WO2017155170A1 - Microfluidic element and single cell treatment method using same - Google Patents

Abstract

Provided are: a microfluidic element comprising a first well in which a sample solution containing a single cell, as a target cell, or a reagent is accommodated, a second well disposed to be spaced apart from the first well, and two or more microchannels for connecting the first well and the second well by extending from the lower surface of the first well to the lower surface of the second well, wherein the width of the microchannels is smaller than the size of the single cell; and a single cell treatment method using the same.

Description

Microfluidic device, and single cell treatment method using the same

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

Cancer is currently the number one cause of death from diseases not only in Korea, but throughout the world. The significant impact on mortality in cancer patients depends on the presence of metastatic cancer cells. In other words, the technology to accurately and without loss of single cells, such as circulating tumor cells (CTCs), present in one of the hundreds of millions of blood cells in the blood, improves the survival rate of patients before and after cancer treatment. It is essential.

 For example, less than 5 in about 7.5 ml of blood for breast cancer, less than 3 for colorectal cancer, and less than 5 for prostate cancer, must be found in a single throughput that can be separated per unit time. Micros meeting three basic conditions: cell count), cell recovery (recovery, ratio of single cells harvested / collected to single cell injected), and collection efficiency (purity, purity of single cells harvested / collected). Cell separation techniques are required.

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

However, the conventional methods may result in the loss of a single cell while undergoing a process of removing a large number of blood cells contained in blood for CTC detection. In addition, in the process of performing a treatment such as staining by injecting the single cells from which the blood cells are removed again into a separate experimental device, there is a possibility that a single cell loss occurs.

Provided is a microfluidic device capable of minimizing the loss of a single cell during various processing of a single cell.

The present invention also provides a single cell processing method capable of continuously processing a single cell through a microfluidic device.

According to one embodiment, a target solution containing a single cell as a target cell, or a reagent containing a first well, a second well disposed spaced apart from the first well, and a second from the lower surface of the first well A microfluidic device including two or more microchannels extending to the bottom surface of the well and connecting the first well and the second well, the width of the microchannel being smaller than the size of the single cell.

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

The first reservoir may further include a first reservoir formed between the two or more microchannels and the second well, and the two or more microchannels and the second well may be connected to the first reservoir.

The two or more microchannels may connect the first well and the second well in parallel.

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

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

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

The bottom surface diameter of the first well may be 1 mm to 50 mm.

The microfluidic device may include two or more single cell processing units 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.

On the other hand, according to another embodiment, as a single cell treatment method using the microfluidic device, injecting a sample solution containing a single cell into the first well, injecting a fixed solution into the first well to fix the single cell A single cell treatment method comprising the steps of: injecting a permeate solution into the first well to pretreat the fixed single cell; and injecting a dye solution into the first well to stain the pretreated single cell. Is provided.

A negative force may be applied to the second well to remove the sample solution, the fixative solution, the penetrating solution, and the dye solution from the first well.

The sample solution, the fixer, the penetrant, and the dye solution removed from the first well may be accommodated in the second well through the microchannel.

The flow rate of the sample solution, the fixed solution, the penetrating solution, and the dye solution passing through the microchannel by the negative pressure applied may be greater than the moving speed of the single cell induced by the negative pressure.

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

When the washing step is completed, the washing liquid may be removed from the first well by applying a negative force to the second well.

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

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

1 is a perspective view of a microfluidic device according to one embodiment;

2 is a plan view from above of the microfluidic device of FIG. 1;

3 is a cross-sectional view taken along line III-III of FIG.

4 to 6 show various modifications of the single cell treatment unit according to one embodiment,

7 is a perspective view showing a microfluidic device including two or more single cell processing units according to another embodiment;

8 to 18 are views sequentially showing a single cell treatment method according to one embodiment.

Hereinafter, an embodiment will be described in detail so that a person of ordinary skill in the art may easily implement the present embodiment. However, they may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

In the drawings, the thickness of layers, films, panels, regions, etc., are exaggerated for clarity. Like parts are designated by like reference numerals throughout the specification. When a portion of a layer, film, region, plate, etc. is said to be "on top" of another part, this includes not only when the other part is "right over" but also when there is another part in the middle. On the contrary, when a part is "just above" another part, there is no other part in the middle.

