KR102112416B1 - microfluidics chip for filtering extracellular vesicles - Google Patents

Abstract

The microfluidic chip for extracting nanovesicles according to an embodiment of the present invention for solving the above problems includes: a first substrate formed with a first inlet portion through which a mixed fluid containing nanovesicles flows; A filter substrate stacked on the lower surface of the first substrate and filtering the mixed fluid flowing into the first inlet; And a second substrate stacked on the filter substrate, a second inlet portion into which a buffer solution flows, and a first discharge portion through which the nanovesicles filtered from the filter substrate are mixed with the buffer liquid and discharged. It includes.

Description

Microfluidics chip for filtering extracellular vesicles

The present invention relates to a microfluidic chip for extracting nanovesicles, and to a microfluidic chip for extracting nanovesicles that can rapidly extract nanovesicles from a mixed fluid containing nanovesicles and increase the purity of the extracted nanovesicles. .

Nanocellular vesicles (Extracellular vesicles, EVs) are vesicles that cells release outside the cell, and include microvesicles (MVs), apoptotic bodies, exosomes, and the like.

These nanovesicles can be distinguished from each other according to size and role, but function as a transport medium for information exchange between cells. Nano vesicles are derived from cells and act to stimulate or inhibit metabolism of other cells.

The nano vesicles reflect the approximate characteristics and state of the hair cells that release them. The substances that the nano vesicles move from the parent cell to other cells are bio-derived substances such as proteins, lipids, metabolites, and nucleic acids (DNA / RNA). It is transmitted to cells (Recipient cell) and performs the role of signaling between cells.

Exosome (Exosome) is one of the nano-vesicles, among them is the smallest vesicular particle having a diameter of about 20 ~ 200 nm, and serves to transport bio-derived substances including proteins. Exosomes have the same genetic and protein information as parent cells, and are packaged with lipid membranes, which are structurally stable and exist in abundant amounts in the blood, so they can act as biomarkers.

On the other hand, a microfluidics chip (microfluidics chip) is a biochip capable of simultaneously performing various experimental conditions by flowing a fluid through a microfluidic channel. The microfluidic chip uses a substrate (or chip material) made of plastic, glass, silicon, or the like to create a micro channel, and after moving a fluid (for example, a liquid sample) through the channel, the microfluidic chip Sample separation, cell mixing, synthesis, quantitative analysis, cell proliferation observation, etc. can be performed in the inner chamber. As described above, the microfluidic chip is also referred to as a lab on a chip in that experiments performed in a laboratory are performed in a small chip.

The microfluidic chip not only creates cost and time saving effects in the fields of pharmaceutical, biotechnology, medicine, biomedical, food, environment, and fine chemistry, but also can increase accuracy, efficiency, and reliability. For example, the use of microfluidic chips can significantly reduce the amount of expensive reagents used in cell culture, proliferation, and differentiation, compared to conventional methods, thereby reducing significant costs. In addition, in the analysis of biological samples such as proteins, genes, DNA, cells, neurons, enzymes, and antibodies, a much smaller amount is used than conventional methods, and images can also be used. As analysis is possible, it is possible to reduce the amount of sample consumption or consumption and analysis time.

In particular, the development of a microfluidic chip capable of extracting nanovesicles such as exosomes, which have recently attracted attention as a biomarker, enables diagnosis of diseases from exosomes having the same genetic and protein information as parent cells.

The problem to be solved by the present invention is to provide a microfluidic chip for extracting nanovesicles that can rapidly extract nanovesicles from a mixed fluid containing nanovesicles and increase the purity of the extracted nanovesicles.

The problems of the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The microfluidic chip for extracting nanovesicles according to an embodiment of the present invention for solving the above problems includes: a first substrate formed with a first inlet portion through which a mixed fluid containing nanovesicles flows; A filter substrate stacked on the lower surface of the first substrate and filtering the mixed fluid flowing into the first inlet; And a second substrate stacked on the filter substrate, a second inlet portion into which a buffer solution flows, and a first discharge portion through which the nanovesicles filtered from the filter substrate are mixed with the buffer liquid and discharged. It includes.

In addition, the filter substrate, the first membrane to transmit the nano-vesicles of the mixed fluid flowing into the first inlet, the first material of a predetermined size is impermeable; And a second membrane through which the nanovesicles mixed in the buffer solution introduced into the second inlet part are impermeable and permeate a second material having a predetermined size. It may include.

Further, the first membrane and the second membrane may be disposed on the same plane.

In addition, the first substrate, the first trap is connected to the first inlet and disposed to face the first membrane; And a second discharge part connected to the first trap to discharge the mixed fluid containing the first material that is impermeable from the first membrane. It may include.

In addition, the second discharge part may be connected to the first inlet part and the first circulation channel.

