weight parts of mannitol in the cleaning solution can be 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.

[0020] In a specific embodiment, the weight parts of tris(hydroxymethyl)aminomethane hydrochloride in the cleaning solution can be 4 parts, 5.6 parts, 6.8 parts, 8.2 parts, 8.8 parts.

[0021] In some specific embodiments, the weight parts of tris(hydroxymethyl)aminomethane hydrochloride in the cleaning solution can be 4 - 5.6 parts, 4 - 6.8 parts, 4 - 8.2 parts, 5.6 - 6.8 parts, 5.6 - 8.8 parts, 6.8 - 8.2 parts, 6.8 - 8.8 parts, 8.2 - 8.8 parts.

[0022] In a specific embodiment, the weight parts of fatty acid diethanolamide in the cleaning solution can be 3.5 parts, 4.4 parts, 4.7 parts, 5.2 parts, 6.2 parts.

[0023] In some specific embodiments, the weight parts of fatty acid diethanolamide in the cleaning solution can 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.

[0024] Mannitol is a sugar alcohol and an isomer of sorbitol. It is readily soluble in water, is a white crystalline powder, and has a sweet taste similar to sucrose. Tris(hydroxymethyl)aminomethane hydrochloride is commonly used in biological buffers. Fatty acid diethanolamide belongs to non - ionic surfactants and is readily soluble in water.

[0025] The present application further optimizes the components of the cleaning solution. The microfluidic chip is cleaned with a cleaning solution composed of mannitol, tris(hydroxymethyl)aminomethane hydrochloride, and fatty acid diethanolamide to obtain exosomes. Through experimental analysis, by controlling the addition amounts of mannitol, tris(hydroxymethyl)aminomethane hydrochloride, and fatty acid diethanolamide in the cleaning solution within the above - mentioned ranges, the content of exosomes obtained by separation can be further effectively increased.

[0026] Preferably, the time for the cleaning solution to clean the microfluidic chip is 0.5 - 3.5 min.

[0027] Preferably, the time for the cleaning solution to clean the microfluidic chip is 1 - 3 min.

[0028] In a specific embodiment, the time for the cleaning solution to clean the microfluidic chip can be 0.5 min, 1 min, 2 min, 3 min, 4 min, 5 min.

[0029] In some specific embodiments, the time for the cleaning solution to clean the microfluidic chip can be 0.5 - 1 min, 0.5 - 2 min, 0.5 - 3 min, 1 - 2 min, 1 - 3.5 min, 2 - 3 min, 2 - 3.5 min.

[0030] Through experimental analysis, the present application controls the time for the cleaning solution to clean the microfluidic chip within the above range, which can further effectively improve the content of exosomes obtained by separation.

[0031] Preferably, the injection flow rate of the labeled sample is 3 - 5.5 μL / min.

[0032] Preferably, the injection flow rate of the labeled sample is 4 - 5 μL / min.

[0033] In a specific embodiment, the injection flow rate of the labeled sample can be 3 μL / min, 4 μL / min, 4.5 μL / min, 5 μL / min, 5.5 μL / min.

[0034] In some specific embodiments, the injection flow rate of the labeled sample can be 3 - 4 μL / min, 3 - 4.5 μL / min, 3 - 5 μL / min, 4 - 4.5 μL / min, 4 - 5.5 μL / min, 4.5 - 5 μL / min, 4.5 - 5.5 μL / min, 5 - 5.5 μL / min.

[0035] Through experimental analysis, the present application controls the injection flow rate of the labeled sample within the above range, which can further effectively improve the content of exosomes obtained by separation.

[0036] Preferably, the voltage of the alternating current is 5 - 8.5 V and the frequency is 15 kHz.

[0037] Preferably, the exosome-labeled fluorescent molecule is PKH26.

[0038] In a second aspect, the present application provides a system for exosome separation and detection using the above method, adopting the following technical solution:

[0039] A system for exosome separation and detection using the above method, the system specifically includes an injection part, a separation and optical imaging part, and a detection part that are connected in sequence.

[0040] Preferably, the separation and optical imaging part includes a separation part of exosomes and an optical imaging part during the exosome separation process.

[0041] Preferably, the detection part includes a transmission scanning electron microscope and NTA (nanoparticle tracking analysis) for analyzing and detecting exosomes.