In one embodiment a "single-cell", which is a target cell, is a very rare cell, for example with only a few to several dozens present in a sample, for example, a drug in the blood. It refers to a circulating tumor cell (CTC), etc. present in one of the billion cells of blood cells.

In one embodiment, "reagent" means various kinds of drugs for biochemical treatment of the single cell, and includes, for example, washing solution such as buffer solution, fixed solution, penetrating solution, dye solution, and the like. to be.

Meanwhile, in one embodiment, "sample liquid" means that the "single cell" is contained in a reagent such as a buffer solution.

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

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

1 and 2, the microfluidic device 100 according to the embodiment is disposed on the substrate 10 and the substrate 10, and is a single cell processor configured to receive and process a single cell as a target cell. And 20.

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

The single cell processing unit 20 is disposed directly on the substrate 10. The single cell processing unit 20 includes a body 21, a first well 22, a second well 23, and two or more microchannels 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 so as to confirm the presence of a single cell in the sample. For example, the body 21 may be made of a material such as glass or plastic.

Meanwhile, the body 21 may be formed of a material having hydrophobicity, for example, polydimethylsiloxane (PDMS). In general, since the sample liquid and various reagents are hydrophilic fluids, the body 21 is formed of a hydrophobic material, so that a strong fluid resistance is applied to the inside of the microchannel 24 so that a first external force is not applied. Reagents in the well 22 can be prevented from flowing into the microchannel 24 and the second well 23.

However, one embodiment is not necessarily limited thereto, and may be formed of a material having hydrophilicity, and at least, the surface forming the sidewalls of the first well 22 and the second well 23 and the fine flow path 24 will be described. The surfaces to be formed may be controlled to have hydrophobicity through surface modification or the like.

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

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

As illustrated in FIG. 2, the first well 22 and the second well 23 may be formed to have a circular shape. However, one embodiment is not necessarily limited thereto, and the shape of the first well 22 and the second well 23 may have a polygonal shape such as a triangular shape, a square shape, a hexagonal shape, an octagonal shape, an elliptic shape, or the like.

The microchannel 24 extends from the bottom surface of the first well 22 to the bottom surface of the second well 23 to connect the first well 22 and the second well 23 to each other. The micro-channel 24 according to the exemplary embodiment may be a space formed between the substrate 10 and the body 21 by contacting the substrate 10 after pattern etching the lower surface of the body 21.

On the other hand, two or more micro-channels 24 may extend from the first well 22 toward the second well 23. In one embodiment, as shown in FIGS. 1 and 2, two or more microchannels 24 extend from the first well 22 toward the second well 23 in parallel, respectively, and then the first well. At one point between the 22 and the second well 23 may be formed in a shape that is combined into one path and connected to the second well 23.

The microchannel 24 serves as a passage for supplying the sample liquid or the reagent contained in the first well 22 to the second well 23. That is, the microchannel 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 the microchannel 24 is smaller than the size of a single cell. As a result, a single cell may not pass through the microchannel 24 and remain inside the first well 22. That is, the micro channel 24 may discharge only the sample liquid or the reagent except the single cell inside the first well 22 to the second well 23.

On the other hand, since the micro-channel 24 is formed to be smaller than the size of a single cell having a width of several tens to hundreds of micrometers in general, the sample liquid or reagent contained in the first well 22 has a predetermined pressure or more. Only to be discharged to the second well 23 through the micro-channel 24.

That is, the microfluidic device 100 according to the exemplary embodiment may be formed of a sample liquid including a single cell injected into the first well 22, or a sample fluid containing only a single cell except for a single cell. Can be removed by moving to two wells (23).

In more detail, the sample liquid or reagent contained in the first well 22 may have a predetermined positive pressure to overcome the high hydraulic resistance of the microchannel 24. 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 Equation 1 below.

In Equation 1, R _h denotes a hydraulic resistance applied to the fine passage 24, ΔP denotes a pressure change amount to be applied, and Q denotes a flow rate through the fine passage 24, respectively.

In one embodiment, a first pressure may be applied to the second well 23 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 using a pipette or the like. The sample liquid contained in 22) or the sample can be moved to the second well 23.

