KR101970646B1 - Microfluidic device, and treating method of single-cell using the same - Google Patents

Microfluidic device, and treating method of single-cell using the same Download PDF

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KR101970646B1
KR101970646B1 KR1020160028958A KR20160028958A KR101970646B1 KR 101970646 B1 KR101970646 B1 KR 101970646B1 KR 1020160028958 A KR1020160028958 A KR 1020160028958A KR 20160028958 A KR20160028958 A KR 20160028958A KR 101970646 B1 KR101970646 B1 KR 101970646B1
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well
single cell
solution
sample
microfluidic device
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KR20170105825A (en
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문희성
김연정
유창은
한경연
박웅양
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삼성전자주식회사
사회복지법인 삼성생명공익재단
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Priority to KR1020160028958A priority Critical patent/KR101970646B1/en
Priority to PCT/KR2016/009420 priority patent/WO2017155170A1/en
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    • 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
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • 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

Abstract

A sample liquid containing a single cell as a target cell or a first well containing a reagent, a second well disposed apart from the first well, and a second well extending from a lower surface of the first well to a lower surface of the second well, A microfluidic device including at least two microfluidic channels connecting a first well and a second well, wherein a width of the microfluidic channel is smaller than that of a single cell, and a single cell treatment method using the microfluidic device.

Description

Technical Field [0001] The present invention relates to a microfluidic device and a single cell processing method using the microfluidic device,

A microfluidic device, and a method for treating a single cell using the same.

Cancer is currently the leading cause of morbidity and mortality worldwide, not only in Korea. The greatest impact on the mortality rate of cancer patients depends on the presence or absence of metastatic cancer cells. In other words, the technique of accurately and accurately detecting single cells such as circulating tumor cells (CTC), which is present in one of the blood cell's hundreds of millions of blood cells, improves the survival rate before and after cancer treatment It is essential.

 For example, in the case of breast cancer, blood should be found at less than 5 in 7.5 ml, less than 3 in colorectal cancer, and less than 5 in prostate cancer, and the throughput, Micro cells that meet three basic conditions such as cell count, recovery (ratio of single cell count to the number of single cells injected / collected), and collection efficiency (purity, purity of separated single cells) Cell separation technology is required.

CTC capture methods that have been published so far include gene detection using polymerase chain reaction (PCR), centrifugation, meter reading using magnetophoresis, and fluorescence staining or using a filter.

However, in the conventional methods, a single cell may be lost through a process of removing a large number of blood cells contained in blood for CTC detection. In addition, there is a possibility that a single cell may be lost in the course of performing a treatment such as dyeing by injecting a single cell from which the hemocyte has been removed into a separate experimental device.

One embodiment provides a microfluidic device capable of minimizing the loss of single cells during various processing steps of a single cell.

In addition, a single cell treatment method capable of continuously treating single cells through a microfluidic device according to an embodiment is provided.

According to one embodiment, a sample liquid containing a single cell as a target cell, or a first well containing a reagent, a second well disposed apart from the first well, and a second well from the lower surface of the first well, The microfluidic device is provided with two or more microfluidic channels extending to the lower surface of the well and connecting the first well to the second well, wherein the width of the microfluidic channel is smaller than the size of the single cell.

The ratio of the area of the lower surface of the first well to the cross-sectional area in the width direction of the micro channel may be 1000: 1 to 400000000: 1.

The micro flow path may further include a first reservoir portion formed between the at least two micro flow paths and the second well, and the at least two micro flow paths and the second well may be connected to the first reservoir portion, respectively.

The at least two micro flow paths may connect the first well and the second well in parallel.

The width of the fine flow path may be 0.5 탆 to 15 탆.

The lower surface of the first well and the lower surface of the second well may be coplanar.

The lower surface of the first well viewed from above may have a circular shape.

The lower surface diameter of the first well may be between 1 mm and 50 mm.

The microfluidic device may include two or more single cell processing units each including the first well, the second well, and the two or more microchannels.

The two or more single cell processing units may be arranged to have a matrix form.

According to another aspect of the present invention, there is provided a method of treating a single cell using the microfluidic device, comprising: injecting a sample solution containing a single cell into the first well; injecting a fixer into the first well, Treating the fixed single cells by injecting a permeate into the first well, and injecting a staining solution into the first well to stain the pre-treated single cells. / RTI >

A negative force may be applied to the second well to remove the sample solution, the fixation solution, the penetration solution, and the staining solution from the first well.

The sample solution, fixer solution, permeate solution, and dye solution removed from the first well may be passed through the micro channel to be accommodated in the second well.

