CN116875455A - Microfluidic chip for exosome separation and exosome separation method - Google Patents

Microfluidic chip for exosome separation and exosome separation method Download PDF

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CN116875455A
CN116875455A CN202310890238.5A CN202310890238A CN116875455A CN 116875455 A CN116875455 A CN 116875455A CN 202310890238 A CN202310890238 A CN 202310890238A CN 116875455 A CN116875455 A CN 116875455A
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cavity
chamber
capturing
air flow
oil
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彭年才
郭晓牛
雍张
胡飞
赵书豪
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Xian Jiaotong University
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Xian Jiaotong University
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    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/06—Magnetic means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Abstract

The invention discloses a microfluidic chip for separating exosomes and an exosome separating method, comprising a chip body, wherein a capturing cavity, a first oil cavity, a washing cavity, a second oil cavity and a recovery cavity are distributed on the chip body at intervals; the capturing cavity, the first oil cavity, the washing cavity, the second oil cavity and the recycling cavity are all communicated through a micro door; the bottoms of the capturing cavity and the recycling cavity are communicated with a gas channel, and the air channel is used for conveying external gas into the capturing cavity or the recycling cavity. The device disclosed by the invention combines the bubble micromixer with the non-miscible phase technology, realizes high-efficiency mixing of magnetic beads and samples, replaces the traditional manual pipette to blow and mix uniformly, solves the problem that the non-miscible phase technology is difficult to realize automatic mixing, reduces the pollution risk of reagents and samples, and improves the purity of the obtained exosomes.

Description

Microfluidic chip for exosome separation and exosome separation method
Technical Field
The invention belongs to the technical field of exosome purification, and relates to a microfluidic chip for exosome separation and an exosome separation method.
Background
Exosomes are vesicles 30-150nm in diameter secreted by most mammalian cells, and are typically cupped in morphology. Exosomes are widely found in body fluids such as blood, saliva, urine, etc., and contain a large amount of genetic information and biomolecules derived from parent cells. A great deal of research shows that exosomes play a key role in physiological or pathological processes of human bodies, and are closely related to the occurrence and progress of various diseases such as tumors, alzheimer's disease, skin and the like. These inherent properties give exosomes unique opportunities in cancer diagnosis, immunotherapy, drug delivery, etc., attracting scientists to great research interests.
Enrichment of high purity exosomes from body fluids is a necessary prerequisite for research of biological functions and clinical applications. However, due to the nanoscale size of exosomes, the origin, size, composition, functional heterogeneity that they exist in themselves, so far, the rapid separation of high purity exosomes from complex body fluids remains a great technical challenge. The conventional exosome separation method, including ultracentrifugation, ultrafiltration, size exclusion chromatography and the like, generally cannot effectively distinguish exosomes from other impurities such as vesicles and protein aggregates close to the physical properties of the exosomes, and has the defects of complicated operation steps, long time consumption, low separation efficiency and the like. The immunomagnetic separation method realizes exosome capture by utilizing high-specificity affinity between antigen and antibody, is the exosome separation method with highest purity at present, has the advantages of simple operation, homogenization in the reaction process and the like, and has been widely applied to exosome purification and analysis. However, the method still needs more manual operation, reagents need to be transferred among test tubes for multiple times, an open reaction system increases the possibility of sample pollution, and the separation and extraction effect depends on the operation level of laboratory staff to a certain extent, so that the repeatability and accuracy of laboratory researches are greatly different. Therefore, it is highly desirable to build an automated magnetic separation system that achieves a unified combination of enrichment, purification and recovery functions and improves separation performance stability.
