CN111172009A - Integrated exosome separation chip and preparation method and application thereof - Google Patents
Integrated exosome separation chip and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an integrated exosome separation chip and a preparation method and application thereof. The sample introduction unit is positioned in front of the exosome capturing unit, and the enrichment unit is positioned behind the exosome capturing and releasing unit. According to the invention, the sample is introduced, the exosome is captured and released, the non-exosome substance is cleaned, and the exosome is collected and purified on the microfluidic chip, so that the exosome extraction process is optimized, the exosome extraction and purification efficiency is greatly improved, the manual operation procedure is simplified, and a better exosome separation and purification effect is obtained.
Description
Technical Field
The invention relates to the field of exosome separation, in particular to an integrated exosome separation chip.
Technical Field
The exosome is an extracellular nanoscale vesicle formed by cells through a series of regulation processes such as endocytosis, fusion and efflux. It is widely distributed in human body, and urine, sweat, blood, milk, etc. of human body all contain exosome. The role of exosomes in the human body mainly plays two roles, the first is that of immunologically active exosomes, which play a role mainly in antigen presentation and co-stimulation and have an information transfer function. The second is an exosome containing a considerable amount of RNA and mediating the exchange of genetic material between cells, and has a material-transferring function. With the progress of research, it is found that exosomes play an important role in adaptive immunity, inflammation process, embryogenesis, and tumor generation and development process. In the case of tumors, over a hundred years ago, it was discovered through dissection that specific tumor cells always tend to metastasize to specific tissue organs, and thus a well-known "seed and soil" metastasis hypothesis was proposed that tumor cells could only form metastases in the appropriate tissue organ environment. With the development of the technology, the metastasis mechanism of the tumor is continuously improved, people find that the tumor can actively change the microenvironment of a metastasis focus by secreting exosomes, and the exosomes can promote tumor angiogenesis and tumor metastasis by regulating the immune function, or directly act on tumor cells to influence the tumor development. Therefore, research on exosomes is expected to provide a new idea for early diagnosis of tumors, inhibition of tumor development and the like.
To study exosomes, the first question is how to isolate exosomes from a sample. The microfluidic chip technology is an important technology in the 21 st century, and has the characteristics of micro-size, automation, small amount of used reagents, high flux, realization of multifunctional integration and the like, so that the microfluidic chip technology becomes a potential platform for exosome research. At present, two types of technologies for separating exosomes by using a microfluidic chip are available, one is a separation technology based on size, and the separation technology mainly comprises the step of directly acting on a sample by using device structures such as a nanopore membrane, a nano array, a microfilter and the like to separate exosomes. The other is separation technology based on immunocapture, which mainly comprises planar immunocapture and microbead immunocapture. However, it is still a problem to obtain high-purity exosomes efficiently.
Disclosure of Invention
The invention aims to provide an integrated exosome separation chip.
An integrated exosome separation chip mainly comprises a top channel chip, a porous filter membrane and a bottom channel chip;
the top channel chip consists of three identical independent pentagonal chambers, and each top channel chamber comprises an outlet; an inlet hole is arranged opposite to the outlet;
the porous filter membrane is sealed between the upper channel layer and the lower channel layer through PDMS (polydimethylsiloxane) glue;
the bottom channel chip consists of three pentagonal chambers opposite to the direction of the top channel, each bottom channel chamber comprises an inlet, and each inlet is connected with an inlet hole at the corresponding position of the top channel chip through a polytetrafluoroethylene tube;
the top channel chip and the bottom channel chip form a unit by an upper chamber, a lower chamber, a corresponding chamber and a middle filter membrane, and the unit sequentially comprises a sample introduction unit, an exosome capture and release unit and an exosome enrichment unit;
the exosome capturing and releasing unit is positioned behind the sample feeding unit, and the exosome enriching unit is positioned behind the exosome capturing and releasing unit; the exosome sample introduction unit, the exosome capturing and releasing unit and the exosome enrichment unit are connected with an outlet of the last unit and an inlet of the next unit through polytetrafluoroethylene tubes to control the on-off of the units.