In addition, the first substrate may include a first chamber formed on the first substrate and disposed to face the second membrane; And a third discharge part connected to the first chamber to discharge the second material transmitted from the second membrane. It may include.

In addition, the second substrate is connected to the second inlet, the second chamber through which the nanovesicles transmitted from the first membrane are mixed with the buffer solution; And a second trap connected to the second chamber, connected to the first discharge portion, and disposed to face the second membrane; It may include.

Also, the first discharge part may be connected to the second inlet part and the second circulation passage.

In addition, the filter substrate, the first filter substrate is in contact with the lower surface of the first substrate is provided with the fifth membrane; And a second filter substrate contacting the lower surface of the second substrate and having the sixth membrane. It may include.

In addition, the first substrate may include a fifth chamber connected to the first inlet and disposed to face the fifth membrane.

In addition, the second substrate is connected to the second inlet and the first outlet, a sixth chamber in which the nanovesicles permeated in the fifth membrane are mixed with the buffer solution; And a flow channel connected to the sixth chamber and flowing the buffer solution to the sixth membrane. It may include.

In addition, the third substrate may further include a third substrate contacting the lower surface of the second filtering substrate.

In addition, the third substrate may include: a seventh chamber disposed opposite to the sixth membrane and opposite to the sixth chamber; And a sixth discharge part connected to the seventh chamber and discharging the second material transmitted from the sixth membrane. It may include.

In addition, the nanovesicles may be exosomes (exosomes).

In addition, the first material may include cell debris.

In addition, the second substance may include serum protein.

In addition, the diameter of the pores of the first membrane may be 200 nm or less, and the diameter of the pores of the second membrane may be less than 20 nm.

Details of other embodiments are included in the detailed description and drawings.

According to the microfluidic chip for extracting nanovesicles according to an embodiment of the present invention, nanovesicles containing exosomes are extracted from a microfluidic chip as a first discharge part, and impurities such as cell debris and serum proteins are discharged to the outside. Therefore, it is possible to quickly extract the nano vesicles.

In addition, since the first circulation flow path and the second circulation flow path are provided, the nanovesicles remaining in the mixed fluid are supplied back to the first inlet to increase the recovery rate of the nanovesicles, so that the buffer solution containing the high purity nanovesicles can be extracted. Can be.

In addition, since the overall configuration is simple and can be configured to extend in the longitudinal direction, it is possible to reduce the manufacturing cost of the microfluidic chip.

The effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the description of the claims.

1 is a perspective view of a microfluidic chip for nano-vesicle extraction according to an embodiment of the present invention.
Figure 2 is an exploded perspective view of a microfluidic chip for nano-vesicle extraction according to an embodiment of the present invention.
3 is a cross-sectional view of the filter substrate 30 according to an embodiment of the present invention shown in FIG. 2.
Figure 4 is a schematic view showing the mechanism of the microfluidic chip for nano-vesicle extraction according to an embodiment of the present invention.
5 is a prototype photograph of a microfluidic chip for nano-vesicle extraction according to an embodiment of the present invention.
6 is a view showing that the microfluidic chip for extracting nano vesicles according to an embodiment of the present invention is used by extending in the longitudinal direction according to an embodiment.
7 is an exploded perspective view of a microfluidic chip for nano-vesicle extraction according to another embodiment of the present invention.
8 is a schematic view showing the mechanism of the microfluidic chip for nano-vesicle extraction according to another embodiment of the present invention.

In describing the present invention, when it is determined that a detailed description of related known functions or configurations may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted. Even if the terms are the same, it is said in advance that the reference numerals do not match if the parts displayed are different.

In addition, the terms to be described below are terms set in consideration of functions in the present invention, which may vary according to an operator's intention or practice, such as an experimenter and a measurer, and thus the definition should be made based on the contents throughout the present specification.

In the present specification, terms such as first and second may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from other components. For example, without departing from the scope of the present invention, the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component. The term and / or includes a combination of a plurality of related described items or any one of a plurality of related described items.

The terms used herein are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Terms such as those defined in a commonly used dictionary should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

Also, when a part is said to "include" a certain component, this means that other components may be further included instead of excluding other components, unless specifically stated to the contrary.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The same reference numerals in each drawing denote the same members.

1 is a perspective view of a microfluidic chip for extracting nanovesicles according to an embodiment of the present invention, FIG. 2 is an exploded perspective view of a microfluidic chip for extracting nanovesicles according to an embodiment of the present invention, and FIG. 3 is FIG. 2 It is a cross-sectional view of the filter substrate 30 according to an embodiment of the present invention shown in, Figure 4 is a schematic view showing the mechanism of the microfluidic chip for nano-vesicle extraction according to an embodiment of the present invention, Figure 5 Is a prototype photograph of a microfluidic chip for nano-vesicle extraction according to an embodiment of the present invention.