The microfluidic technology integrates a plurality of biochemical analysis processes into one chip, has the characteristics of high analysis speed, high automation and integration degree, less consumption of samples and reagents, high flux and the like, and is an important analysis platform in the field of life science. In recent years, researchers have developed a variety of microfluidic platforms based on immunomagnetic separation for exosome enrichment and molecular characterization, most of which are dynamic continuous flow systems. The Batini et al integrate the polypeptide Vn96 functionalized magnetic beads into a microfluidic device, and the capturing efficiency of the cell culture supernatant source exosomes reaches 90% in about 20 minutes. The microfluidic platform ExoSearch proposed by Zhao et al integrates immunomagnetic separation and in-situ surface marker multiple detection, and improves the separation efficiency of magnetic beads on exosomes by continuous flow mixing. However, the continuous flow system requires an external energy source to precisely control the movement of the fluid, and the micro-flow channel has a complex structure and high manufacturing and processing difficulty. So far, few studies have been reported on exosome separation platforms based on static microcavity systems.
Disclosure of Invention
The invention aims to solve the problems of low automation degree, high experimental result repeatability, high variability and the like of a manual-based immunomagnetic separation method, and is different from the high requirements of a dynamic continuous flow microfluidic system on aspects of fluid motion control, a micro-channel structure, a manufacturing process and the like.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
the microfluidic chip for separating the exosome comprises a chip body, wherein a capturing cavity, a first oil cavity, a washing cavity, a second oil cavity and a recovery cavity are distributed on the chip body at intervals;
the capturing cavity, the first oil cavity, the washing cavity, the second oil cavity and the recycling cavity are all communicated through a micro door;
the bottoms of the capturing cavity and the recycling cavity are communicated with an air flow channel, and the air flow channel is used for conveying external air into the capturing cavity or the recycling cavity.
The invention further improves that:
the lower part of the capturing chamber is communicated with a first air flow passage;
the first air flow passage is of an L-shaped structure, the inlet end of the first air flow passage is higher than the bottom of the capturing chamber, and the outlet end of the first air flow passage is arranged below the capturing chamber.
A first sub-flow passage is vertically distributed between the first air flow passage and the capturing chamber;
one end of the first sub-runner is communicated with the outlet end of the first air runner, and the other end of the first sub-runner is communicated with the inlet at the bottom of the capturing cavity.
The lower part of the recovery cavity is communicated with a second air flow passage which is of an L-shaped structure;
the inlet end of the second air flow passage is higher than the bottom of the recovery cavity, and the outlet end of the second air flow passage is positioned below the recovery cavity;
the lower extreme of retrieving the chamber sets up a plurality of interval distribution's second sub-runner, second sub-runner one end intercommunication retrieves the bottom of chamber, the exit end of the second air runner of other end intercommunication.
The micro-door is of a trapezoid structure, and the length of the inlet end of the micro-door is larger than that of the outlet end of the micro-door.
The distance from the inlet end to the outlet end of the micro-door is 2-4mm;
the height of the cavity in the micro-door is 300-500 mu m;
the length of the outlet end of the micro-gate is 500-800 mu m.
The width of the inner cavity of the air flow channel is 50-200 mu m.
The end surfaces of the chamber ports of the capturing chamber, the first oil chamber, the washing chamber, the second oil chamber and the recycling chamber are vertically distributed with the end surfaces of the air flow channel inlet ends.
An exosome separation method comprising the steps of:
adding a biological sample and nano magnetic beads into the capturing cavity, and sequentially adding silicone oil, washing buffer, silicone oil and elution buffer into the first oil cavity, the washing cavity, the second oil cavity and the recovery cavity respectively;
continuously introducing gas into the capturing cavity at a constant speed, wherein the gas generates bubbles to drive the nano magnetic beads and the biological sample to be uniformly mixed;
after the uniform mixing is finished, placing a permanent magnet on the side surface of the chip body, and enabling the permanent magnet to move along the first oil cavity, the washing cavity, the second oil cavity and the recovery cavity of the capturing cavity in sequence;
continuously introducing gas into the recovery cavity at a constant speed to promote the mixing of the magnetic bead-exosome and the elution buffer;
after stopping mixing, collecting the eluent rich in exosomes in the recovery cavity.