If the pressure capable of overcoming the hydraulic resistance of the micro-channel 24 (for example, a negative pressure greater than or equal to a threshold pressure due to the hydraulic resistance of the micro-channel 24) is equal to the first well 22 or the second well 23. ), The sample liquid or reagent contained in the first well 22 remains entirely in the first well 22 due to the high hydraulic resistance of the microchannel 24. That is, the micro flow path 24 may serve as a kind of check valve.

Therefore, the microfluidic device 100 according to the exemplary embodiment selectively removes only the sample solution including the single cells injected into the first well 22 or only the remainder except the single cells in the sample from 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.

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

3 is a cross-sectional view taken along line III-III of FIG. 2.

Referring to FIG. 3, the microchannel 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 single cell to be a target cell, for example, 0.5 μm to 15 μm, for example 1 μm to 15 μm, eg For example, 1 μm to 12 μm, for example 1 μm to 10 μm.

When the width of the micro-channel 24 is less than 0.5 μm, the removal time of the sample liquid or reagent for treating single cells is excessively increased, thereby reducing the treatment efficiency, and the width of the micro-channel 24 exceeds 15 μm. In this case, the size of the microchannel 24 is about the same as that of the single cell into which the microchannel 24 is inserted, and the single cell is sucked into the microchannel 24 by the negative pressure applied to the second well 23. By closing or moving to the second well 23 to be removed, a single cell loss can occur.

The cross section in the width direction of the micro-channel 24 may be variously designed in consideration of the ease of processing of the body 21, for example, may be formed in a semicircle, an ellipse, a square shape, or the like.

On the other hand, the length of the micro-channel 24 is the interval between the first well 22 and the second well 23, the amount of sample liquid or sample injected into the first well 22, the second well 23 It can be variously designed according to the sound pressure applied to, for example, 5 ㎛ to 1 mm, for example 5 ㎛ to 800 ㎛, for example 5 ㎛ to 600 ㎛, for example 5 ㎛ to 500 ㎛ have.

When the length of the micro-channel 24 is less than 5 μm, the flow rate of the fluid passing through the micro-channel 24 is excessively increased due to the negative pressure applied to the second well 23, so that the sample liquid in the first well 22, or It is difficult to precisely control the amount of sample. On the other hand, when the length of the micro-channel 24 exceeds 1mm, the magnitude of the negative pressure to be applied to the second well 23 increases, while the removal of the sample liquid or the sample slows down, so that the single cell treatment process time This may increase excessively.

According to an embodiment, the heights of the first well 22 and the second well 23 may have the same value H1. The height of the first well 22 and the second well 23 according to one embodiment can be variously set, for example 1 mm to 50 mm, 1 mm to 20 mm, for example 1 mm to 10 mm Can be. When the height of the first well 22 and the second well 23 is less than 1 mm, the amount of the sample liquid or reagent injected into the first well 22 may be insufficient, and the first well 22 and the second well may be insufficient. If the height of the well 23 exceeds 50 mm, an unnecessary amount of reagent may be injected into the first well 22, and after the single cell treatment, the sample liquid or reagent may be removed through the second well 23. Is difficult, and the single cell processing process time can be long.

The bottom surface of the first well 22 and the bottom surface of the second well 23 according to one embodiment may be coplanar, as shown in FIG. 3, that is, in one embodiment, The bottom surface of the first well 22, the bottom surface of the second well 23, and the bottom surface of the microchannel 24 may all be coplanar. As such, the lower surface of the first well 22, the lower surface of the second well 23, and the lower surface of the fine flow path 24 are positioned on the same plane without the step, thereby providing the first well 22 with the first well 22. The sample liquid or reagent stored therein can be easily moved to the fine flow path 24 by the negative pressure applied to the second well 23.

Meanwhile, referring to FIG. 3, in one embodiment, the bottom surface diameter D1 of the first well 22 is larger than the bottom surface diameter D3 of the second well 23, but the bottom surface of the first well 22 Diameters of the lower surfaces of the second wells 23 may be the same as or different from each other, and the amount of the sample liquid or reagent injected or discharged, the diameter of the sample liquid or the pipette for injecting the reagent, and the like. It can be set in various ways.