The flow rate of the sample solution, fixing solution, permeation solution, and dyeing solution passing through the microchannel by the applied negative pressure may be larger than the movement speed of the single cell induced by the negative pressure.

The single cell processing method may further include washing the first well by injecting a washing solution into the first well.

Upon completion of the washing step, a negative force may be applied to the second well to remove the washing liquid from the first well.

It is possible to provide a microfluidic device capable of minimizing the loss of single cells during various processes of single cells.

In addition, it is possible to provide a single cell treatment method capable of continuously treating single cells through a microfluidic device.

1 is a perspective view illustrating a microfluidic device according to an embodiment,
FIG. 2 is a top plan view of the microfluidic device of FIG. 1,
3 is a cross-sectional view taken along line III-III in Fig. 2,
4 to 6 show various modifications of the single cell processing unit according to one embodiment,
7 is an image showing a microfluidic device including two or more single cell processing units according to another embodiment,
FIGS. 8 to 18 are views sequentially illustrating a single cell processing method according to one embodiment,
19 is a fluorescence microscope image showing single cells stained according to one embodiment.

Hereinafter, exemplary embodiments will be described in detail so that those skilled in the art can easily carry out the present invention. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein.

In the drawings, the thickness is enlarged to clearly represent the layers and regions. Like parts are designated with like reference numerals throughout the specification. Whenever a portion of a layer, film, region, plate, or the like is referred to as being "on" another portion, it includes not only the case where it is "directly on" another portion, but also the case where there is another portion in between. Conversely, when a part is "directly over" another part, it means that there is no other part in the middle.

A " single-cell ", which in one embodiment is a target cell, is a very rare cell in which only a few to a few tens of cells are present, for example, And circulating tumor cells (CTC), which are present in one of hundred million blood cells.

In one embodiment, the term " reagent " refers to various kinds of drugs for biochemical treatment of the single cell. For example, the reagent means a washing solution such as a buffer solution, a fixing solution, a permeator, to be.

On the other hand, in one embodiment, the "sample liquid" means that the "single cell" is contained in a reagent such as a buffer solution or the like.

First, a schematic structure of a microfluidic device according to an embodiment will be described with reference to FIGS. 1 and 2. FIG.

FIG. 1 is a perspective view illustrating a microfluidic device according to one embodiment, and FIG. 2 is a plan view of the microfluidic device of FIG. 1 viewed from above.

1 and 2, a microfluidic device 100 according to an embodiment includes a substrate 10, a single cell processing unit (not shown) disposed on the substrate 10 and serving as a target cell, (20).

In one embodiment, the substrate 10 may be made of a material that does not chemically react with various reagents. In addition, the substrate 10 may be made of an optically transparent material so as to confirm existence of single cells in the sample. In one embodiment, the substrate 10 may be made of, for example, glass, plastic, or the like. For example, a slide glass may be used as the substrate 10.

The single cell processing portion 20 is disposed directly above the substrate 10. [ The single cell processing section 20 includes a body 21, a first well 22, a second well 23, and at least two micro channels 24.

The body 21 is formed on the substrate 10 and may have a cross-sectional area corresponding to the cross-sectional area of the substrate 10. The body 21 may be made of an optically transparent material, for example, glass, plastic, or the like, so as to confirm existence of a single cell in the sample.

Meanwhile, the body 21 may be formed of a material having hydrophobicity, for example, polydimethylsiloxane (PDMS). Generally, since the sample liquid or various reagents are fluids having hydrophilicity, the body 21 is formed of a hydrophobic material, so that strong fluid resistance is applied to the inside of the microchannel 24, It is possible to prevent the reagent of the well 22 from flowing into the micro channel 24 and the second well 23.

However, the present invention is not limited to this embodiment, and it may be formed of a material having hydrophilic property, and at least a surface of the first well 22 and the second well 23, which will be described later, The surface to be treated may be controlled to have hydrophobicity through a method such as surface modification.

The first well 22 is a semi-closed space in which the upper surface formed by the contact with the upper surface of the substrate 10 is opened while the body 21 is opened up and down. In other words, the side wall of the first well 22 is the body 21, and the lower surface of the first well 22 is the upper surface of the substrate 10. In one embodiment, the sample or reagent may be injected through the first well 22.

The second well 23 is also a semi-enclosed space with an open top surface formed by the same method as the first well 22. The second well 23 can receive and remove the sample or reagent contained in the first well 22 through the micro channel 24.