1 to 5, the microfluidic chip for extracting nanovesicles according to an embodiment of the present invention is a first substrate 10 having a first inlet 11 through which a mixed fluid containing nanovesicles is introduced. , It is laminated on the lower surface of the first substrate 10, the filter substrate 30 for filtering the mixed fluid flowing into the first inlet 11, and the filter substrate 30, the buffer liquid is introduced 2, the inlet portion 21 is formed, the second substrate 20 is formed on the filter substrate 30, the nano-vesicles filtered and mixed with the buffer solution to form a first discharge portion 23 is discharged.

Nanocellular vesicles (Extracellular vesicles, EVs) are vesicles that the cells release to the outside of the cells as described in the prior art, micro vesicles (MVs), apoptotic bodies, exosomes, etc. Includes.

In general, microvesicles (MVs) may have a diameter of 50 to 1000 nm (nano meter), apoptotic bodies having a diameter of 50 to 5000 nm, and exosomes having a diameter of 20 to 200 nm. Hereinafter, the nano vesicle according to an embodiment of the present invention is implemented as an exosome, but the embodiment is not limited thereto. According to an embodiment of the present invention, when the nanovesicles are performed as exosomes, the diameter of the nanovesicles may be 20 to 200 nm.

The first substrate 10 may be formed of polydimethylsiloxane (PDMS) or silicone resin material, but embodiments are not limited thereto. Hereinafter, the first substrate 10 is described as being formed of a PDMS material, but embodiments are not limited thereto.

A first inlet 11 is formed in the first substrate 10. The first inlet portion 11 may be formed by being recessed or penetrated into the first substrate 10. The mixed fluid is introduced into the first inlet part 11. Hereinafter, the mixed fluid will be described as referring to the fluid flowing into the first inlet 11 unless otherwise described.

The mixed fluid may be composed of distilled water, culture medium, phosphate buffer, or a mixture thereof. The mixed fluid contains nano vesicles. The mixed fluid may include one or more microvesicles, apoptotic bodies, exosomes, serum proteins having a smaller diameter than exosomes, cell debris having a larger diameter than exosomes, and proteins and minerals contained in the culture medium.

A tube through which the mixed fluid is supplied may be connected to the first inlet part 11. A syringe pump is provided in the tube to press the mixed fluid to the first inlet 11 to allow the mixed fluid to flow into the first inlet 11.

A first trap 17 connected to the first inlet 11 through the micro channel C may be formed on the first substrate 10. The micro channel C may be formed by being recessed in each substrate. The width or depth of the micro channel C may be formed in millimeter (mm) or micrometer (um) units. Hereinafter, each component is described as being connected to the micro channel C unless otherwise described.

The first trap 17 is formed by bending the micro channel C multiple times. The first trap 17 serves to increase the flow time and filtration area of the mixed fluid in the first membrane 31 of the filter substrate 30 to be described later.

A second discharge portion 13 connected to the first trap 17 may be formed on the first substrate 10. The second discharge portion 13 is discharged through the filter substrate 30, the mixed fluid flowing into the first inlet 11 will be described later. The second discharge part 13 discharges the mixed fluid containing the first material that is not permeated from the first membrane 31 to be described later.

The filter substrate 30 is stacked on the lower surface of the first substrate 10. The filter substrate 30 according to the present invention can be variously implemented. The filter substrate 30 according to an embodiment of the present invention shown in FIGS. 1 to 4 is disposed between the first substrate 10 and the second substrate 20 to be described later.

The filter substrate 30 may be made of various materials. The filter substrate 30 according to an embodiment of the present invention may be formed of a plastic material, and preferably may be formed of a PC (polycarbonate) material, but the embodiment is not limited thereto.

The filter substrate 30 filters the mixed fluid flowing into the first inlet 11. In this case, the filter substrate 30 may be provided with one or more membranes (membrane) for filtering the mixed fluid. The membrane may permeate or imperme materials contained in the mixed fluid depending on the size of the pore diameter. The membrane will be described later.

The filter substrate 30 includes a first membrane 31 that transmits the nanovesicles of the mixed fluid flowing into the first inlet 11 and impermeable to a first material of a predetermined size.

The first membrane 31 filters the mixed fluid flowing into the first inlet 11. The mixed fluid flowing into the first trap 17 may be filtered in the first membrane 31. At this time, the first membrane 31 transmits the nanovesicles. That is, the diameter of the pores of the first membrane 31 is performed at 200 nm or less to form a size through which the nanovesicles can pass. In this case, exosomes having a diameter of 200 nm or less are transmitted through the pores of the first membrane 31.