The pore diameter of the porous filter membrane of the sample introduction unit is 100nm-200nm, the pore diameter of the porous filter membrane of the exosome capture and release unit is 2 μm-5 μm, and the pore diameter of the porous filter membrane of the exosome enrichment unit is 20nm-40 nm.
The exosome-separating chip is prepared according to the following steps: preparing an upper channel template and a lower channel template by using SU8 glue through a soft lithography technology, performing reverse molding by using mixed PDMS (PDMS: initiator: 10:1), sealing the porous filter membrane on the lower channel by using PDMS glue, curing the glue by using an oven at 80 ℃, and sealing the upper channel and the porous filter membrane by using an oxygen plasma device.
When an exosome separation chip is adopted to separate exosomes in a sample, the method comprises the following steps:
(1) performing hydrophobic modification on the chip, specifically, placing the chip in a vacuum drier for about ten minutes, adding hexadecane containing 2-5% by volume of octadecyl trichlorosilane into all channels, and incubating for three minutes at room temperature; sucking up liquid in the channel, cleaning the channel for at least 5min by using ethanol and water in sequence, and placing the chip in an oven at 80 ℃ for more than 1 h;
(2) injecting a certain amount of exosome capture microbeads into a lower channel chamber of a chip exosome capture and release unit through a pipette gun;
(3) the chip is rinsed by PBS (phosphate buffer solution), and then a sample to be separated is led into a bottom channel chamber of an exosome sample injection unit through a polytetrafluoroethylene tube; the sample entering the sample introduction unit flows into the top channel chamber inlet of the capture and separation unit from the sample introduction unit outlet through the polytetrafluoroethylene tube;
(4) injecting a DTT (dithiothreitol) solution from the capture and release unit bottom layer channel chamber inlet through a polytetrafluoroethylene tube and inlet port; the released exosome flows into the exosome enrichment unit from the top layer channel chamber outlet of the capture and release unit through the bottom layer channel chamber inlet of the exosome enrichment unit through a polytetrafluoroethylene tube;
(5) and (3) enabling the PBS to flow in through a polytetrafluoroethylene tube through an inlet of a bottom channel chamber of the exosome enrichment unit to realize the enrichment and cleaning of the exosome.
The exosome capture microbead needs to be modified before use, and the modification steps are as follows: incubating for half an hour at 85 ℃ by using 1-2% of silane coupling agent, and washing by using water; adding 2-10 μ g/mL DTSSP (3, 3-dithiobis (sulfosuccinimidyl propionate)) solution, incubating at 4 deg.C for 12-16 h, and washing with PBS; adding 2-10 μ g/mL biotinylated BSA (bovine serum albumin) at 4 deg.C, incubating for more than 2h, and washing with PBS; adding 2-10 μ g/mL avidin, incubating at 4 deg.C for 1-2 h, and washing with PBS; biotinylated exosome-associated antibody was added at 2-10 μ g/mL and incubated at 4 ℃ for about 2h for future use.
When the integrated exosome separation chip is used, the flow velocity in all channels is 2-10 mu L/min. The exosome capturing and releasing unit adopts the principle that DTT (dithiothreitol) can reduce disulfide bonds in DTSSP (3, 3-dithiobis (sulfosuccinimidyl propionate)), so as to realize reversible capturing and releasing of exosomes.
The invention has the advantages that:
1. the invention adopts two modes of separating exosomes based on size and immunocapture, and can obtain exosomes with higher purity.
2. The invention introduces a sample, captures and releases exosomes, and concentrates exosome enrichment on one chip, thereby optimizing an exosome extraction process.
3. According to the invention, the silica beads are intercepted by the filter membrane, so that the manual operation is simplified, the error is reduced, and the spatial-temporal resolution which is difficult to realize by the traditional method is realized.
Drawings
FIG. 1 is a front view of an integrated exosome-separation chip;
FIG. 2 is a schematic diagram of a top-level chip of an integrated exosome-separation chip;
FIG. 3 is a schematic diagram of a bottom chip of an integrated exosome-separation chip;
FIG. 4 is a schematic perspective view of an integrated exosome-separation chip;
FIG. 5 is a schematic diagram of a process of separating exosomes by ② integrated exosome separation chip, including ② exosome sample introduction process, ② exosome capture process, ② exosome release process, and ② exosome enrichment process.