The lower surface diameter D1 of the first well 22 and the diameter D3 of the lower surface of the second well 23 are each, for example, 1 mm to 50 mm, 1 mm to 20 mm, for example 1 mm to 10 mm. Can be. When the diameters of the lower surfaces of the first well 22 and the second well 23 are less than 1 mm, the amount of the sample liquid or reagent injected into the first well 22 may be somewhat insufficient. It is difficult to induce drag force. In addition, when the diameter of the bottom surface of the first well 22 and the second well 23 exceeds 50 mm, the amount of reagent to be injected for single cell treatment may be excessively increased.

In one embodiment, as the sample solution is injected into the first well 22 and over time, the majority of single cells sink to the lower surface of the first well 22. At this time, when the sample liquid is drawn out to the micro-channel 24 by applying a negative pressure to the second well 23, the flow rate of the sample liquid passing through the micro-channel 24 is rapidly changed by the negative pressure. Will be moved to. On the other hand, the single cells are hardly affected by the negative pressure applied to the second well 23 and show little movement in the lower surface of the first well 22.

As the negative pressure is applied to the second well 23, drag force due to fluid movement is applied in addition to gravity and buoyancy as a force applied to the single cells existing in the sample liquid. This is because the sample liquid moves at a slower speed than the flow rate passing through the microchannel 24.

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

The behavior of these single cells within the first well 22 is influenced by the ratio of the bottom surface area of the first well 22 to the widthwise cross-sectional area of the microchannel 24.

In one embodiment, the ratio of the area of the lower surface of the first well 22 to the widthwise cross-sectional area of the microchannel 24 is the width D2 of the microchannel 24 described above and the bottom of the first well 22. It can be variously adjusted according to the surface diameter D1, 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 width-wise cross-sectional area 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. It may remain on the bottom surface of one well 22.

In a process for removing only a specific fluid from a general microfluidic device, valves formed in a microchannel or multiple pumped devices are used. Such a general microfluidic device is troublesome to precisely control valves or to connect a fluid adapter or a syringe pump each time a specific fluid removal process is performed.

On the other hand, when the microfluidic device is centrifuged by another method for concentrating a single cell, loss of the single cell may occur in the course of aspiration of the sample liquid or reagent after centrifugation.

In addition, even if a single cell obtained through the above method is attached to a substrate (smear) and then further processed, loss of a single cell may occur in the process of washing, and then the single cell may be recovered and further processed. Is required, for example, when a single cell has to be recovered after characterization and extracted from DNA, etc., loss of a single cell may occur during recovery.

 However, in the case of the microfluidic device 100 according to an embodiment, the body 21 is formed of a hydrophobic material so that a single cell may be formed in the microchannel 24 and the second well in a situation in which no external force is applied. 23) can be prevented from entering. In addition, by adjusting the ratio of the area of the lower surface of the first well 22 to the widthwise cross-sectional area of the microchannel 24, the high hydraulic resistance of the microchannel 24 itself and the drag applied to a single cell can be controlled.

Accordingly, the microfluidic device 100 according to an embodiment may more easily remove only a specific fluid from the first well 22 through a simple tool such as a pipette to continuously remove a sample liquid or various reagents. Even after removal into one well 22, no single cell loss occurs.

That is, according to the exemplary embodiment, the single cell treatment process may be continuously performed in the microfluidic device 100 while minimizing the loss of the single cell during various treatments 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.

Referring to FIG. 4, the single cell processing unit may be arranged such that two or more microchannels 24 ′ connect the first well 22 and the second well 23 in parallel. The fine flow passage 24 ′ can move the sample liquid or the reagent independently of the fine flow passage 24 of FIGS. 1 to 3 described above.

In FIG. 4, the fine flow passage 24 ′ has a structure in which five flow passages are arranged in parallel to each other, but are not necessarily limited thereto, and the magnitude of the hydraulic resistance that varies according to the quantity of the fine flow passage 24 ′, The diameter of the first well 22, the cross-sectional area, and the magnitude of the sound pressure applied to the second well 23 may be set in various ways.