[0042] The exosome separation and detection system provided by the present application includes a separation part and a detection part of exosomes. After the exosome separation is completed, the exosomes can be detected in a timely manner, and the detection process does not affect the application of exosomes. In the system provided by the present application, the separation and optical imaging part includes a separation part of exosomes and an optical imaging part during the exosome separation process, so that the separation situation of exosomes can be monitored in a timely manner. At the same time, the detection part includes a transmission scanning electron microscope and NTA (nanoparticle tracking analysis) for analyzing and detecting exosomes. The transmission scanning electron microscope is used to observe the morphology of exosomes, and NTA is used to analyze the concentration and particle size of exosomes. Therefore, the separation and detection of exosomes can be completed by using the above system, and the morphology, concentration and particle size parameters of exosomes can be obtained.

[0043] In summary, the present application has the following beneficial effects:

[0044] (1) The method for separating and detecting exosomes provided by the present application can effectively shorten the separation time of exosomes, and achieve quantitative detection while separating exosomes, so as to realize the one-step separation and quantification of exosomes from body fluids.

[0045] (2) The method provided by the present application realizes the separation of exosomes from other substances in the sample under the action of dielectrophoresis provided by alternating current. Without diluting the sample (serum, plasma or blood sample), it can strictly limit the mechanical damage to exosomes, and at the same time can effectively reduce the processing steps of sample and exosome separation, and shorten the separation time of exosomes.

[0046] (3) The method provided by the present application also includes a detection part of exosomes, which can detect exosomes in a timely manner after separation, and the detection process does not affect the later application of exosomes.

[0047] (4) The method provided by the present application does not rely on antibody affinity binding, and can maximally maintain the viability of exosome RNA and protein biomarkers for subsequent detection, identification and analysis. Description of the Drawings

[0048] Figure 1 It is a diagram showing the morphology, concentration and particle size distribution of exosomes obtained by the method provided in Example 3 of the present application.

[0049] Figure 2The fluorescence distribution on the microfluidic chip before and after applying alternating current in the method provided in Embodiment 3 of this application.

[0050] Figure 3 The quantitative results after separating exosomes in rabbit plasma with different concentrations by using the method provided in Embodiment 3 of this application. Detailed implementation manners

[0051] This application provides a method for separating and detecting exosomes based on a microfluidic chip. The method specifically includes the following steps:

[0052] (1) Fluorescent labeling of exosomes: Using an exosome-labeled fluorescent molecule to perform fluorescent labeling on a sample to obtain a labeled sample.

[0053] Among them, the exosome-labeled fluorescent molecule is PKH26.

[0054] (2) Separation of exosomes: Adding the labeled sample obtained in step (1) into the microfluidic chip, and using alternating current to provide dielectrophoresis to separate the exosomes in the labeled sample; and using a cleaning solution to clean the microfluidic chip under the same conditions to obtain exosomes.

[0055] The cleaning solution includes mannitol, tris(hydroxymethyl)aminomethane hydrochloride, and fatty acid diethanolamide. Among them, based on 1000 parts by weight, the cleaning solution includes the following components in 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, based on 1000 parts by weight, the cleaning solution includes the following components in 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.

[0056] Among them, the voltage of the alternating current is 5 - 8.5 V, and the frequency is 15 kHz. The injection flow rate of the labeled sample is 3 - 5.5 μL / min. The time for the cleaning solution to clean the microfluidic chip is 0.5 - 3.5 min.

[0057] (3) Detection of exosomes: Using a transmission scanning electron microscope to observe the morphology of the exosomes obtained in step (2), and using NTA to measure the concentration and particle size distribution of the exosomes obtained in step (2).

[0058] This application also provides a system for separating and detecting exosomes by using the above method. The system specifically includes an injection part, a separation and optical imaging part, and a detection part that are connected in sequence. Among them, the separation and optical imaging part includes a separation part of exosomes and an optical imaging part during the exosome separation process. The detection part includes a transmission scanning electron microscope and NTA for analyzing and detecting exosomes.

[0059] Specifically, a system for exosome isolation and detection using the above method specifically includes an injection part, a separation and optical imaging part, and a detection part that are connected in sequence. The labeled sample enters the separation and optical imaging part from the injection part. The separation and optical imaging part includes an exosome separation part and an optical imaging part during the exosome separation process. The separation includes a microfluidic chip. The labeled sample enters the microfluidic chip, and under the action of dielectrophoresis provided by alternating current, the microfluidic chip is cleaned with a cleaning solution under the same conditions to obtain exosomes. The displacement change of exosomes in the labeled sample can be observed through the optical imaging part. The obtained exosomes enter the detection part. The detection part includes a transmission scanning electron microscope and NTA for analyzing and detecting exosomes. The morphology of the obtained exosomes is observed using the transmission scanning electron microscope, and the concentration and particle size distribution of the obtained exosomes are measured using NTA.