FIG. 6 is a graph showing the change in fluorescence on silica beads observed after exosomes are captured using the chip and subjected to fluorescent staining and released;
FIG. 7 is a graph comparing the relative content of exosomes captured using the chip and ultracentrifugation;
FIG. 8 shows the expression of exosome-associated genes isolated from MCF-7 cell culture medium;
FIG. 9 shows the expression of the relevant genes in blood exosomes of breast cancer patients.
Wherein: 1, a top-layer channel chip 2, a porous filter membrane 3, a bottom-layer channel chip 4, a top-layer channel chamber 5, an outlet 6, an inlet hole 7, an inlet 8, a bottom-layer channel chamber 9, a polytetrafluoroethylene tube 10 for capturing silica beads, 11 to-be-separated large-volume impurities (such as cell fragments and large vesicles) in a sample, 12 to-be-separated small-volume impurities (such as protein molecules) in the sample, 13 to-be-separated exosomes in the sample;
A. exosome sample introduction unit B, exosome capture and release unit C, exosome enrichment and cleaning unit
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
Example 1
An integrated exosome separation chip is mainly composed of a top channel chip 1, a porous filter membrane 2 and a bottom channel chip 3; the top channel chip 1 consists of three identical independent pentagonal chambers, and each top channel chamber 4 comprises an outlet 5; an inlet opening 6 is arranged opposite to the outlet 5; the porous filter membrane 2 is sealed between the top layer channel layer and the bottom layer channel layer through PDMS (polydimethylsiloxane) glue;
the bottom channel chip 3 consists of three pentagonal chambers opposite to the direction of the top channel, each bottom channel chamber 8 comprises an inlet 7, and each inlet 7 is connected with an inlet hole 6 at the corresponding position of the top channel chip through a polytetrafluoroethylene tube; the top channel chip 1 and the bottom channel chip form a unit by an upper chamber, a lower chamber, a corresponding chamber and a middle filter membrane, and the unit is sequentially provided with an exosome sample introduction unit A, an exosome capture and release unit B and an exosome enrichment unit C; the exosome capturing and releasing unit B is positioned behind the exosome sampling unit A, and the exosome enriching unit C is positioned behind the exosome capturing and releasing unit B; and the exosome sample introduction unit A, the exosome capturing and releasing unit B and the exosome enrichment unit C are connected with an outlet of the last unit and an inlet of the next unit through polytetrafluoroethylene tubes to control the on-off of the units.
The pore diameter of the porous filter membrane of the sample introduction unit is 100nm-200nm, the pore diameter of the porous filter membrane of the exosome capture and release unit is 2 μm-5 μm, and the pore diameter of the porous filter membrane of the exosome enrichment unit is 20nm-40 nm. Preparation of integrated exosome separation chip
(1) Preparation of the chip
Firstly, drawing upper and lower channels as shown in figures 2 and 3 by using autoCAD software, and manufacturing a mask;
preparing an upper layer channel template and a lower layer channel template by using SU8 glue through a soft lithography technology;
the mixed PDMS (PDMS: initiator ═ 10:1) was used for the back mold;
then, PDMS glue is used to seal the filter membranes with the aperture of 200nm, 5 μm and 40nm at the corresponding positions of the lower channel, as shown in FIG. 4, and the glue is cured in an oven at 80 ℃;
sealing the upper channel and the porous filter membrane by using oxygen plasma equipment;
the prepared chip uses hexadecane containing 5 percent of octadecyl trichlorosilane, and is incubated for three minutes at room temperature; and (3) sucking liquid in the channel, washing the channel for 5min by using ethanol and water in sequence, and placing the chip in an oven at 80 ℃ for 1h for later use.