As shown in FIG. 2, the first and second wells 22 and 23 may be formed to have a circular shape in a lower surface of each of the first and second wells 22 and 23. However, the first and second wells 22 and 23 may have a polygonal shape such as a triangular shape, a square shape, a hexagonal shape, an octagonal shape, an elliptical shape, or the like.

The micro channel 24 extends from the lower surface of the first well 22 to the lower surface of the second well 23 to connect the first well 22 and the second well 23 to each other. The micro flow path 24 according to an embodiment may be a space formed between the substrate 10 and the body 21 by patterning the lower surface of the body 21 and then contacting the substrate 10.

On the other hand, two or more fine flow paths 24 may extend from the first well 22 toward the second well 23. In one embodiment, two or more microfluidic channels 24 extend in parallel from the first well 22 toward the second well 23 as shown in FIGS. 1 and 2, May be formed in a shape that is joined to the second well 23 by one path at any point between the second well 22 and the second well 23.

The fine flow path 24 serves as a passage for supplying the sample solution or the reagent stored in the first well 22 to the second well 23. That is, the micro channel 24 may serve as a drainage way for discharging the reagent contained in the first well 22 to the second well 23.

In one embodiment, the width of microchannel 24 is formed smaller than the size of a single cell. Accordingly, the single cell can remain in the first well 22 without passing through the micro channel 24. That is, the micro channel 24 can discharge only the sample solution or the reagent except the single cells in the first well 22 to the second well 23.

Since the micro channel 24 is formed to have a width smaller than the size of a single cell having a size generally ranging from several tens to several hundreds of micrometers, the sample liquid or reagent contained in the first well 22 is not more than a certain pressure It can be discharged to the second well 23 through the micro flow path 24.

That is, in the microfluidic device 100 according to one embodiment, only the sample liquid including the single cells injected into the first well 22 or the sample except for the single cells is selectively supplied through the microchannel 24 2 well 23 to be removed.

More specifically, a sample liquid or reagent contained in the first well 22 is supplied with a predetermined positive pressure through the first well (not shown) so that the hydraulic resistance of the microchannel 24 can be overcome. 22, or a predetermined negative pressure must be applied to the second well 23.

The hydraulic resistance applied to the fine flow path 24 is expressed by the following Equation 1.

Figure 112016023359632-pat00001
............................................ (Equation 1)

In Equation 1, R h denotes a hydraulic resistance applied to the micro flow path 24, ΔP denotes a pressure change amount to be applied, and Q denotes a flow rate through the micro flow path 24.

In one embodiment, by applying a negative pressure greater than the product of the hydraulic resistance of the microchannel 24 and the flow rate through the microchannel 24 to the second well 23 using a pipette or the like, 22 or the sample can be moved to the second well 23.

If a pressure capable of overcoming the hydraulic resistance of the micro channel 24 (for example, negative pressure equal to or higher than a critical pressure due to the hydraulic resistance of the micro channel 24) is supplied to the first well 22 or the second well 23 The sample liquid or reagent contained in the first well 22 is entirely retained in the first well 22 due to the high hydraulic resistance of the microchannel 24. In this case, That is, the fine flow path 24 can serve as a kind of check valve.

Accordingly, in the microfluidic device 100 according to an embodiment, only the remaining sample fluid except for a single cell is selectively removed from the first well 22 in the sample solution or sample containing the single cell injected into the first well 22 It is possible to easily control the flow of the sample liquid or reagent moving from the first well 22 to the microchannel 24 without providing a separate valve in the microchannel.

Hereinafter, the structure of the single cell processing unit 20 according to one embodiment will be described in more detail with reference to FIG. 3 and FIGS. 1 and 2 described above.

3 is a sectional view taken along the line III-III in FIG.

3, the fine flow path 24 has a predetermined width D2 and a predetermined length H2. As described above, the width of the micro channel 24 may have various values depending on the type of a single cell to be a target cell. For example, the width of the micro channel 24 may range from 0.5 탆 to 15 탆, for example, from 1 탆 to 15 탆, For example between 1 탆 and 12 탆, for example between 1 탆 and 10 탆.

When the width of the micro channel 24 is less than 0.5 탆, the removal time of the sample solution or the reagent for single cell treatment is excessively increased to lower the treatment efficiency and the width of the micro channel 24 exceeds 15 탆 A single cell is sucked into the microchannel 24 by the negative pressure applied to the second well 23 and the microchannel 24 is sucked into the microchannel 24, Or may be removed by moving to the second well 23, resulting in loss of single cells.

The cross section in the width direction of the micro flow path 24 can be designed in various ways in consideration of ease of processing of the body 21 and the like. For example, the cross section may be formed in a semicircle, an ellipse or a rectangle.