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

In FIG. 5, the length of the microchannel 24 is reduced to correspond to the area occupied by the first reservoir 25, but is not necessarily limited thereto, and the specific shape of the first reservoir 25, an acceptable volume, and the like are not limited thereto. The interval between the first well 22 and the second well 23, the sample solution that can be accommodated by the first well 22 and the second well 23, or the amount of the reagent may be variously set.

Meanwhile, referring to FIG. 6, the single cell processing unit may further include a second reservoir 26 formed in each of two or more microchannels 24. That is, as compared with the above-described FIGS. 1 to 3, the second reservoir 26 having a width wider than the width of the fine passage 24 may be formed at one side of the fine passage 24. That is, it provides a space in which the sample liquid or reagents that are moving inside the microchannel 24 may be temporarily stored.

In FIG. 5, the second reservoir 26 is formed independently for each of the fine flow paths 24, but is not necessarily limited thereto, and the second reservoirs 26 may be formed to be connected to each other. 2 The specific shape of the reservoir 26, the acceptable volume, etc. are the width and length of the micro-channel 24, the distance between the first well 22 and the second well 23, the first well 22 and the second The amount of the sample liquid or reagent that can be accommodated in the well 23, and the flow rate of the fluid flowing through the microchannel 24 in the presence of the second reservoir 26 may be set in various ways.

As described above, the microfluidic device 100 according to an embodiment may be a single cell, even more specifically, a single cell processing unit 20, more specifically, the micro-channel 24 is designed in various ways. While minimizing the loss, the single cell treatment process may be continuously performed in the microfluidic device 100.

Hereinafter, a microfluidic device according to another embodiment will be described with reference to FIG. 7.

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 may be arranged in a row between neighboring single cell processing units 20. 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 the neighboring single cell processing units 20 of the microfluidic device 200 according to another embodiment, single cell processing of each of the single cell processing units 20 may be performed independently.

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 limited thereto. The single cell processing unit 20 can also be applied to various experimental tools.

As described above, the microfluidic device 200 according to another embodiment may include two or more single cell processing units 20 to enable independent single cell processing, thereby improving single cell processing efficiency.

Hereinafter, a single cell treatment method using a microfluidic device according to an embodiment will be described in detail with reference to FIGS. 8 to 18.

Single cell treatment method according to one embodiment, the step of injecting a sample solution containing a single cell into the first well 22, the step of fixing the single cell by injecting a fixed solution into the first well 22, Injecting the permeate solution into the first well 22 to pretreat the fixed single cell, and injecting the dye solution into the first well 22 to stain the pretreated single cell.

First, in the sample liquid injection step, as shown in FIG. 8, the sample liquid 3 including the single cell 2 is injected into and received in the first well 22. The accommodated sample liquid 3 does not move inside the microchannel 24 due to the hydraulic resistance of the microchannel 24 and remains in the first well 22. In addition, the single cells 2 mostly sink to the bottom surface of the first well 22.

Subsequently, as shown in FIG. 9, when a negative pressure greater than or equal to the threshold pressure due to the hydraulic resistance of the microchannel 24 is applied to the second well 23 using a pipette or the like, the first well 22 may be accommodated. The sample liquid 3 flows along the fine flow path 24 toward the second well 23 at the first speed V1. At this time, the single cells 2 that have sunk on the lower surface of the first well 22 are induced by the negative pressure, and move toward the micro-channel 24 at the second speed V2.

In one embodiment, the widthwise length of the microchannel 24 is formed to be smaller than the size of the single cell 2, so that the single cells 2 do not pass through the microchannel 24 and enter the first well 22. Will remain. 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 inside the first well 22, but a negative pressure greater than the threshold pressure due to the hydraulic resistance of the micro-channel 24 is applied. In the state of FIG. 10, the remaining trace liquid sample 3 does not pass through the fine flow path 24.

On the other hand, in one embodiment, it may further comprise the step of washing the first well 22 by injecting a washing solution into the first well 22, the washing step is the sample solution injection step, fixing step, infiltration In each of the steps and staining steps, the removal of the sample solution, the fixed solution, the penetrating solution and the staining solution except for the single cell 2 may be performed.