The first membrane 31 imparts a first material having a predetermined size. Here, the first material having a non-permeable predetermined size includes cell debris having a diameter of more than 200 nm among materials contained in the mixed fluid.

That is, nano vesicles such as apoptotic bodies and microvesicles having a diameter exceeding 200 nm, and cell debris contained in the mixed fluid are impermeable without passing through the pores of the first membrane 31. Hereinafter, a non-permeable material that has not penetrated the first membrane 31 is defined as a first material.

The first trap 17 is disposed to face the first membrane 31. The first trap 17 connected to the first inlet 11 and the micro channel C may be disposed on the first membrane 31. The mixed fluid flowing into the first trap 17 may be filtered in the first membrane 31. In this case, as described above, in the first membrane 31, a part of the nano vesicles is permeable, and the first material is impermeable.

The second discharge portion 13 is connected to the first trap 17 and the micro channel (C). The second discharge part 13 discharges the mixed fluid containing the first material that is impermeable from the first membrane 31 to the outside.

According to an embodiment, the second discharge part 13 may be connected to the first inlet part 11 and the first circulation passage 16. That is, the first circulation channel 16 branched from the tube provided in the first inlet 11 may be connected to the second outlet 13. At this time, a feedback flow path through which the mixed fluid discharged to the second discharge portion 13 can be introduced back to the first inlet portion 11 is formed.

In this case, the nano vesicles that can pass through the first membrane 31 but fail to pass due to the flow, etc., flow back to the first inlet 11 and permeate the first membrane 31 to finally pass through the second membrane. It is discharged to the first discharge portion 23 of the substrate 20. Accordingly, it is possible to improve the purity of the nanovesicles mixed in the buffer solution discharged to the first discharge portion 23.

A first storage unit 12 may be provided in the first circulation channel 16. The first storage unit 12 functions to temporarily store the mixed fluid discharged from the second discharge unit 13. A first outlet portion 14 may be provided on one side of the first storage portion 12. The first outlet portion 14 functions to completely discharge the mixed fluid to the outside when the user operates.

The second substrate 20 is stacked on the filter substrate 30. Depending on the embodiment of the filter substrate 30, the second substrate 20 may be disposed on the upper or lower surface or the upper or lower surface of the filter substrate 30. In the case of an embodiment of the present invention, the second substrate 20 is disposed to be stacked on the lower surface of the filter substrate 30.

The second substrate 20 may be formed of a silicone resin or a polydimethylsiloxane (PDMS) material in the same manner as the first substrate 10, but embodiments are not limited thereto. Hereinafter, the second substrate 20 is described as being formed of PDMS material, but the embodiment is not limited thereto.

A second inlet 21 is formed on the second substrate 20. The second inlet portion 21 may be formed by being recessed or penetrated into the second substrate 20. Buffer liquid is introduced into the second inlet 21. The buffer solution may be performed with phosphate buffered saline (PBS). Hereinafter, the buffer liquid will be described as referring to the fluid flowing into the second inlet 21 unless otherwise described.

A tube through which the buffer solution is supplied may be connected to the second inlet part 21. A syringe pump is provided in the tube, and the buffer liquid is pressurized to the second inlet 21 to allow the buffer liquid to flow into the second inlet 21.

A first discharge portion 23 is formed on the second substrate 20. The first discharge part 23 may be formed by being recessed or penetrated into the second substrate 20. In addition, as described above, the first discharge portion 23 may be connected to the second inlet portion 21 and the micro channel (C).

In the second substrate 20, the nano vesicles filtered by the filter substrate 30 are mixed with the buffer solution. That is, the nano vesicles that have passed through the first membrane 31 are mixed with the buffer solution flowing into the second inlet 21. The buffer solution mixed with the nano vesicle may be discharged to the first discharge part 23.

A second chamber 25 is formed on the second substrate 20. The second chamber 25 is connected to the second inlet 21 so that the nanovesicles transmitted from the first membrane 31 are mixed with the buffer solution. That is, the nano vesicles that have passed through the first membrane 31 are mixed with the buffer solution, so that solution exchange is performed. In this case, contamination of the nano vesicles can be prevented by solution exchange.

A second trap 27 is formed on the second substrate 20. The second trap 27 has one end connected to the second chamber 25 and the other end connected to the first discharge portion 23. The second trap 27 has the same function as the first trap 17, so a detailed description is omitted.

On the other hand, the filter substrate 30 includes a second membrane 33 for impermeating the nanovesicles mixed in the buffer solution flowing into the second inlet 21 and transmitting a second material having a predetermined size.