On the other hand, the length of the micro flow path 24 is determined by the distance between the first well 22 and the second well 23, the amount of the sample solution or sample to be injected into the first well 22, For example, 5 占 퐉 to 1 mm, e.g., 5 占 퐉 to 800 占 퐉, for example, 5 占 퐉 to 600 占 퐉, for example, 5 占 퐉 to 500 占 퐉. have.

When the length of the micro channel 24 is less than 5 mu m, the flow velocity of the fluid passing through the micro channel 24 becomes excessively fast due to the negative pressure applied to the second well 23, It is difficult to precisely control the amount of the sample. On the other hand, when the length of the micro channel 24 exceeds 1 mm, the negative pressure to be applied to the second well 23 becomes large, while the removal of the sample solution or the sample progresses slowly, Can be increased excessively.

The heights of the first well 22 and the second well 23 according to one embodiment may have the same value H1. The height of the first well 22 and the second well 23 according to one embodiment may be variously set, for example, 1 mm to 50 mm, 1 mm to 20 mm, for example, 1 mm to 10 mm . If the height of the first well 22 and the second well 23 is less than 1 mm, the amount of sample solution or reagent to be injected into the first well 22 may be insufficient, If the height of the well 23 exceeds 50 mm, an unnecessarily large amount of reagent can be injected into the first well 22 and the sample solution or reagent is removed through the second well 23 after the single cell treatment And the single cell treatment process time may be prolonged.

The lower surface of the first well 22 and the lower surface of the second well 23 according to one embodiment may be coplanar, as shown in FIG. 3, i.e., in one embodiment, The lower surface of the first well 22, the lower surface of the second well 23, and the lower surface of the microchannel 24 may all be coplanar. The lower surface of the first well 22, the lower surface of the second well 23 and the lower surface of the microchannel 24 are located on the same plane without step difference, The sample solution or the reagent is easily moved to the micro flow path 24 by the negative pressure applied to the second well 23.

3, the lower surface diameter D1 of the first well 22 is larger than the lower surface diameter D3 of the second well 23, but the lower surface of the lower surface of the first well 22 The diameters of the lower surface of the second well 23 may be the same or different from each other, and the amount of the sample solution or reagent to be injected and discharged, the diameter of the pipette for injecting the sample solution, It can be set variously.

The diameter D1 of the lower surface of the first well 22 and the diameter D3 of the lower surface of the second well 23 may be, for example, 1 mm to 50 mm, 1 mm to 20 mm, . When the diameters of the lower surface of the first well 22 and the lower surface of the second well 23 are less than 1 mm, the amount of the sample solution or reagent to be injected into the first well 22 may be somewhat insufficient, It is difficult to induce a drag force. In addition, when the diameter of the lower surface of the first well 22 and the lower surface of the second well 23 exceeds 50 mm, the amount of the reagent to be injected for single cell processing may excessively increase.

In one embodiment, when the sample solution is injected into the first well 22 and the time has elapsed, the majority of the single cells will sink to the bottom surface of the first well 22. At this time, when negative pressure is applied to the second well 23 to extract the sample liquid into the microchannel 24, the flow velocity of the sample liquid passing through the microchannel 24 is rapidly reduced by the negative pressure, . On the other hand, the single cells do not substantially move on the lower surface of the first well 22 without being greatly affected by the negative pressure applied to the second well 23.

This is because as negative pressure is applied to the second well 23, drag force due to fluid movement is additionally applied in addition to gravity and buoyancy as a force applied to single cells present in the sample liquid , The sample liquid moves at a speed slower than the flow rate through the micro flow path (24).

That is, when negative pressure is applied to the second well 23 for fluid movement, the pressure drop effect applied to the microchannel 24 and the first well 22 is mostly concentrated also in the microchannel 24, A slight pressure drop is applied to the cells inside the first well 22 having a relatively large cross-sectional area.

The behavior of such single cells within the first well 22 is affected by the ratio of the area of the lower surface of the first well 22 to the cross-sectional area in the width direction of the microchannel 24.

The ratio of the area of the lower surface of the first well 22 to the cross sectional area in the width direction of the micro flow path 24 is set so that the width D2 of the micro flow path 24 and the width of the lower surface of the first well 22 For example, 1000: 1 to 800000000: 1, for example, 1000: 1 to 400000000: 1.