That is, as shown in FIG. 11, in the washing step, when the washing liquid 4 is injected into the first well 22 of FIG. 10, the washing liquid 4 is also formed by the hydraulic resistance of the micro-channel 24. (22) It exists inside. Subsequently, when a negative pressure is applied to the second well 23, as shown in FIG. 9 described above, the washing liquid 4 passes through the microchannel 24 and is received in the second well 23, whereas a single cell is used. (2) hardly move inside the first well 22.

As a result, when the washing of the sample liquid 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.

Subsequently, in the fixing step, the fixing solution 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 to the lower surface of the first well 22 by the fixation solution 5 are removed together with a small amount of fixation solution 5 as shown in FIG. 14 even if the fixation solution 5 is removed by negative pressure. It remains inside the one well 22.

Thereafter, the inside of the first well 22 is cleaned by performing the washing process as illustrated in FIG. 11 once, and then a negative pressure is applied to the second well 23 to remain in the first well 22. Remove the fixed solution (5) and wash solution. If some of the fixed single cells 2 are detached from the lower surface of the first well 22 in the course of performing the washing process, the width of the microchannel 24, the hydraulic resistance, and the single cells 2 are described above. Due to the drag induced, the single cell 2 remains inside the first well 22.

Subsequently, in the infiltration step, as shown in FIG. 15, the permeate solution 6 is injected into the first well 22 to pass through the cell membrane of the single cell 2 fixed to the lower surface of the first well 22. After performing the process of infiltrating the permeate solution 6, a negative pressure is applied to the second well 23 to remove the permeate solution 6 as shown in FIG. 16.

Thereafter, the inside of the first well 22 is cleaned by performing the washing process as illustrated in FIG. 11 once, and then a negative pressure is applied to the second well 23 to remain in the first well 22. Remove the permeate (6) and wash solution.

Subsequently, in the staining step, as shown in FIG. 17, the dye solution 7 is injected into the first well 22 to perform staining of the infiltrated single cell 2, and then the second well 23. Negative pressure is applied to remove the dye solution 7 as shown in FIG.

Thereafter, the inside of the first well 22 is cleaned by performing the washing process as illustrated in FIG. 11 once, and then a negative pressure is applied to the second well 23 to remain in the first well 22. Remove the dye solution (7) and wash solution.

As such, in one embodiment, even if a sample cell injected into the first well 22 of the microfluidic device 100, or a cell treatment process for continuously exchanging various reagents is performed, It may remain in the first well 22 without being lost.

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

Hereinafter, specific examples of the present invention are presented. The examples described below are merely to specifically illustrate or explain the present invention, and the present invention is not limited thereto. In addition, the description is not described herein, so those skilled in the art that can be sufficiently technically inferred, the description thereof will be omitted.

Fabrication of Microfluidic Devices

A predetermined groove pattern is formed on the surface of the upper surface of a PDMS (Dow Corning Co., Ltd.) substrate having a width of 75 mm, a length of 25 mm, and a thickness of 5 mm using a soft lithography method. The depth of the formed groove pattern may be 10 μm, and the line width may be 6 μm.

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

Thereafter, the slide glass is attached to the upper surface of the PDMS substrate on which the groove pattern is formed, and then the upper and lower sides of the PDMS substrate to which the slide glass is attached are inverted, thereby manufacturing the microfluidic device as illustrated in FIG. 1.

In the manufactured microfluidic device, the space formed by the first open portion and the slide glass is the first well, the space formed by the second open portion and the slide glass is the second well, and the space formed by the groove and the slide glass is the micro flow path. .

Evaluation 1: Single Cell Treatment via Immunocytochemistry

As a single cell, prepare a sample solution in which MCF7 (breast cancer cells) is dispersed in phosphate buffered saline (PBS). 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 is injected into the first well of the prepared microfluidic device, and then a pipette is placed in the second well and a negative pressure is applied to the pipette at a flow rate of about 100 μL / min or less. Adjust to move toward

Thereafter, 20 μL of PBS is injected into the first well to wash the first well, and then a pipette is placed in the second well and a negative pressure is applied to the PBS remaining in the first well, about 100 μL / min. Adjust to move toward the pipette at the following flow rates.

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

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

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

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.

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

Then, after drying the microfluidic device containing the dyed MCF7, the stained MCF7 is observed through a fluorescence microscope.