[0060] The present application will be further described in detail below with reference to Preparation Examples 1-16, Examples 1-24, and Comparative Examples 1-8.

[0061] Preparation Example

[0062] Preparation Examples 1-13

[0063] Preparation Examples 1-13 respectively provide a cleaning solution.

[0064] The differences between the above preparation examples are as follows: the addition amounts of the components in the cleaning solution are different, as specifically shown in Table 1.

[0065] The preparation method of the above cleaning solution is as follows: dissolve each component according to the addition amount of each component in 1000 ml of ddH 2 O water.

[0066] Table 1 Addition of components in Preparation Examples 1-13

[0067]

[0068] Preparation Examples 14-16

[0069] Preparation Examples 14-16 respectively provide a cleaning solution.

[0070] The differences between the above preparation examples are as follows: the addition types of the components in the cleaning solution are different, as specifically shown in Table 2.

[0071] Table 2 Addition of components in Preparation Example 3, Preparation Examples 14-16

[0072]

[0073]

[0074] Example

[0075] Examples 1 - 13

[0076] Examples 1 - 13 respectively provide a method for separating and detecting exosomes based on a microfluidic chip. Among them, the sample is 1 ml of rabbit plasma.

[0077] The differences in the above examples are as follows: the types of cleaning solutions used in the method are different, as shown in Table 3 specifically.

[0078] The above method specifically includes the following steps:

[0079] (1) Fluorescent labeling of exosomes: Use the exosome labeling fluorescent molecule PKH26 to perform fluorescent labeling on the sample to obtain the labeled sample.

[0080] (2) Separation of exosomes: Add the labeled sample obtained in step (1) to a microfluidic chip (ACE chip), and use alternating current to provide dielectrophoresis to separate the exosomes in the labeled sample. Among them, the voltage of the alternating current is 5 V, and the frequency is 15 kHz. The injection flow rate of the labeled sample is 4.5 μL / min.

[0081] And use the cleaning solution to clean the microfluidic chip under the same conditions to obtain the captured exosomes. Among them, the time for the cleaning solution to clean the microfluidic chip is 2 min.

[0082] (3) Detection of exosomes: Use PBS buffer to elute the exosomes captured in step (2) at a flow rate of 5 μL / min for 2 min to obtain an exosome solution. Use a transmission scanning electron microscope to observe the morphology of the exosomes, and use NTA (nanoparticle tracking analysis) to measure the concentration and particle size distribution of the exosomes.

[0083] A system for separating and detecting exosomes using the above method specifically includes an injection part, a separation and optical imaging part, and a detection part that are connected in sequence. The labeled sample obtained in step (1) enters the separation and optical imaging part from the injection part. The separation and optical imaging part includes a separation part of exosomes and an optical imaging part during the exosome separation process, and the separation includes a microfluidic chip. The labeled sample enters the microfluidic chip, and under the action of alternating current providing dielectrophoresis, and use the cleaning solution to clean the microfluidic chip under the same conditions to obtain exosomes. The displacement change of the exosomes in the labeled sample can be observed through the optical imaging part. The obtained exosomes enter the detection part. The detection part includes a transmission scanning electron microscope and NTA for analyzing and detecting exosomes. Use a transmission scanning electron microscope to observe the morphology of the obtained exosomes, and use NTA to measure the concentration and particle size distribution of the obtained exosomes.

[0084] Table 3 Types of cleaning solutions used in the methods provided by Examples 1 - 13

[0085]

[0086] Examples 14 - 17

[0087] Examples 14 - 17 respectively provide a method for separating and detecting exosomes based on a microfluidic chip.

[0088] The differences between the above - mentioned examples are as follows: the cleaning time of the microfluidic chip with the cleaning solution in the method is different, as shown in Table 4 specifically.

[0089] Table 4 Cleaning time of the microfluidic chip with the cleaning solution in the methods provided by Example 3 and Examples 14 - 17

[0090] Example Serial Number Washing Time (min) 3 2 14 0.5 15 1 16 3 17 3.5 18 4 19 5

[0091] Examples 20 - 23

[0092] Examples 20 - 23 respectively provide a method for separating and detecting exosomes based on a microfluidic chip.