By adjusting the ratio of the area of the lower surface of the first well 22 to the cross-sectional area in the width direction of the micro channel 24 within the above range, the single cells are not affected by the negative pressure applied to the second well 23 Lt; RTI ID = 0.0 > well 22 < / RTI >

In a process for removing only a specific fluid from a general microfluidic device, valves formed in a microfluidic channel are used, or a multiplexed pump device is used. Such a typical microfluidic device has the troubles of precision control of the valves or connection of a fluidic adapter or a syringe pump every time a specific fluid removal process is performed.

As another method for concentrating single cells, when a microfluidic device is centrifuged, loss of single cells may occur during aspiration of a sample solution or a reagent after centrifugation.

In addition, even when the single cells obtained through the above method are smeared on the substrate and then the subsequent treatment is performed, loss of single cells may occur in the process of washing, etc., and then the single cells are recovered and further processed (For example, when DNA must be extracted after recovery of a single cell line), single cell loss may occur during recovery.

 However, in the case of the microfluidic device 100 according to one embodiment, when the body 21 is formed of a material having hydrophobic property and no single external force is applied, a single cell is formed in the microchannel 24 and the second well 23 can be prevented from flowing. The high hydraulic resistance of the microchannel 24 itself and the drag force applied to the single cell can be controlled by controlling the ratio of the area of the lower surface of the first well 22 to the cross-sectional area of the microchannel 24 in the width direction.

Accordingly, the microfluidic device 100 according to an embodiment can more easily remove only the specific fluid from the first well 22 through a simple tool such as a pipette, and can continuously remove the sample fluid or various reagents The single cell is not lost even if it is put in the 1 well 22 and then removed.

That is, according to one embodiment, it is possible to continuously perform a single cell treatment process in the microfluidic device 100 while minimizing the loss of single cells during various processes of the single cell.

Hereinafter, various modifications of the single cell processing unit according to one embodiment will be described with reference to FIGS. 4 to 6. FIG.

Referring to FIG. 4, the single cell processing unit may be arranged such that two or more microchannel 24 'are connected in parallel to the first well 22 and the second well 23, respectively. The micro channel 24 'is capable of independently moving the sample solution or the reagent with respect to the micro channel 24 of FIGS. 1 to 3 described above.

4, the fine flow path 24 'has a structure in which five flow paths are arranged in parallel to each other in parallel, but the present invention is not limited thereto. The size of the hydraulic pressure resistance varies depending on the number of the fine flow paths 24' The diameter and the cross-sectional area of the first well 22, the magnitude of the negative pressure applied to the second well 23, and the like.

5, the single-cell processing unit may further include a first reservoir 25 formed between the second micro-channel 24 and the second well 23. That is, the first reservoir 25 having a larger area as compared with the above-described FIG. 1 to FIG. 3, so that the sample solution or reagents are temporarily stored and finally discharged through the second well 23, May be formed.

5, the length of the microchannel 24 is reduced according to the area occupied by the first reservoir 25, but the present invention is not limited thereto. The specific shape of the first reservoir 25, The distance between the first well 22 and the second well 23, the amount of the sample solution that can accommodate the first well 22 and the second well 23, or the amount of the reagent.

6, the single-cell processing unit may further include a second reservoir 26 formed in each of the two or more microchannels 24. That is, in contrast to the above-described Figs. 1 to 3, the second flow path portion 26 having a width wider than the width of the micro flow path 24 may be formed on one side of the micro flow path 24. [ That is, it provides a space in which the sample liquid or reagents, which are moving in the microchannel 24, can be temporarily stored.

5, the second reservoir portion 26 is formed independently for each micro flow path 24, but the present invention is not limited thereto. The second reservoir portions 26 may be connected to each other, The specific shape and acceptable volume of the reservoir 26 can be determined by the width and length of the microchannel 24, the distance between the first well 22 and the second well 23, the distance between the first well 22 and the second well 23, The amount of sample solution or reagent that can be accommodated in the well 23 and the flow rate of the fluid flowing through the micro flow path 24 in the presence of the second reservoir portion 26,

As described above, even if the microfluidic device 100 according to an embodiment has various designs of the single cell processing unit 20, more specifically, the microfluidic channel 24, The single cell processing process can be continuously performed in the microfluidic device 100 while minimizing the loss of the cell.

Hereinafter, a microfluidic device according to another embodiment will be described with reference to FIG.

7 is an image showing a microfluidic device including two or more single cell processing units according to another embodiment.

Referring to FIG. 7, the microfluidic device 200 according to another embodiment includes two or more single-cell processing units 20, and neighboring single-cell processing units 20 may be arranged to heat each other. That is, the two or more single cell processing units 20 may be arranged to have a matrix form.

That is, since no separate flow path is formed between neighboring single cell processing units 20 of the microfluidic device 200 according to another embodiment, single cell processing independent of each single cell processing unit 20 is possible.