The second membrane 33 filters the buffer liquid flowing into the second inlet 21. The buffer liquid flowing through the second chamber 25 to the second trap 27 may be filtered in the second membrane 33. At this time, the second membrane 33 makes the nanovesicles impermeable. That is, the diameter of the pores of the second membrane 33 is performed to be less than 20 nm to form a size that the nanovesicles cannot pass. In this case, exosomes having a diameter of 20 nm or more do not pass through the pores of the second membrane 33 and are impermeable.

The second membrane 33 transmits the second material having a predetermined size. Here, the second material having a predetermined size transmitted includes serum protein having a diameter of less than 20 nm among materials contained in the mixed fluid.

That is, serum proteins and other inorganic materials having a diameter of less than 20 nm are transmitted through the pores of the second membrane 33. Hereinafter, a material that has passed through the second membrane 33 is defined as a second material.

The second trap 27 is disposed to face the second membrane 33. The second trap 27 connected to the second chamber 25 and the micro channel C may be disposed under the second membrane 33. The buffer liquid flowing into the second trap 27 may be filtered in the second membrane 33 described above. In this case, as described above, in the second membrane 33, the nanovesicles are impermeable, and the second material is permeable.

A first chamber 19 is formed on the first substrate 10. The first chamber 19 may be disposed to face the second membrane 33. In this case, a buffer material containing a second material and a second material that has passed through the second membrane 33 in the second trap 27 may be accommodated in the first chamber 19.

A third discharge portion 15 is formed on the first substrate 10. The third discharge unit 15 may be connected to the first chamber 19 by a micro channel (C). One or more third discharge units 15 may be formed. In the case of an embodiment of the present invention, it is described that two third discharge parts 15 are formed, but the embodiment is not limited thereto. The third discharge unit 15 discharges the buffer solution permeated from the second membrane 33 and the second material contained in the buffer solution to the outside.

Accordingly, the above-described first material and the second material are discharged to the outside, the nano-vesicles containing exosomes are discharged to the first discharge part 23, and finally the purity of the nano-vesicles mixed and discharged in the buffer solution (purity) becomes higher.

According to an embodiment, the first discharge part 23 may be connected to the second inlet part 21 and the second circulation passage 26. That is, the second circulation passage 26 branched from the tube provided in the second inlet 21 may be connected to the first outlet 23. At this time, a feedback flow path through which the buffer liquid discharged to the first discharge portion 23 can be fed back to the second flow portion 21 is formed.

In this case, since the buffer solution in which the nano vesicles are mixed is filtered through the second chamber 25 and the second trap 27 again in the second membrane 33, the second material is more permeable, and the first discharge portion ( 23) it is possible to improve the purity of the nano vesicles mixed in the buffer solution discharged.

A second storage unit 22 may be provided in the second circulation channel 26. The second storage unit 22 functions to temporarily store the buffer liquid discharged from the first discharge unit 23. A second outlet portion 24 may be provided at one side of the second storage portion 22. The second outlet portion 24 functions to completely discharge the buffer liquid to the outside when the user operates. In this case, the buffer solution having a high purity of the nanovesicles can be obtained by the user.

In an embodiment of the present invention, the first membrane 31 and the second membrane 33 may be disposed on the same plane. That is, as illustrated in FIGS. 1 to 5, the first membrane 31 and the second membrane 33 may be provided on one filter substrate 30.

In this case, as shown in Figure 2, the filter substrate 30 according to an embodiment of the present invention is a first film layer 35, the first film layer 35 is laminated on the lower surface of the first substrate 10 ), A first film layer 31 and a second membrane layer 33 interposed in the second film layer 37, the first film layer 35, and the second film layer 37 stacked on the lower surface.

The first film layer 35 and the second film layer 37 may be formed of a PC material as described above. The first membrane 31 and the second membrane 33 may be formed of a polymer material. In addition, as described above, the first membrane 31 and the second membrane 33 form different pore sizes. The first membrane 31 and the second membrane 33 are disposed between the first film layer 35 and the second film layer 37.

At this time, the first film layer 35, the first membrane 31, the second membrane 33, and the second film layer 37 are combined by a laminating process. In this case, two membranes having different pore sizes may be provided on one filter substrate 30, thereby simplifying the configuration of the microfluidic chip and reducing manufacturing cost.

Based on the above-described configuration, the operation of the microfluidic chip for extracting nanovesicles according to an embodiment of the present invention will be described.

First, the user supplies the mixed fluid to the first inlet 11 of the first substrate 10. The mixed fluid may be supplied to a tube connected to the first inlet part 11 by applying air pressure by a syringe pump.

The mixed fluid flows through the micro channel (C) to the first trap (17). The mixed fluid is filtered in the first membrane 31 of the filter substrate 30. The nano vesicle passes through the pores of the first membrane 31, and the first material does not pass through the pores of the first membrane 31. The first material is discharged to the second discharge unit 13 through the micro-channel C together with the mixed fluid. As described above, when the second discharge part 13 is connected to the first inlet part 11 and the first circulation flow passage 16, the mixed fluid flows back into the first inlet part 11.