[0093] The differences between the above - mentioned examples are as follows: the injection flow rate of the labeled sample in the method is different, as shown in Table 5 specifically.

[0094] Table 5 Injection flow rate of the sample in the methods provided by Example 3 and Examples 20 - 23

[0095] Example Serial Number Sample Injection Flow Rate (μL / min) 3 4.5 20 3 21 4 22 5 23 5.5

[0096] Example 24

[0097] This example provides a method for separating and detecting exosomes based on a microfluidic chip.

[0098] The difference between this example and Example 3 is that the cleaning solution in the method is PBS buffer solution.

[0099] Comparative Example

[0100] Comparative Examples 1 - 6

[0101] Comparative Examples 1 - 6 respectively provide a method for separating and detecting exosomes based on a microfluidic chip.

[0102] The differences between the above - mentioned comparative examples are as follows: the types of the cleaning solutions used in the methods are different, as shown in Table 6 specifically.

[0103] Table 6 Types of the cleaning solutions used in the methods provided by Comparative Examples 1 - 6

[0104] Comparative Example Serial Number Washing Solution Type 1 Preparation Example 14 2 Preparation Example 15 3 Preparation Example 16 4 Preparation Example 17 5 Preparation Example 18 6 Preparation Example 19

[0105] Comparative Example 7

[0106] This comparative example provides a method for separating exosomes using ultracentrifugation.

[0107] The above method specifically includes the following steps:

[0108] (1) Place the cell supernatant at 4°C and centrifuge at 300 g for 10 min to remove cells and dead cells. After centrifugation, take the supernatant for further use.

[0109] (2) Place the supernatant obtained in step (1) at 4°C and centrifuge at 2000 g for 10 min to remove cell debris. After centrifugation, discard the supernatant and take the pellet for further use.

[0110] (3) Place the supernatant obtained in step (2) at 4°C and centrifuge at 10000 g for 30 min to remove large membrane vesicles. After centrifugation, discard the supernatant and take the pellet for further use.

[0111] (4) Transfer the supernatant obtained in step (3) to an ultracentrifuge tube until it reaches 2 - 3 mm from the tube mouth. Place the ultracentrifuge tube in an ultracentrifuge at 4°C and 120000 g for 70 min. After centrifugation, carefully remove as much supernatant as possible (note that it is easy to lose the pellet enriched with exosomes), and then resuspend the pellet at the bottom of the ultracentrifuge tube with DPBS to obtain the primary exosome sample.

[0112] (5) Resuspend the primary exosome sample obtained in step (4) with DPBS and centrifuge again to remove as much supernatant as possible. Resuspend the pellet at the bottom of the ultracentrifuge tube with DPBS to obtain the exosome solution.

[0113] Comparative Example 8

[0114] This comparative example provides a method for separating exosomes using immunoaffinity. Specifically, it is a method for separating CD9 + exosomes from serum using immunomagnetic beads.

[0115] The above method specifically includes the following steps:

[0116] (1) Collect fresh serum, place it in a centrifuge, and centrifuge at 2000 g for 30 min at room temperature. Discard the cell debris pellet and take the centrifuged supernatant.

[0117] (2) Vortex and oscillate the streptavidin-modified MyOne TM StreptavidinT1 magnetic beads. After the magnetic bead suspension is homogeneous, take 5 μl of the magnetic bead suspension and add it to 500 μl of separation buffer. Suspend and wash, separate the magnetic beads with a magnetic stand, and wash three times. After washing, suspend the magnetic beads in 500 μl of separation buffer;

[0118] (3) Add 5 μl of mouse anti-human biotinylated CD9 monoclonal antibody produced by Ancell to the magnetic bead suspension obtained in the above method (2), invert and mix evenly, and place it on the SampleMixer incubator for incubation for 30 min (parameters: invert and rotate 90°, tilt for 5 s, vibrate at 5° for 1 s); after incubation, place the incubation solution on the magnetic stand for 5 min, discard the supernatant, suspend the magnetic beads with 500 μl of separation buffer, suspend and wash, separate the magnetic beads with the magnetic stand, and wash three times. After washing, suspend the magnetic beads with 500 μl of separation buffer.