Further, the gas is injected into the capturing cavity or the recovery cavity through the digital injection pump, and the injection speed is 100-500 mu l/min.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a microfluidic chip for separating exosomes, wherein a static non-miscible 'virtual wall' interface is formed between chambers through a micro-gate, hydrophilic impurities and lipophilic impurities can be respectively retained in a water phase chamber and an oil phase chamber in the process of transferring magnetic beads, washing and purifying of the magnetic beads are realized, the magnetic beads are transferred by virtue of external magnet driving in the whole process, solution in the chambers is always static, compared with the purification mode based on continuous flow, pumping solution is not required to repeatedly wash the magnetic beads, reagent consumption and magnetic bead loss caused by repeated washing are reduced, purification time is shortened, meanwhile, a gas channel is communicated in a capturing chamber and a recovery chamber, gas is mixed with the solution to form bubbles, the rising, expanding, cracking and other processes of the bubbles are realized, the movement of the fluid and the magnetic beads in the chambers is disturbed to form micro-eddies, so that the collision probability of the magnetic beads and biological samples is increased, the magnetic separation efficiency of the exosomes is further improved.
Further, in the invention, the outlet end of the first air flow channel is arranged below the capturing cavity, so that bubbles formed in the cavity can rise, expand and break from the bottom, and disturbance is generated from the bottom to the top, thereby improving the mixing effect.
Further, in the invention, if the first dry sub-flow channels are vertically distributed between the first air flow channel and the capturing chamber, the uniformity of the distribution of bubbles in the chamber is ensured.
The invention discloses an exosome separation method, which can be carried out only through an external magnet in the transferring process of magnetic beads, and the solution of each cavity in a chip is static, so that pumping solution is not required to repeatedly wash the magnetic beads, reagent consumption and magnetic bead loss caused by repeated washing are reduced, purification time is shortened, and when preliminary mixing and final recovery are carried out, the internal mixing is realized by utilizing the disturbance effect of bubbles, a bubble micromixer and a non-miscible phase technology are combined, so that the high-efficiency mixing of the magnetic beads and a sample is realized, the traditional manual pipette is replaced for blowing and beating the mixing, the problem that the non-miscible phase technology is difficult to realize automatic mixing is solved, the pollution risk of reagents and the sample is reduced, the purity of the obtained exosome is improved, the whole processes of exosome capturing, purifying and eluting recovery are completed in one microfluidic chip, the requirement on manual operation is reduced, the stable experimental result is more facilitated, the downstream functional analysis and clinical research are facilitated, the exosome obtained by the method disclosed by the invention can be realized rapidly and efficiently, the exosome enrichment is easy and convenient to obtain the exosome with high purity under the premise of not influencing the exosome activity.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic diagram of the overall structure of the present invention.
Wherein: 1-a capture chamber; 2-a first oil chamber; 3-a wash chamber; 4-a second oil chamber; 5-a recovery chamber; 6-micro gate; 7-a first air flow passage; 8-a first sub-flow path; 9-a second air flow path; 10-a second sub-flow path.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In the description of the embodiments of the present invention, it should be noted that, if the terms "upper," "lower," "horizontal," "inner," and the like indicate an azimuth or a positional relationship based on the azimuth or the positional relationship shown in the drawings, or the azimuth or the positional relationship in which the inventive product is conventionally put in use, it is merely for convenience of describing the present invention and simplifying the description, and does not indicate or imply that the apparatus or element to be referred to must have a specific azimuth, be configured and operated in a specific azimuth, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like, are used merely to distinguish between descriptions and should not be construed as indicating or implying relative importance.
Furthermore, the term "horizontal" if present does not mean that the component is required to be absolutely horizontal, but may be slightly inclined. As "horizontal" merely means that its direction is more horizontal than "vertical", and does not mean that the structure must be perfectly horizontal, but may be slightly inclined.
In the description of the embodiments of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "mounted," "connected," and "connected" should be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
The invention is described in further detail below with reference to the attached drawing figures:
referring to fig. 1, the embodiment of the invention discloses a microfluidic chip for exosome separation, a capture module for exosome immunomagnetic separation, a purification module based on an immiscible phase technology and a recovery module for exosome elution, wherein a pressure or flow controller is not needed in the extraction process, and a small permanent magnet can be used for realizing driving without multiple washing.