Meanwhile, a buffer solution is supplied to the second inlet 21 of the second substrate 20. The buffer solution flows into the second chamber 25 along the micro channel C. In the second chamber 25, nano vesicles that have passed through the first membrane 31 flow. The buffer solution is mixed with nano vesicles in the second chamber 25 to perform solution exchange and flows to the second trap 27 along the micro channel C.

The buffer solution mixed with the nanovesicles contacts the second membrane 33 in the second trap 27. In this case, the buffer solution in which the nano vesicles are mixed is filtered in the second membrane 33. The nano vesicle does not pass through the pores of the second membrane 33, and the second material passes through the pores of the second membrane.

The second material that has passed through the pores of the second membrane 33 is discharged to the third discharge unit 15 along with some buffer liquid that has passed through the second membrane 33. The nano vesicles that do not pass through the pores of the second membrane 33 are discharged to the first discharge portion 23 together with the buffer solution. As described above, when the first discharge part 23 is connected to the second inlet part 21 and the second circulation passage 26, the buffer solution flows back into the second inlet part 21.

Hereinafter, an experimental example of the microfluidic chip for extracting nanovesicles of the present invention will be described. In general, tangential flow filtration (TFF) is used to extract exosomes. In the case of the TFF method, 100 ml mixed fluid containing exosomes is filtered by supplying it to a filtration module with one membrane attached with a peristaltic pump. At this time, if the remaining mixed fluid becomes 15 ml or less as the filtration time passes, bubbles are generated in the device equipped with the filtration module, and filtration efficiency and filtration performance are remarkably deteriorated.

In addition, the substances contained in the mixed fluid filtered by the filtration module include not only exosomes, but also serum proteins of a smaller size than exosomes. Therefore, in order to separate the serum protein, it is necessary to put the mixed fluid back into a separate separation device and separate the serum protein. Accordingly, it usually takes more than 2 hours to extract the exosomes at the target content level.

On the other hand, in the case of the microfluidic chip for extracting nanovesicles of the present invention, even a small amount of mixed fluid can be filtered. Since it is formed in a micro-channel (C) structure, it is possible to extract exosomes from the first outlet (23) even if 1 ml of mixed fluid is supplied to the first inlet (11). Since no bubbles are generated, filtration performance is maintained. In addition, the working time for extracting the exosome at the target content level is also about 1 hour, and the filtration time is reduced by more than 2 times compared to the conventional TFF method.

As described above, the microfluidic chip for extracting nanovesicles according to an embodiment of the present invention extracts nanovesicles containing exosomes from one microfluidic chip into the first discharge part 23 and extracts cell debris and serum proteins. Since the impurities are discharged to the outside, the nano vesicles can be quickly extracted.

In addition, the first circulation flow path 16 and the second circulation flow path 26 are provided to supply the nano vesicles remaining in the mixed fluid to the first inlet 11 again, thereby increasing the recovery rate of the nano vesicles. The buffer solution containing the nano vesicles can be extracted.

In addition, since the overall configuration is simple, it is possible to reduce the manufacturing cost of the microfluidic chip.

6 is a view showing that the microfluidic chip for extracting nano vesicles according to an embodiment of the present invention is used by extending in the longitudinal direction according to an embodiment.

Referring to FIG. 6, a microfluidic chip for extracting nano vesicles according to an embodiment of the present invention may be implemented by extending in a longitudinal direction.

That is, as shown in Figure 6, the first substrate 10, the filter substrate 30, the second substrate 20 is disposed. At this time, the first chamber 19 of the first substrate 10 is extended to the third trap 17a, which is the same as the first trap 17, through the connection channel L, and the first discharge of the second substrate 20 The portion 23 may be extended to the third chamber 25a, which is the same as the second chamber 25, through the connection channel L. In this case, the third membrane 31a provided between the third trap 17a and the third chamber 25a functions the same as the first membrane 31.

Thereafter, the fourth trap 27a, the fourth chamber 19a, and the rest of the components are connected to the micro channel C, and the fourth membrane 33a, which is the same as the second membrane 33, is configured to extend the portion. It can be configured in the same manner as one embodiment of the present invention. In this case, the buffer solution in which the nano vesicles are mixed is discharged to the fourth discharge part 23a, and the buffer solution containing the second material and the second material is discharged to the fifth discharge part 15a.

7 is an exploded perspective view of a microfluidic chip for extracting nanovesicles according to another embodiment of the present invention, and FIG. 8 is a side view showing the mechanism of a microfluidic chip for extracting nanovesicles according to another embodiment of the present invention It is a schematic.