(2) Modification of silica beads
Take 5X 107100 mu L/mL silica beads, incubating for half an hour at 85 ℃ by using 1-2% silane coupling agent, and washing by using water;
adding 2 mu g/mL DTSSP solution, incubating for 16h at 4 ℃, and washing by using PBS;
add 2 u g/mL biotinylated BSA at 4 degrees C were incubated for 2h, using PBS washing;
adding 2-10 μ g/mL avidin, incubating at 4 deg.C for 2h, and washing with PBS;
biotinylated EpCAM antibody at 2. mu.g/mL was added, incubated at 4 ℃ for about 2h, washed with PBS, and finally resuspended to 100. mu.L in PBS for use.
Example 2
Isolation of exosomes in MCF-7 (human breast cancer cell) medium
Separating exosomes in MCF-7 culture medium by using the chip prepared in the embodiment 1 and modified silica beads;
firstly, using PBS to rinse a chip to fill liquid in a channel;
then 10 mul of silica beads were added from the inlet of the exosome capture and release unit;
then, 10mL of cell culture medium to be separated is introduced into the sample injection unit from an inlet through a polytetrafluoroethylene tube at the flow rate of 2 muL/min, as shown in figure 5 (r), larger impurities such as cell fragments, large vesicles and the like in the unit are intercepted;
the sample entering the sample introduction unit flows into the inlet of the capturing and separating unit from the outlet of the sample introduction unit through the polytetrafluoroethylene tube, in the process, exosomes are captured by the immune microbeads, and impurities with small volume flow into the waste liquid vessel from the outlet of the capturing and separating unit through the polytetrafluoroethylene tube, and the process is shown as ② in fig. 5;
then 10 muM/L DTT solution is injected from the inlet of the capture and release unit through a polytetrafluoroethylene tube at the flow rate of 2 muL/min, and exosome is released from the silica beads in the process, as shown in the third step in FIG. 5;
the released exosomes flow into the exosome-enriching unit from the outlet of the capturing and releasing unit through the inlet of the exosome-enriching unit at a rate of 2 μ L/min through the polytetrafluoroethylene tube, and in this unit, the exosomes are enriched on the porous filtration membrane, and finally, 1mL of PBS flows in from the exosome-enriching unit through the inlet of the exosome-enriching unit through the polytetrafluoroethylene tube at a rate of 2 μ L/min, and then flows out into the waste liquid tank from the outlet, so that the exosomes are enriched, and washed, the process of which is shown as ④ in fig. 5.
FIG. 6 is a graph showing the immunofluorescence staining of exosomes on silica beads after the exosomes are captured by the silica beads in the process and the fluorescence change on the silica beads during release, and the release efficiency of the chip after capture can reach 90% by using gray level analysis.
Example 3
Isolation of exosomes in MCF-7 (human breast cancer cell) medium
Separating exosomes in MCF-7 culture medium by using the chip prepared in the embodiment 1 and modified silica beads;
firstly, using PBS to rinse a chip to fill liquid in a channel;
then 10 mul of silica beads were added from the inlet of the exosome capture and release unit;
then, 10mL of cell culture medium to be separated is introduced into the sample injection unit from the inlet through a polytetrafluoroethylene tube at the flow rate of 10 muL/min, as shown in figure 5 (r), larger impurities such as cell fragments, large vesicles and the like in the unit are intercepted;
the sample entering the sample introduction unit flows into the inlet of the capturing and separating unit from the outlet of the sample introduction unit through the polytetrafluoroethylene tube, in the process, exosomes are captured by the immune microbeads, and impurities with small volume flow into the waste liquid vessel from the outlet of the capturing and separating unit through the polytetrafluoroethylene tube, and the process is shown as ② in fig. 5;
then 5 muM/L DTT solution is injected from the inlet of the capture and release unit through a polytetrafluoroethylene tube at the flow rate of 10 muL/min, and exosome is released from the silica beads in the process, as shown in the third step in figure 5;
the released exosomes flow into the exosome-enriching unit from the outlet of the capturing and releasing unit through the inlet of the exosome-enriching unit at a speed of 10 muL/min through a polytetrafluoroethylene tube, and the exosomes are enriched on the porous filter membrane in the unit, and finally 1mL of PBS flows into the exosome-enriching unit from the exosome-enriching unit through the inlet of the exosome-enriching unit through the polytetrafluoroethylene tube at a speed of 10 muL/min and flows out of the outlet into a waste liquid pool to realize the enrichment and cleaning of the exosomes, wherein the process is shown as (r) in FIG. 5;
finally, the exosomes on the filter membrane are resuspended by 100 μ L of PBS to realize the separation of the exosomes.