The number of units of the capturing module, the purifying module and the recovering module is not limited. The material of the microfluidic chip is not particularly limited, and materials well known in the art may be used. In the embodiment of the present invention, the material of the microfluidic chip is polymethyl methacrylate (PMMA), and specifically includes the following structures:
the microfluidic chip for separating the exosome comprises a chip body, wherein a capturing cavity 1, a first oil cavity 2, a washing cavity 3, a second oil cavity 4 and a recovery cavity 5 are distributed on the chip body at intervals; the capturing chamber 1, the first oil chamber 2, the washing chamber 3, the second oil chamber 4 and the recovery chamber 5 are all communicated through a micro door 6; the bottoms of the capturing chamber 1 and the recycling chamber 5 are communicated with a gas channel, and the air channel is used for conveying external gas into the capturing chamber 1 or the recycling chamber 5.
Further, in the embodiment of the present invention, the capturing chamber 1 is used for mixing exosomes in the cell supernatant with magnetic beads to achieve capturing of free exosomes, the capturing chamber 1 has a rectangular structure with a volume of 350 μl, the capturing chamber 1 is added with nano magnetic beads which are commercially purchased or prepared by itself, for example, strep-Tactin magnetic beads of IBA company, cat No. 6-5510-050 are adopted in the specific embodiment of the present invention, and the surface of the magnetic beads is modified in a functional manner.
Further, the purification module comprises a first oil cavity 2, a washing cavity 3 and a second oil cavity 4, wherein silicone oil is placed in the first oil cavity 2 and the second oil cavity 4, a washing buffer solution is placed in the washing cavity 3, a non-miscible phase interface is constructed by the silicone oil and the washing buffer solution through a micro gate 6 between the silicone oil and the washing buffer solution, and the magnetic bead-exosome is washed and purified to remove residual lipophilic impurities or hydrophilic impurities on the surface of the magnetic bead-exosome, so that the purity of the enriched exosome is improved.
Further, the structures of the first oil chamber 2, the washing chamber 3 and the second oil chamber 4 disclosed in the embodiment of the present invention are all rectangular, and the shapes and volumes of the oil phase chamber and the washing chamber are not particularly limited, and the common shapes in the art can be adopted.
Further, in the embodiment of the invention, the volumes of the first oil chamber 2, the washing chamber 3 and the second oil chamber 4 are all set to 90 μl; in the present invention, the silicone oil is commercially available, for example, in the embodiment of the present invention, a silicone oil available from BBI company under the trade designation CA11 BA0011 is used, and the washing buffer composition is preferably 0.1M Tris-cl,0.15M NaCl and 1mM EDTA.
Further, in the embodiment of the present invention, the recovery chamber 5 is used for adding an elution buffer, and is used for releasing and recovering high-purity exosomes from the surface of the magnetic beads based on a competitive binding mechanism, the recovery chamber 5 is rectangular in structure, the volumes of the recovery chamber 5 are all set to 150 μl, and the elution buffer in the recovery chamber 5 is preferably a 100mM biotin solution.
Further, in the embodiment of the present invention, the micro-gate 6 is distributed between every two adjacent chambers, the micro-gate 6 has a trapezoid structure, and the thickness of the inner cavity of the micro-gate 6 is 300-500 μm, more preferably 400 μm; the length of the "micro gate" structure is 2-4mm, more preferably 3mm, and the length of the shorter end of the trapezoid structure is 500-800 μm, more preferably 600 μm.
Further, in the embodiment of the present invention, the first air flow channel 7 and the second air flow channel 9 are both L-shaped structures, the inlet ends of the first air flow channel 7 and the second air flow channel 9 are used for connecting a digital injection pump, and the widths of the inner cavities of the first air flow channel 7 and the second air flow channel 9 are both 50-200 μm.