Hereinafter, description will be made mainly on components having differences, and components that are not described or illustrated will be replaced with descriptions and illustrated contents of the above-described components.

7 to 8, the first substrate 10 according to another embodiment of the present invention is formed with a fifth chamber 17b connected to the first inlet 11. In this case, the fifth chamber 17b may be implemented as a trap. The fifth chamber 17b is disposed to face the fifth membrane 41 to be described later.

In addition, the first substrate 10 is formed with a second discharge portion 13 connected to the fifth chamber 17b. Since the second discharge unit 13 is the same as the second discharge unit 13 according to an embodiment of the present invention, a detailed description is omitted.

In another embodiment of the present invention, the filter substrate 30 may be implemented in two or more. In the case of FIG. 7 or less, the filter substrate 30 is in contact with the lower surface of the first substrate 10 and is provided on the lower surface of the first filtering substrate 40 and the second substrate 20 provided with the fifth membrane 41. It includes a second filter substrate 60 in contact with the sixth membrane 61 is provided.

The first filtering substrate 40 contacts the lower surface of the first substrate 10. One membrane is provided on the first filter substrate 40. In this case, the first filter substrate 40 is provided with a fifth membrane 41, the diameter of the pores of the fifth membrane 41 can be carried out to 200 nm or less. Since the fifth membrane 41 is the same as the first membrane 31, detailed description is omitted.

The second filtering substrate 60 contacts the lower surface of the second substrate 20. In this case, the second substrate 20 is disposed between the first filter substrate 40 and the second filter substrate 60.

The second filter substrate 60 is provided with one membrane. In this case, the second filter substrate 60 is provided with a sixth membrane 61, and the diameter of the pores of the sixth membrane 61 may be less than 20 nm. Since the 6th membrane 61 is the same as the 2nd membrane 33, detailed description is abbreviate | omitted.

The first filter substrate 40 or the second filter substrate 60 may be manufactured by laminating two film layers as described above. In this case, a membrane may be interposed between two film layers and then produced by performing a laminating process.

First, the mixed fluid is introduced into the first inlet 11 as in the embodiment of the present invention. The mixed fluid is filtered in the fifth membrane (41). In the fifth membrane 41, the nanovesicles of the mixed fluid are permeated, and the first material is impermeable and discharged to the second discharge part 13.

The sixth chamber 25b is formed in the second substrate 20. The sixth chamber 25b is connected to the second inlet portion 21 and is connected to the first outlet portion 23 in the same way as one embodiment of the present invention. In the sixth chamber 25b, nanovesicles transmitted from the fifth membrane 41 are mixed with the buffer solution. Since the sixth chamber 25b is the same as the second chamber 25, detailed description is omitted.

A flow channel 28 is formed under the sixth chamber 25b. The flow channel 28 is connected to the sixth chamber 25b. The flow channel 28 is formed through the second substrate 20. The flow channel 28 flows the buffer solution accommodated in the sixth chamber 25b to the sixth membrane 61.

The sixth membrane 61 is provided on the second filter substrate 60. The sixth membrane 61 is the same as the second membrane 33 described above. The sixth membrane 61 filters the buffer liquid flowing through the flow channel 28. The sixth membrane 61 impermeable to the nano vesicle and permeates the second material.

The third substrate 70 is in contact with the lower surface of the second filter substrate 60. The third substrate 70 is formed so as not to interfere with the second inlet 21 and the first outlet 23 of the second substrate 20. In the present invention, the length of the third substrate 70 is formed smaller than the length of the second substrate 20, so that the second inlet portion 21 and the first outlet portion 23 of the second substrate 20 are Exposure. Since the material and characteristics of the third plate are the same as those of the first substrate 10, detailed descriptions are omitted.

The third substrate 70 is disposed to face the sixth membrane 61 and the seventh chamber 71 disposed opposite to the sixth chamber 25b, and the seventh chamber 71 connected to the sixth membrane 71 ( 61) includes a sixth discharge unit 73 for discharging the second material permeated.

The seventh chamber 71 is formed on the third substrate 70. The seventh chamber 71 is disposed opposite to the sixth membrane 61 and the sixth chamber 25b to receive a buffer solution containing the second substance and the second substance transmitted from the sixth membrane 61. .

A sixth discharge part 73 is formed under the seventh chamber 71. The sixth discharge part 73 is connected to the seventh chamber 71. The sixth discharge portion 73 is formed through the third substrate 70. The sixth discharge unit 73 discharges the buffer liquid containing the second material and the second material accommodated in the seventh chamber 71 to the outside.

Based on the above-described configuration, the operation of the microfluidic chip for nano-vesicle extraction according to another embodiment of the present invention will be described.