FIG. 7 shows the relative amounts of exosomes in 10mL of medium, which was measured by BSA method, after the exosomes separated by ultracentrifugation and integrated exosome separation chip were finally resuspended in 100. mu.L, and it can be seen from the results that the amount of exosomes separated by the integrated exosome separation chip was more than 75% of that in ultracentrifugation. FIG. 8 shows the results of the identification of genes related to isolated exosomes, which shows that miR-21 is expressed well, and the other genes are expressed, consistent with the literature results.
Example 4
Separation of exosomes from blood sample of breast cancer patient
The chip prepared in the embodiment 1 and the modified silica beads are selected to separate exosomes in the blood sample of the breast cancer patient;
firstly, using PBS to rinse a chip to fill liquid in a channel;
then 10 mul of silica beads were added from the inlet of the exosome capture and release unit;
taking 500 mu L of blood samples of breast cancer patients, and diluting the blood samples into 5mL of PBS;
5mL of sample to be separated is introduced into the sample introduction unit from an inlet through a polytetrafluoroethylene tube at a flow rate of 5 muL/min, as shown in figure 5 (r), larger impurities such as cell fragments, large vesicles and the like in the unit are intercepted;
the sample entering the sample introduction unit flows into the inlet of the capturing and separating unit from the outlet of the sample introduction unit through the polytetrafluoroethylene tube, in the process, exosomes are captured by the immune microbeads, and impurities with small volume flow into the waste liquid vessel from the outlet of the capturing and separating unit through the polytetrafluoroethylene tube, and the process is shown as ② in fig. 5;
then 10 muM/L DTT solution is injected from the inlet of the capture and release unit through a polytetrafluoroethylene tube at the flow rate of 5 muL/min, and exosome is released from the silica beads in the process, as shown in the third step in figure 5;
the released exosomes flow into the exosome-enriching unit from the outlet of the capturing and releasing unit through the inlet of the exosome-enriching unit at a speed of 5 mul/min through a polytetrafluoroethylene tube, and in the exosome-enriching unit, the exosomes are enriched on a porous filter membrane, and finally, 5mL of PBS flows into the exosome-enriching unit from the exosome-enriching unit through the inlet of the exosome-enriching unit through the polytetrafluoroethylene tube at a speed of 5 mul/min, and then flows out of the outlet into a waste liquid pool to realize the enrichment and cleaning of the exosomes, and the process is shown as (r) in FIG. 5;
finally, the exosomes on the filter membrane are resuspended by 1mL of PBS to realize the separation of the exosomes.
FIG. 9 shows the results of the identification of genes related to exosomes in the blood of breast cancer patients, which shows that miR-21 is well expressed, and other genes are expressed, consistent with the results in the literature.
The integrated exosome separation chip can meet different purposes by changing the pore size of a membrane and capturing an antibody modified by silica beads. For example, when vesicle or substance with specific size is separated, the sizes of the sample introduction unit and the enrichment unit filter membrane can be controlled, the antibody modified by the silica beads can be changed, and the separation requirement on the specific vesicle can be met.