Further, in the embodiment of the present invention, the outlet end of the first air flow channel 7 is below the capturing chamber 1, the first air flow channel 7 is communicated with the capturing chamber 1 through a plurality of distributed first sub-flow channels 8, the first sub-flow channels 8 are vertically distributed between the first air flow channel 7 and the capturing chamber 1, one end is communicated with the first air flow channel 7, and the other end is communicated with the capturing chamber 1.
Further, in the embodiment of the present invention, the outlet end of the second air flow channel 9 is located below the recovery cavity 5, a plurality of second sub-flow channels 10 are distributed between the recovery cavity 5 and the second air flow channel 9, one end of the second sub-flow channel 10 is communicated with the recovery cavity 5, and the other end is communicated with the second air flow channel 9.
Further, in the embodiment of the invention, the chip body is composed of two layers of organic glass (polymethyl methacrylate, PMMA), one layer is a basal layer with the thickness of 1mm, and the other layer is a runner layer with the thickness of 5 mm.
The embodiment of the invention also discloses an exosome separation method, which comprises the following steps:
step 1: adding a biological sample and superparamagnetic nano magnetic beads into the capturing chamber 1, and sequentially adding silicone oil, washing buffer solution and elution buffer solution into the first oil cavity 2, the washing cavity 3, the second oil cavity 4 and the recovery cavity 5 respectively;
step 2: continuously introducing gas into the capturing cavity at a constant speed for 10min by using a digital injection pump so as to promote the uniform mixing and combination of the magnetic beads and the biological sample;
step 3: after the uniform mixing is finished, the permanent magnet is arranged on the side surface of the micro-fluidic chip and moves from the capturing cavity 1 to the first oil cavity 2, the washing cavity 3, the second oil cavity 4 and the recovery cavity 5 in sequence at a fixed speed;
step 4: continuously introducing gas into the capturing chamber 1 at a constant speed for 15min by using a digital injection pump so as to promote the mixing of the magnetic bead-exosome and the elution buffer;
step 5: after stopping mixing, collecting the eluent rich in exosomes in the recovery cavity 5.
Further, in step 1, the volume of the loaded biological sample was 200. Mu.l, and the volume of the added silicone oil was 50. Mu.l; the volume of wash buffer added was 50 μl; the addition volume of elution buffer was 100 μl; the volume of the added superparamagnetic nano-beads was 50. Mu.l.
Further, in the method, the injection speed of the digital injection pump is set to be 100-500 mu l/min, more preferably, the injection speed is 400 mu l/min, gas is pumped into the air flow channel, and the purpose of pumping the gas is to generate micro bubbles at the bottom of the capturing chamber 1, so that the movement of fluid is accelerated and the mixing of magnetic beads and a sample is promoted based on the processes of rising, expanding, breaking and the like of the micro bubbles.
After the bubble is uniformly mixed, a permanent magnet is arranged on the side surface of the micro-fluidic chip, and the capturing chamber 1 moves towards the first oil chamber 2, the washing chamber 3, the second oil chamber 4 and the recovery chamber 5 in sequence, so that the magnetic beads are dragged to pass through an immiscible phase interface, and purification is completed and the magnetic beads are moved to the recovery chamber 5. In the present invention, the moving speed of the magnet is preferably 1 to 5mm/s, more preferably 2mm/s, and the moving method of the magnet includes manual movement and mechanical movement.
In the present invention, the purpose of the mixing of the recovery chamber 5 is to accelerate the elution process based on the biotin competition mechanism, and to detach the exosomes adsorbed on the surface of the magnetic beads.
After the elution is completed, the method for collecting the eluent is preferably pipetting. The collected eluent is enriched with high-concentration exosomes, which can be studied by means of subsequent analytical characterization.
By utilizing the microfluidic chip and the separation method provided by the invention to enrich the exosomes, the separation time is only about 26min, the capturing efficiency of the exosomes can reach about 75%, the release efficiency can reach about 60%, and the integrity and the physiological activity of the exosomes can not be influenced.