The microfluidic device 200 according to another embodiment may be, for example, a well-plate having a plurality of single cell processing units 20, but is not necessarily limited to, The single cell processing unit 20 can be applied to various experimental tools.

As described above, the microfluidic device 200 according to another embodiment includes two or more single cell processing units 20, and can perform independent single cell processing, thereby improving single cell processing efficiency.

Hereinafter, a single cell processing method using a microfluidic device according to one embodiment will be sequentially described with reference to FIGS. 8 to 18. FIG.

The single cell treatment method according to an embodiment includes injecting a sample solution containing a single cell into the first well 22, fixing the single cell by injecting a fixing solution into the first well 22, Injecting a permeate into the first well 22 to pretreat the fixed single cells, and injecting a staining solution into the first well 22 to stain the pretreated single cells.

First, as shown in Fig. 8, a sample liquid 3 containing a single cell 2 is injected into and accommodated in the first well 22 in the sample liquid injecting step. The accommodated sample liquid 3 can not move into the microchannel 24 due to the hydraulic pressure resistance of the microchannel 24 and stays in the first well 22. [ Further, the single cells 2 are mostly sitting on the lower surface of the first well 22.

9, when a negative pressure equal to or higher than the critical pressure due to the hydraulic pressure resistance of the microchannel 24 is applied to the second well 23 using a pipette or the like, The sample liquid 3 flows along the micro flow path 24 toward the second well 23 at the first velocity V1. At this time, the single cells (2) sitting on the lower surface of the first well (22) are dragged by the negative pressure and move toward the microchannel (24) at the second velocity V2.

The length of the microchannel 24 in the width direction is smaller than the size of the single cell 2 so that the single cells 2 can not pass through the microchannel 24 and can be inserted into the first well 22 . Further, by adjusting the cross-sectional area of the micro channel 24 in the width direction cross-sectional area and a first well 22 of the second speed V2 than the first speed V1 at least 10 - it shows a low speed of about six times. Accordingly, even if the sample liquid 3 is removed from the first well 22 as shown in Fig. 10, the single cells 2 still remain in the first well 22.

On the other hand, even after the removal of the sample liquid 3 is completed, a small amount of the sample liquid 3 may remain in the first well 22, but a negative pressure higher than the critical pressure due to the hydraulic resistance of the microchannel 24 is applied 10, the remaining small amount of the sample liquid 3 can not pass through the micro flow path 24. [

Meanwhile, in one embodiment, the cleaning step may further include washing the first well 22 by injecting a washing solution into the first well 22, and the washing step may include a step of injecting the sample solution, Step, and dyeing steps, respectively, after the removal of the sample solution, fixation solution, permeate, and dye solution except for the single cells (2).

11, when the cleaning liquid 4 is injected into the first well 22 of FIG. 10, the cleaning liquid 4 is also supplied to the first well 22 by the hydraulic resistance of the fine flow path 24, (22). 9, when the negative pressure is applied to the second well 23, the cleaning liquid 4 passes through the microchannel 24 and is accommodated in the second well 23, while the single cell (2) do not substantially move within the first well (22).

As a result, when washing of the sample solution is completed, only a small amount of the washing liquid 4 and the single cells 2 remain in the first well 22 as shown in FIG.

Thereafter, in the fixing step, fixing liquid 5 is injected into the first well 22 of FIG. 12 to fix the single cell 2 to the lower surface of the first well 22. The single cells 2 fixed on the lower surface of the first well 22 by the fixer 5 can be separated from the fixer 5 by a small amount of fixing liquid 5 as shown in FIG. Lt; RTI ID = 0.0 > well < / RTI >

Thereafter, the cleaning process as shown in FIG. 11 is performed once to clean the inside of the first well 22, and negative pressure is applied to the second well 23 to remove residual Remove the fixing liquid (5) and the cleaning liquid. Even if some of the fixed single cells 2 are desorbed from the lower surface of the first well 22 during the washing process, the width of the microfluidic channel 24, the hydraulic resistance, The single cell 2 is left in the first well 22 by the drag force induced in the first well 22.

15, the infiltrating liquid 6 is injected into the first well 22 to pass through the cell membrane of the single cell 2 fixed on the lower surface of the first well 22, After the infiltration liquid 6 is infiltrated, negative pressure is applied to the second well 23 to remove the infiltrant 6 as shown in FIG.

Thereafter, the cleaning process as shown in FIG. 11 is performed once to clean the inside of the first well 22, and negative pressure is applied to the second well 23 to remove residual Remove the permeate (6) and wash solution.