[0119] (4) Add 10 μl of the serum obtained in method (1) to the magnetic bead suspension obtained in method (3), invert and mix evenly, and place it on the SampleMixer incubator for incubation for 2 h (parameters: invert and rotate 90°, tilt for 5 s, vibrate at 5° for 1 s); after incubation, place the incubation solution on the magnetic stand, discard the supernatant, suspend the magnetic beads with 500 μl of separation buffer, suspend and wash, separate the magnetic beads with the magnetic stand, and wash three times; after washing, the complex separated with CD9+ exosomes, namely magnetic bead-antibody-CD9 + exosomes, is obtained.

[0120] Performance detection test

[0121] Detection test one

[0122] The exosome solutions obtained by the methods provided in the above Examples 1-24 and Comparative Examples 1-8 were respectively subjected to the following detections, and the detection results are shown in Table 7.

[0123] The specific detection method is as follows: respectively extract RNA from the above-prepared exosome solution using the miRNeasy Serum / Plasma Advanced Kit (RNA extraction kit of QIAGEN), then reverse transcribe the RNA into cDNA, add 8 μL of the reverse-transcribed cDNA obtained to each ddPCR well, 1 μL of the B2M (a housekeeping gene) primer and probe mixture, 1 μL of enzyme-free water, and 10 μL of ddPCR TM Supermix for Probes (NodUTP). After PCR amplification, use Bio-Rad QX200 TM to quantify the housekeeping gene B2M in exosomes. The content of exosomes is reflected according to the content of the housekeeping gene B2M.

[0124] Table 7 Detection results of Examples 1-24 and Comparative Examples 1-8

[0125]

[0126] Compared with the methods of separating exosomes by ultracentrifugation and immunoaffinity separation, the method for separating and detecting exosomes provided by this application can not only effectively shorten the separation time of exosomes, but also integrate the separation and detection of exosomes. As can be seen from Table 7, by comparing the detection results of Examples 1-24 and Comparative Examples 7-8, the content of B2M obtained by separating using the method provided by this application is greater than that obtained by separating using the ultracentrifugation method, and is almost close to the content of B2M obtained by immunoaffinity separation. The content of B2M can reflect the content of exosomes obtained by treatment. Therefore, as described above, on the basis of effectively shortening the separation time of exosomes and integrating the separation and detection of exosomes, the content of exosomes obtained by separating using the method provided by this application can also be close to the content of exosomes obtained by immunoaffinity separation of exosomes.

[0127] By comparing the detection results of Examples 1-23 and Example 24, it can be seen that compared with the PBS buffer solution, the cleaning solution provided by this application can further increase the content of B2M, that is, it can effectively increase the content of exosomes obtained by separation.

[0128] By comparing the detection results of Example 3 and Comparative Examples 1-6, it can be seen that when mannitol, tris(hydroxymethyl)aminomethane hydrochloride or fatty acid diethanolamide is used alone, or when a cleaning solution prepared by using any two of them is used in the method for separating and detecting exosomes, the detected content of B2M is relatively low, and is much lower than the content of B2M obtained when a cleaning solution prepared by using all three of them is used in the method for separating and detecting exosomes. It can be seen from this that using mannitol, tris(hydroxymethyl)aminomethane hydrochloride and fatty acid diethanolamide alone or in any combination of two of them cannot achieve effective separation of exosomes.

[0129] By comparing the detection results of Examples 1-5, it can be seen that controlling the addition amount of mannitol in the cleaning solution to 12-18.4 parts can increase the content of exosomes obtained by separation. Further, controlling the addition amount of mannitol in the cleaning solution to 14.5-16.2 parts can further increase the content of exosomes obtained by separation.

[0130] By comparing the detection results of Example 3 and Examples 6-9, it can be seen that controlling the addition amount of tris(hydroxymethyl)aminomethane hydrochloride in the cleaning solution to 4-8.8 parts can increase the content of exosomes obtained by separation. Further, controlling the addition amount of tris(hydroxymethyl)aminomethane hydrochloride in the cleaning solution to 5.6-8.2 parts can further increase the content of exosomes obtained by separation.

[0131] By comparing the detection results of Example 3 and Examples 10 - 13, it can be seen that controlling the addition amount of fatty acid diethanolamide in the cleaning solution within 3.5 - 6.0 parts can increase the content of exosomes obtained by separation. Further, controlling the addition amount of fatty acid diethanolamide in the cleaning solution within 4.4 - 5.2 parts can further increase the content of exosomes obtained by separation.