The invention discloses a specific embodiment:
example 1
The method for enriching exosomes by using the microfluidic chip comprises the following steps:
1) Centrifuging and filtering the cell culture supernatant to be tested at 300g, 2000g and 10000g for 10min, 20min and 30min, removing cell fragments and microvesicles, and taking the supernatant;
2) 200 μl of filtered cell culture supernatant sample and 50 μl of superparamagnetic nano-beads are added into the capture chamber 1, and 50 μl of silicone oil and washing buffer, 100 μl of elution buffer are sequentially added into the corresponding chamber;
3) Continuously introducing gas into the capturing cavity by the digital injection pump at a constant speed of 400 mu l/min, continuously injecting for 10min, and fully enriching exosomes;
4) The permanent magnet is utilized to drag the nano magnetic beads to sequentially pass through a capturing cavity 1, a first oil cavity 2, a washing cavity 3 and a second oil cavity 4 which are distributed on the chip body at intervals, and reach a recovery cavity 5, and the washing and purification of the magnetic beads-exosome are realized by virtue of the virtual wall formed by water and oil phases and the action of interfacial tension;
5) The digital syringe pump continuously introduces gas into the capture chamber at a constant speed of 400 μl/min for 15min, and releases recovered exosomes.
Comparative example 1 separation of exosomes by manual magnetic bead method
1) Centrifuging and filtering the cell culture supernatant to be tested at 300g, 2000g and 10000g for 10min, 20min and 30min, removing cell fragments and microvesicles, and taking the supernatant;
2) The filtered cell culture supernatant samples were coupled to CD63 antibodies in 500 μl of nanomagnetic beads: mixing at a ratio of 0.5mg, incubating at 4deg.C for 15min, and enriching exosomes;
3) Separating the incubated mixture by using an external magnetic field, carrying out solid-liquid separation, discarding supernatant to obtain a magnetic separation object, and washing the magnetic separation object for 3 times by using a washing buffer solution;
4) The washed magnetic separation was washed with elution buffer at 0.5mg: mixing in proportion of 1mL, incubating for 20min on a constant temperature oscillator, eluting thoroughly, and repeating for 3 times to obtain exosome extractive solution.
TABLE 1 comparison of different exosome separation methods
According to the exosome separation method realized by the chip provided by the embodiment of the invention, the adopted static immiscible phase technology is used for respectively retaining hydrophilic impurities and lipophilic impurities in the water phase chamber and the oil phase chamber by means of a virtual wall interface formed by water and oil phases, so that the on-chip washing and purification of the magnetic bead-exosome are realized. Compared with a purification mode based on continuous flow, the method does not need pumping solution to repeatedly wash the magnetic beads, reduces reagent consumption and magnetic bead loss caused by repeated washing, and remarkably shortens the purification time.
The bubble micromixer is integrated at the bottoms of the capturing chamber and the recycling chamber, and the movement of fluid and magnetic beads in the chamber is disturbed through the processes of rising, expanding, cracking and the like of bubbles to form micro-eddies, so that the collision probability of the magnetic beads and biological samples is increased, and the magnetic separation efficiency of an external body is further improved. According to the invention, the bubble micromixer is combined with the non-miscible phase technology, so that the high-efficiency mixing of the magnetic beads and the sample is realized, the traditional manual pipette is replaced for blowing and mixing, the problem that the non-miscible phase technology is difficult to realize automatic mixing is solved, and the pollution risk of the reagent and the sample is reduced.
The microfluidic chip provided by the invention can be used for rapidly and efficiently realizing exosome enrichment, and obtaining the high-purity complete exosome on the premise of not influencing the physiological activity of the exosome.