17, the dyeing solution 7 is injected into the first well 22 to dye the infiltrated single cells 2, and then the second well 23 ) To remove the dyeing solution 7 as shown in Fig.

Thereafter, the cleaning process as shown in FIG. 11 is performed once to clean the inside of the first well 22, and negative pressure is applied to the second well 23 to remove residual Remove the dyeing solution (7) and washing solution.

As described above, even when the sample is injected into the first well 22 of the microfluidic device 100 or a cell treatment process in which various reagents are successively exchanged, the single cell 2 Can remain in the first well 22 without loss.

 That is, one embodiment can provide a single cell treatment method capable of continuously treating a single cell 2 through the microfluidic device 100.

Hereinafter, specific embodiments of the present invention will be described. The embodiments described below are only intended to illustrate or explain the present invention, and thus the present invention should not be limited thereto. In addition, contents not described here can be inferred sufficiently technically if they are skilled in the art, and a description thereof will be omitted.

Fabrication of microfluidic devices

A predetermined groove pattern is formed on the surface of a PDMS (manufactured by Dow Corning) substrate having a width of 75 mm, a length of 25 mm and a thickness of 5 mm by using a soft lithography method. The depth of the formed groove pattern may be 10 [mu] m and the line width may be 6 [mu] m.

Thereafter, the PDMS substrate is punched to form a first opening portion and a second opening portion that open the PDMS substrate in the up and down direction. The first opening and the second opening may have a circular cross-sectional shape, the cross-sectional diameter of the first opening may be 4 mm, and the cross-sectional diameter of the second opening may be 0.3 mm. The first opening portion and the second opening portion formed are connected to the groove pattern, respectively.

Thereafter, a slide glass is attached to the upper surface of the PDMS substrate having the groove pattern formed thereon, and then the PDMS substrate having the slide glass attached thereto is reversed upside down to produce a microfluidic device as shown in Fig.

The manufactured microfluidic device has a space formed by the first opening and the slide glass forming the first well, a space formed by the second opening and the slide glass forming the second well, and a space formed by the groove and the slide glass forming the microfluidic channel .

Evaluation 1: Immunocytochemistry ( immunocy tochemistry ) For single cell treatment

Prepare a sample solution in which MCF7 (breast cancer cells) is dispersed in phosphate buffered saline (PBS) as a single cell. MCF7 is based on the sample solution of about 30 μ L can contain from about 10 to about 20.

30 μL of the prepared sample solution was injected into the first well of the prepared microfluidic device, and then the pipette was placed in the second well. The negative pressure was applied to the sample, and the PBS was pipetted at a flow rate of about 100 μL / min or less Lt; / RTI >

Then, 20 μL of PBS was injected into the first well to wash the first well, the pipette was placed in the second well, and the PBS remaining in the first well was applied at about 100 μL / min To the pipette at the following flow rate.

Then, 20 μL of 4% paraformaldehyde (PFA) is injected into the first well as a fixative, and MCF7 is immobilized on the slide glass for 10 minutes. Thereafter, the pipette is placed in the second well and negative pressure is applied to remove the PFA remaining in the first well.

Then, 20 μL of PBS is injected into the first well to wash the first well, the pipette is placed in the second well, and negative pressure is applied to remove the remaining PBS in the first well.

Then, the injection of 0.1% saponin (saponin) of 20 μ L into chimtuaek the first well, and performs the task of penetrating the cell membrane of the chimtuaek MCF7 fixed on a slide glass over a period of 10 minutes.

Then, 20 μL of PBS is injected into the first well to wash the first well, and then the pipette is placed in the second well, and the saponin remaining in the first well is removed by applying negative pressure.

Then, the first and the Hoechst (blue), FITC (green) and PE (red), 0.1% saponin, and a mixed solution 20 μ L of PBS to the staining solution to the first well injection, and performs MCF7 dyeing of over 90 minutes.

Then, 20 μL of PBS was injected into the first well to wash the first well, the pipette was placed in the second well, and negative pressure was applied to remove the remaining dye in the first well.

Thereafter, the microfluidic device containing the stained MCF7 was dried, and the stained MCF7 was observed through a fluorescence microscope. The results are shown in Fig.

19 is a fluorescence microscope image showing single cells stained according to one embodiment.

Referring to FIG. 19, it can be seen that the nucleus portion of MCF7 was blue by Hoechst, the cytokeratin portion of MCF7 was green by FITC, and the EpCAM portion of MCF7 was red by PE.