[0132] By comparing the detection results of Example 3 and Examples 14 - 19, it can be seen that when the cleaning time of the cleaning solution is 4 min and 5 min, the content of exosomes obtained by separation is basically the same, while controlling the cleaning time of the cleaning solution within 0.5 - 3.5 min can increase the content of exosomes obtained by separation. Further, controlling the cleaning time of the cleaning solution within 1 - 3 min can further increase the content of exosomes obtained by separation.

[0133] By comparing the detection results of Example 3 and Examples 20 - 23, it can be seen that controlling the sample injection flow rate within 3 - 5.5 μL / min can increase the content of exosomes obtained by separation. Further, controlling the sample injection flow rate within 4 - 5 μL / min can further increase the content of exosomes obtained by separation.

[0134] Detection Test Two

[0135] Using the method provided in Example 3, the results observed by transmission scanning electron microscopy and NTA detection are as Figure 1 shown.

[0136] Figure 1 This is the diagram of the morphology, concentration, and particle size distribution of exosomes obtained by the method provided in Example 3 of this application.

[0137] From Figure 1 it can be seen that the exosomes obtained by the method provided in Example 3 have a particle size distribution in the range of 150 - 200 nm, meeting the requirements of exosome distribution, and the obtained exosomes have high purity and no contaminating proteins.

[0138] Detection Test Three

[0139] Using a macro - optical zoom microscope to observe the change in the fluorescence distribution on the microfluidic chip before and after applying alternating current in Example 3.

[0140] Figure 2 This is the fluorescence distribution on the microfluidic chip before and after applying alternating current in the method provided in Example 3 of this application.

[0141] From Figure 2 it can be seen that after applying alternating current, the red - fluorescent exosomes are separated and concentrated around the edge of the micro - electrode with the strongest DEP high - field region, indicating that this microfluidic chip can achieve the separation of exosomes.

[0142] Detection Test Four

[0143] The method provided in Example 3 was used to separate and quantify exosomes from rabbit plasma at different concentrations. Meanwhile, the signal intensity of PKH26 fluorescent molecules on the microfluidic chip was monitored through an optical imaging system. The results are as Figure 3 shown.

[0144] Figure 3 The quantitative results of exosomes separated from rabbit plasma at different concentrations by using the method provided in Example 3 of this application.

[0145] It can be seen from Figure 3 that the concentrations of exosomes obtained by separation using the above method are 1.034×10 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 there is a good correlation between the fluorescence signal detected by the optical imaging system and the concentration of the separated exosomes. The detection limit of the method provided in this application can reach 1.034×10 4 particles / mL.

[0146] In this application, an exosome separation and detection system integrating separation and detection was constructed by combining the dielectric field generated by alternating current with the characteristics of exosomes and fluorescently labeled molecules for exosome detection, realizing the separation and quantification of non-destructive exosomes from a sample in one step, which can be used for further biological applications.

[0147] This specific embodiment is only an explanation of this application, and it is not a limitation of this application. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as it is within the scope of the claims of this application, it is protected by the patent law.

Claims

1. A method for separating and detecting exosomes based on a microfluidic chip, characterized in that, the method specifically comprises the following steps: (1) Fluorescent labeling of exosomes: Using an exosome-labeling fluorescent molecule to perform fluorescent labeling on a sample to obtain a labeled sample; (2) Separation of exosomes: Adding the labeled sample obtained in step (1) into a microfluidic chip, and using an alternating current to provide dielectrophoresis to separate the exosomes in the labeled sample; and using a cleaning solution to clean the microfluidic chip under the same conditions to obtain exosomes; Based on 1000 parts by weight, the cleaning solution comprises the following components in 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; (3) Detection of exosomes: Using a transmission scanning electron microscope to observe the morphology of the exosomes obtained in step (2), and using NTA to measure the concentration and particle size distribution of the exosomes obtained in step (2).

2. The method for separating and detecting exosomes based on a microfluidic chip according to claim 1, characterized in that, Based on 1000 parts by weight, the cleaning solution comprises the following components in 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.

3. The method for separating and detecting exosomes based on a microfluidic chip according to claim 1, characterized in that, the time for the cleaning solution to clean the microfluidic chip is 0.5 - 3.5 min.

4. The method for separating and detecting exosomes based on a microfluidic chip according to claim 1, characterized in that, the injection flow rate of the labeled sample is 3 - 5.5 μL / min.

5. The method for separating and detecting exosomes based on a microfluidic chip according to claim 1, characterized in that, the voltage of the alternating current is 5 - 8.5 V, and the frequency is 15 kHz.

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