As a result, it can be confirmed that the nuclear nucleus portion of MCF7 is well colored by Hoechst, the cytokeratin portion of MCF7 is green by FITC, and the EpCAM portion of MCF7 is red by PE.

On the other hand, one embodiment is not necessarily limited thereto, prostate cancer cells (PC3), melanoma (M14), colorectal cancer cells (HT29), gastric cancer cells (HGC27, NUGC2, MKN7, MKN28), not ovary MCF7 as a single cell Cancer cells (OVCA433) or the like may be used, and in the case of using blood such as whole blood in which the blood cells are not removed from the MCF7 as a sample solution, in addition to the above-described treatment process, a process of dissolving and removing red blood cells Additional processes such as may also be carried out.

As the dye, the cytokeratin portion may be dyed red using PE, and the CD45 portion may be dyed green using Alexa488.

As such, using the microfluidic device according to the embodiment, it is possible to continuously perform various cell treatments of a single cell in the microfluidic device.

Evaluation 2: Continuous Reagent Exchange

Using MCF7 as a single cell, about 10 to 40 pre-fixed MCF7s are injected into the first well of the microfluidic device, 20 μL of PBS is injected into the first well, and the pipette is placed in the second well. The process of removing the PBS remaining in the first well by applying negative pressure is repeated a total of 32 times. The quantity of MCF7 remaining in the first well is recorded for each iteration step, and the number of times the MCF7 is lost is determined. The results are shown in Table 1 below.

	MCF7 Loss Occurred	No MCF7 Loss
Count
Episode 2	30 times

Referring to Table 1, as a result of 32 repeated experiments, the number of times MCF7 loss occurred was only 2 out of 32 times. You can check it. That is, by using the microfluidic device according to the embodiment, it is possible to minimize the loss of a single cell even when undergoing repeated cell treatment step unlike the general centrifugation method.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to the scope of the invention.

Claims (16)

1. A first well containing a sample solution or a reagent containing a single cell as a target cell,

   A second well disposed spaced apart from the first well, and

   At least two micro-channels extending from the lower surface of the first well to the lower surface of the second well connecting the first well and the second well,

   The microfluidic device having a width smaller than the size of the single cell.
2. In claim 1,

   And a ratio of the area of the lower surface of the first well to the widthwise cross-sectional area of the microchannel is in a range of 1000: 1 to 400000000: 1.
3. In claim 1,

   Further comprising a first reservoir formed between the two or more micro-channel and the second well,

   The two or more micro-channel and the second well is connected to the first reservoir portion, respectively.
4. In claim 1,

   And the two or more microchannels connect the first well and the second well in parallel.
5. In claim 1,

   The microfluidic device has a width of 0.5 μm to 15 μm.
6. In claim 1,

   And a bottom surface of the first well and a bottom surface of the second well are coplanar.
7. In claim 1,

   A microfluidic device having a circular shape at a lower surface of the first well as viewed from the top.
8. In claim 7,

   The lower surface diameter of the first well is 1 mm to 50 mm microfluidic device.
9. In claim 1,

   A microfluidic device comprising two or more single cell processing units having the first well, the second well, and the two or more microchannels.
10. In claim 1,

    Wherein said two or more single cell processing units are arranged to have a matrix form.
11. A single cell treatment method using the 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 a fixed solution into the first well,

    Pretreatment of the fixed single cell by injecting the infiltration solution into the first well, and

    Dyeing the pretreated single cell by injecting a stain solution into the first well

    Single cell treatment method comprising a.
12. In claim 11,

    Applying a negative force to the second well to remove the sample solution, the fixative solution, the permeate solution, and the dye solution from the first well.
13. In claim 12,

    The sample solution, the fixing solution, the penetrating solution, and the staining solution removed from the first well are received in the second well through the micro-channel.
14. In claim 12,

    The flow rate of the sample solution, the fixed solution, the penetrating solution, and the dye solution passing through the microchannel by the negative pressure applied is greater than the moving speed of the single cell induced by the negative pressure.
15. In claim 12,

    Injecting the washing solution into the first well to wash the first well further comprising the step.
16. The method of claim 15,

    When the washing step is completed, a single cell treatment method of applying a negative force to the second well to remove the wash solution from the first well.

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