In one embodiment, the present invention is not necessarily limited to this, and the single cell may be a prostate cancer cell (PC3), a melanoma (M14), a colon cancer cell (HT29), a gastric cancer cell (HGC27, NUGC2, MKN7, MKN28) Cancer cell (OVCA433) or the like may be used. If blood such as whole blood in which hemocyte cells have not been removed from MCF7 is used as a sample fluid, in addition to the above-described treatment process, a step of dissolving and removing red blood cells And the like.

Also, as a staining agent, the cytokeratin moiety can be stained red with PE and the CD45 moiety can be stained with green using Alexa488.

As described above, by using the microfluidic device according to one embodiment, various cell processes of a single cell can be continuously performed in the microfluidic device.

Evaluation 2: Continuous reagent exchange

Approximately 10 to 40 pre-fixed MCF7 were injected into the first well of the microfluidic device using MCF7 as a single cell, 20 [ mu] L of PBS was injected into the first well, and a pipette was placed in the second well And the negative pressure is applied to remove the PBS remaining in the first well is repeated 32 times in total. The number of MCFs remaining in the first well was recorded for each repeated execution step to determine the number of times at which the MCF7 was lost. The results are shown in Table 1 below.

MCF7 loss occurred No loss of MCF7 Number of times Episode 2 30 times

Referring to Table 1, it was found that MCF7 was lost in the first cell at a high probability of about 94%, and only 32 of the total 32 times were lost. Can be confirmed. That is, when the microfluidic device according to one embodiment is used, loss of a single cell can be minimized even when a repetitive cell treatment step is performed unlike a general centrifugation method or the like.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, And falls within the scope of the invention.

2: single cell 3: sample
4: Washing solution 5: Fixing solution
6: Penetrating solution 7: Staining solution
10: substrate 20: single cell processing unit
21: body 22: first well
23: second well 24: fine flow path
25: first bottom part 26: second bottom part
100, 200: microfluidic device

Claims (16)

As a target cell, a sample liquid containing a single cell, or a first well containing a reagent,
A second well disposed apart from the first well,
And two or more microchannels extending from a lower surface of the first well to a lower surface of the second well to connect the first well to the second well,
Wherein a diameter of a lower surface of the first well is larger than a diameter of a lower surface of the second well,
The width of the micro channel is smaller than the size of the single cell,
And the ratio of the area of the lower surface of the first well to the cross-sectional area in the width direction of the micro channel is 1000: 1 to 400000000: 1. delete The method of claim 1,
Further comprising a first reservoir portion formed between the at least two micro flow paths and the second well,
Wherein the at least two micro flow paths and the second well are connected to the first reservoir portion, respectively. The method of claim 1,
Wherein the at least two micro flow paths connect the first well and the second well in parallel. The method of claim 1,
And the width of the fine flow path is from 0.5 mu m to 15 mu m. The method of claim 1,
Wherein the lower surface of the first well and the lower surface of the second well are coplanar. The method of claim 1,
Wherein the lower surface of the first well viewed from above has a circular shape. 8. The method of claim 7,
And the lower surface of the first well has a diameter of 1 mm to 50 mm. The method of claim 1,
Wherein at least two of the first wells, the second wells, and the at least two microfluidic channels include at least two single cell processing units. The method of claim 1,
Wherein the at least two single cell processing units are arranged to have a matrix form. 11. A method for treating a single cell using a microfluidic device according to any one of claims 1 to 10,
Injecting a sample solution containing a single cell into the first well,
Fixing the single cell by injecting fixative into the first well,
Injecting a permeate into the first well to pre-treat the fixed single cells, and
Injecting a staining solution into the first well to stain the pretreated single cells
, ≪ / RTI &
Wherein the sample liquid, the fixing liquid, the penetrating liquid, and the dyeing liquid injected into the first well are applied to the second well in a state where a negative force exceeding a hydraulic pressure resistance applied to the microchannel is applied to the first well, Lt; / RTI > delete 12. The method of claim 11,
Wherein the sample solution, fixer solution, permeate solution, and dye solution each of which is removed from the first well by the applied negative pressure are received in the second well through the micro channel. 12. The method of claim 11,
Wherein the velocity of each of the sample liquid, the fixing liquid, the penetrating liquid, and the dyeing solution passing through the micro channel by the applied negative pressure is greater than the moving velocity of the single cell induced by the negative pressure. 12. The method of claim 11,
Further comprising injecting a wash solution into the first well to wash the first well. 16. The method of claim 15,
Wherein the cleaning solution is removed from the first well by applying a negative pressure in excess of the hydraulic resistance applied to the microchannel to the second well.
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