The exosome separation method based on the microfluidic chip has simple operation, can complete separation within about 26 minutes, and has good separation performance repeatability and strong separation specificity
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The microfluidic chip for separating the exosomes is characterized by comprising a chip body, wherein capture chambers (1), a first oil cavity (2), a washing cavity (3), a second oil cavity (4) and a recovery cavity (5) are distributed on the chip body at intervals;
the capturing cavity (1), the first oil cavity (2), the washing cavity (3), the second oil cavity (4) and the recycling cavity (5) are communicated through a micro-door (6);
the bottoms of the capturing chamber (1) and the recycling chamber (5) are communicated with an air flow channel, and the air flow channel is used for conveying external air into the capturing chamber (1) or the recycling chamber (5).
2. Microfluidic chip for exosome separation according to claim 1, characterized in that the capture chamber (1) communicates below with a first air flow channel (7);
the first air flow passage (7) is of an L-shaped structure, the inlet end of the first air flow passage (7) is higher than the bottom of the capturing chamber (1), and the outlet end of the first air flow passage (7) is arranged below the capturing chamber (1).
3. Microfluidic chip for exosome separation according to claim 2, characterized in that the first air flow channel (7) and the capturing chamber (1) are vertically distributed with a first sub-flow channel (8);
one end of the first sub-runner (8) is communicated with the outlet end of the first air runner (7), and the other end of the first sub-runner is communicated with the inlet at the bottom of the capturing chamber (1).
4. The microfluidic chip for exosome separation according to claim 1, wherein a second air flow channel (9) is communicated below the recovery cavity (5), and the second air flow channel (9) has an L-shaped structure;
the inlet end of the second air flow passage (9) is higher than the bottom of the recovery cavity (5), and the outlet end of the second air flow passage (9) is positioned below the recovery cavity (5);
the lower end of the recovery cavity (5) is provided with a plurality of second sub-runners (10) which are distributed at intervals, one end of each second sub-runner (10) is communicated with the bottom of the recovery cavity (5), and the other end of each second sub-runner is communicated with the outlet end of each second air runner (9).
5. The microfluidic chip for exosome separation according to claim 1, wherein the micro gate (6) has a trapezoid structure, and the length of the inlet end of the micro gate (6) is greater than the length of the outlet end.
6. Microfluidic chip for exosome separation according to claim 5, characterized in that the inlet end to outlet end distance of the micro gate (6) is 2-4mm;
the height of the cavity inside the micro-gate (6) is 300-500 mu m;
the length of the outlet end of the micro-gate (6) is 500-800 mu m.
7. The microfluidic chip for exosome separation according to claim 1, wherein the width of the air flow channel lumen is 50-200 μm.
8. The microfluidic chip for separation of exosomes according to claim 1, wherein the end surfaces of the chamber ports of the capture chamber (1), the first oil chamber (2), the wash chamber (3), the second oil chamber (4) and the recovery chamber (5) are perpendicular to the end surfaces of the air flow channel inlet end.
9. A method for separating exosomes, comprising the steps of:
adding a biological sample and nano magnetic beads into the capturing cavity (1), and sequentially adding silicone oil, washing buffer, silicone oil and elution buffer into the first oil cavity (2), the washing cavity (3), the second oil cavity (4) and the recovery cavity (5) respectively;
continuously introducing gas into the capturing chamber (1) at a constant speed, wherein the gas generates bubbles to drive the nano magnetic beads and the biological sample to be uniformly mixed;
after the mixing is finished, placing a permanent magnet on the side surface of the chip body, and sequentially moving the permanent magnet along the first oil cavity (2), the washing cavity (3), the second oil cavity (4) and the recovery cavity (5) of the capturing cavity (1);
continuously introducing gas into the recovery cavity (5) at a constant speed so as to promote the mixing of the magnetic bead-exosome and the elution buffer;
after stopping mixing, collecting the eluent rich in exosomes in the recovery cavity.
10. An exosome separation method according to claim 9, wherein the injection of gas into the capture chamber (1) or the recovery chamber (5) is performed by a digital syringe pump at a rate of 100-500 μl/min.
CN202310890238.5A 2023-07-19 2023-07-19 Microfluidic chip for exosome separation and exosome separation method Pending CN116875455A (en)

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