First, the user supplies the mixed fluid to the first inlet 11 of the first substrate 10. The mixed fluid may be supplied to a tube connected to the first inlet part 11 by applying air pressure by a syringe pump.

The mixed fluid flows through the micro channel (C) to the fifth chamber (17b). The mixed fluid is filtered in the fifth membrane 41 of the first filter substrate 40. The nano vesicle passes through the pores of the fifth membrane 41, and the first material does not pass through the pores of the fifth membrane 41. The first material is discharged to the second discharge unit 13 through the micro channel C together with the mixed fluid. As described above, when the second discharge part 13 is connected to the first inlet part 11 and the first circulation flow passage 16, the mixed fluid flows back into the first inlet part 11.

Meanwhile, a buffer solution is supplied to the second inlet 21 of the second substrate 20. The buffer liquid flows into the sixth chamber 25b along the micro-channel C. In the sixth chamber 25b, nanovesicles that have passed through the fifth membrane 41 flow. The buffer solution is mixed with the nano vesicles in the sixth chamber 25b to perform solution exchange.

The buffer solution in which the nanovesicles are mixed is also flowed into the flow channel 28. The buffer liquid flowing into the flow channel 28 contacts the sixth membrane 61. The sixth membrane 61 impermeable to the nano vesicle and permeates the second material.

The second material flows into the seventh chamber 71 of the third substrate 70. The buffer liquid accommodated in the seventh chamber 71 is discharged to the sixth discharge unit 73 together with the second material. The buffer solution that is accommodated in the sixth chamber 25b and the second material is transmitted to the sixth membrane 61 is discharged to the first discharge portion 23 together with the remaining nanovesicles.

In addition, as in one embodiment of the present invention, the first circulation passage 16 and the second circulation passage 26 may be provided, and the purity of the nanovesicles may be increased to improve purity.

As described above, a person having ordinary knowledge in the technical field to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical spirit or essential characteristics. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive.

The scope of the present invention is indicated by the scope of the claims, which will be described later, rather than the detailed description, and all the modified or modified forms derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. Should be interpreted.

10: first substrate 20: second substrate
30: filter substrate

Claims (17)

A first substrate having a first inlet portion through which a mixed fluid containing nano vesicles flows; A filter substrate stacked on the lower surface of the first substrate and filtering the mixed fluid flowing into the first inlet; And a second substrate stacked on the filter substrate, a second inlet portion into which a buffer solution flows, and a first discharge portion through which the nano-vesicles filtered from the filter substrate are mixed with the buffer liquid and discharged. Including,
The filter substrate may include a fifth membrane that permeates the nanovesicles of the mixed fluid introduced into the first inlet and impermeable to a first material having a predetermined size; And a sixth membrane through which the nanovesicles mixed in the buffer solution introduced into the second inlet part are impermeable and permeate a second material of a predetermined size. It includes,
The filter substrate is in contact with the lower surface of the first substrate, the first filter substrate is provided with the fifth membrane; And a second filter substrate contacting a lower surface of the second substrate and having the sixth membrane. Microfluidic chip for nano-vesicle extraction comprising a.
delete delete delete delete delete delete According to claim 1,
The first discharge portion is a microfluidic chip for extracting nano vesicles connected to the second inlet and the second circulation flow path.
delete According to claim 1,
The first substrate,
A microfluidic chip for extracting nano vesicles comprising a fifth chamber connected to the first inlet and disposed to face the fifth membrane.
According to claim 1,
The second substrate,
A sixth chamber connected to the second inlet part and connected to the first outlet part, and in which the nanovesicles transmitted through the fifth membrane are mixed with the buffer solution; And
A flow channel connected to the sixth chamber and flowing the buffer liquid to the sixth membrane;
Microfluidic chip for nano-vesicle extraction comprising a.
The method of claim 11,
A microfluidic chip for extracting nano vesicles further comprising a third substrate contacting a lower surface of the second filtering substrate.
The method of claim 12,
The third substrate,
A seventh chamber disposed opposite to the sixth membrane and opposite to the sixth chamber; And
A sixth discharge part connected to the seventh chamber and discharging the second material transmitted from the sixth membrane;
Microfluidic chip for nano-vesicle extraction comprising a.
According to claim 1,
The nanovesicles are microfluidic chips for extracting nanovesicles that are exosomes.
According to claim 1,
The first material is a microfluidic chip for extracting nano vesicles containing cell debris.
According to claim 1,
The second material is a microfluidic chip for extracting nano vesicles containing serum protein.
According to claim 1,
The microfluidic chip for extracting nano vesicles, wherein the pore diameter of the fifth membrane is 200 nm or less, and the pore diameter of the sixth membrane is less than 20 nm.

Images