Claims (7)
1. An integrated exosome-separating chip, characterized by: the micro-fluidic chip mainly comprises a top channel chip, a porous filter membrane and a bottom channel chip;
the top channel chip consists of three identical independent pentagonal chambers, and each top channel chamber comprises an outlet; an inlet hole is arranged opposite to the outlet;
the porous filter membrane is sealed between the upper channel layer and the lower channel layer through PDMS glue;
the bottom channel chip consists of three pentagonal chambers opposite to the direction of the top channel, each bottom channel chamber comprises an inlet, and each inlet is connected with an inlet hole at the corresponding position of the top channel chip through a polytetrafluoroethylene tube;
the top channel chip and the bottom channel chip form a unit by an upper chamber, a lower chamber, a corresponding chamber and a middle filter membrane, and the unit sequentially comprises a sample introduction unit, an exosome capture and release unit and an exosome enrichment unit;
the exosome capturing and releasing unit is positioned behind the sample feeding unit, and the exosome enriching unit is positioned behind the exosome capturing and releasing unit;
the exosome sample introduction unit, the exosome capturing and releasing unit and the exosome enrichment unit are connected with an outlet of the last unit and an inlet of the next unit through polytetrafluoroethylene tubes to control the on-off of the units.
2. Exosome-separating chip according to claim 1, characterized in that: the pore diameter of the porous filter membrane of the sample introduction unit is 100nm-200nm, the pore diameter of the porous filter membrane of the exosome capture and release unit is 2 μm-5 μm, and the pore diameter of the porous filter membrane of the exosome enrichment unit is 20nm-40 nm.
3. The method for preparing an exosome-separating chip according to claim 1, characterized in that: the preparation method comprises the following steps: preparing an upper channel template and a lower channel template by using SU8 glue through a soft lithography technology, performing reverse molding by using mixed PDMS (PDMS: initiator: 10:1), sealing the porous filter membrane on the lower channel by using PDMS glue, curing the glue by using an oven at 80 ℃, and sealing the upper channel and the porous filter membrane by using an oxygen plasma device.
4. The use of an exosome-separating chip according to claim 1, wherein the exosome-separating chip is used for separating exosomes from a sample according to the following steps:
(1) performing hydrophobic modification on the chip, specifically, placing the chip in a vacuum drier for about ten minutes, adding hexadecane containing 2-5% by volume of octadecyl trichlorosilane into all channels, and incubating for three minutes at room temperature; sucking up liquid in the channel, cleaning the channel for at least 5min by using ethanol and water in sequence, and placing the chip in an oven at 80 ℃ for more than 1 h;
(2) injecting a certain amount of exosome capture microbeads into a lower channel chamber of a chip exosome capture and release unit through a pipette gun;
(3) the chip is rinsed by PBS, and then a sample to be separated is introduced into a bottom channel chamber of an exosome sample introduction unit through a polytetrafluoroethylene tube; the sample entering the sample introduction unit flows into the top channel chamber inlet of the capture and separation unit from the sample introduction unit outlet through the polytetrafluoroethylene tube;
(4) injecting a DTT solution from the inlet of the bottom channel chamber of the capture and release unit through a polytetrafluoroethylene tube and an inlet hole; the released exosome flows into the exosome enrichment unit from the top layer channel chamber outlet of the capture and release unit through the bottom layer channel chamber inlet of the exosome enrichment unit through a polytetrafluoroethylene tube;
(5) and (3) enabling the PBS to flow in through a polytetrafluoroethylene tube through an inlet of a bottom channel chamber of the exosome enrichment unit to realize the enrichment and cleaning of the exosome.
5. Use of an exosome-separating chip according to claim 4, characterized in that the exosome-capturing microbeads used need to be modified before use by the following steps: incubating for half an hour at 85 ℃ by using 1-2% of silane coupling agent, and washing by using water; adding 2-10 mug/mL DTSSP solution, incubating at 4 ℃ for 12-16 h, and washing with PBS; adding 2-10 μ g/mL biotinylated BSA at 4 deg.C, incubating for more than 2h, and washing with PBS; adding 2-10 μ g/mL avidin, incubating at 4 deg.C for 1-2 h, and washing with PBS; biotinylated exosome-associated antibody was added at 2-10 μ g/mL and incubated at 4 ℃ for about 2h for future use.
6. Use of an exosome-separating chip according to claim 4, characterised in that: the flow rate in all channels is 2-10 muL/min.
7. Use of an exosome-separating chip according to claim 4, characterised in that: the exosome capturing and releasing unit adopts the principle that DTT can reduce disulfide bonds in DTSSP, and reversible capturing and releasing of exosomes are achieved.
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