KR101533230B1 - Multistage microfluidic chip and method for selective isolation of sample using the same - Google Patents

Multistage microfluidic chip and method for selective isolation of sample using the same Download PDF

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KR101533230B1
KR101533230B1 KR1020140031141A KR20140031141A KR101533230B1 KR 101533230 B1 KR101533230 B1 KR 101533230B1 KR 1020140031141 A KR1020140031141 A KR 1020140031141A KR 20140031141 A KR20140031141 A KR 20140031141A KR 101533230 B1 KR101533230 B1 KR 101533230B1
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South Korea
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sample
microfluidic chip
channel
cells
antibody
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KR1020140031141A
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Korean (ko)
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정효일
현경아
이태윤
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연세대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/4915—Blood using flow cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells

Abstract

The present invention relates to a multi-stage microfluidic chip and a method for selectively separating samples using the multi-stage microfluidic chip. More particularly, the present invention relates to a multi-stage microfluidic device having a multi- The present invention relates to a multi-stage microfluidic chip capable of effectively separating target cells of various characteristics, including circulating rare cells including tumor cells, in a relatively short period of time, and a method for selectively separating samples using the same.
According to the present invention, it is possible to separate target cells of various characteristics not only from limited cell groups, but also to improve separation efficiency even at a small flow rate in a channel.

Description

TECHNICAL FIELD [0001] The present invention relates to a multi-stage microfluidic chip and a method for selectively separating a sample using the multi-

The present invention relates to a multistage microfluidic chip and a method for selectively separating a sample using the same, and more particularly, to a technique for selectively separating and extracting a desired substance from a biological sample using a microfluidic chip .

The present invention also relates to a microfluidic device such as a biochip, a MEMS, a lab-on-a-chip, etc., and is applied to bio-related research fields such as pharmacology, medicine, and microbiology that require biological sample analysis Technology.

In general, biochemical samples contain a mixture of two or more substances. Therefore, the separation technique for analyzing only the desired components or for purifying only specific components in the mixture is very important in the pretreatment of the sample. Especially, lab-on-a-chip technology, which is a concept to integrate microchannels, mixers, pumps, valves, and the like into a single chip to process a small amount of samples at high speed and high efficiency, is attracting attention.

Cell-based diagnostics, which are also important for biological or medical analysis, are in the form of blood analysis, cell research, and microbiological analysis. Recent research and analysis of cells and development of protein and DNA analysis techniques are leading to the development and integration of these clinical diagnostic procedures in the form of microfluidic devices.

Circulating rere cells (CRC) are circulating cells that circulate in blood and are very rarely present in less than 1000 cells per ml of blood. These circulating rare cells include circulating tumor cells (CTC), nucleated red blood cells (nRBC), circulating endothelial cells (CEC) and circulating stem cells (CSC) These are indicators of various diseases and can be used for early diagnosis and prognosis of diseases.

Recently, there have been a growing number of studies for early detection of metastatic cancer or monitoring the outcome of cancer treatment by detecting blood tumor cells which are epithelial cells that have fallen from the tumor. This method has the advantage of not requiring biopsy such as removing the cancer tissue directly in the body, which is an absolutely advantageous method especially for patients with difficult lung biopsy. However, blood tumor cells (1 CTC / 10 < 9 > blood cells) are present in the blood of patients at extremely low concentrations, and thus it has been difficult to efficiently capture and detect them.

In order to effectively isolate the circulating tumor cells, the throughput (the number of cells that can be separated per unit time), the cell recovery rate (the ratio of the number of target cells injected and the number of target cells separated and separated) And the purity of the recovered target cells), and the like.

Korean Patent Publication No. 10-2013-0107583 ("Composition for Diagnosis of Blood Tumor Cells and Method for Detecting Tumor Tumor Cells Using the Same ", published on Oct. 20, 2013) discloses a method for enhancing capture efficiency of blood tumor cells and non- In order to minimize the binding and increase the purity of detection, nanoparticles having a primary antibody such as an EpCAM antibody specifically binding to blood tumor cells and a secondary antibody such as protein A binding to the primary antibody are attached And a magnetic bead having a size of 100 nm to 1 탆.

However, as a method for separating blood tumor cells into target cells using magnetic nanoparticles as the immuno-magnetic platform, there is a disadvantage in that the number of nanoparticles that bind nonspecifically to blood cells must be increased together with a low purity.

In order to overcome this problem, recently, a method using a microfluidic device with an antibody specifically binding to circulating tumor cells has been studied, and this method has the highest purity to date. However, due to the low blood flow (about 2.5 ml / h), the whole blood sample is examined in a long time, and there is still a problem to be solved until it is commercialized in terms of efficiency and purity improvement.

Korean Patent Laid-Open Publication No. 10-2013-0107583 ("Composition for Diagnosis of Blood Tumor Cells and Method for Detecting Blood Tumor Cells Using the Same ", issued October 20, 2013)

"Microfluidic devices for the isolation of circulating rare cells: A focus on affinity-based, dielectrophoresis, and hydrophoresis", Electrophoresis 2013, 34, 1028-1041

SUMMARY OF THE INVENTION The present invention has been devised to solve the above-mentioned problems, and an object of the present invention is to provide a multi-stage microfluidic device having a structure including a primary and a secondary microfluidic chip, The present invention provides a multi-stage microfluidic chip capable of efficiently separating target cells of various characteristics including a plurality of circulating rare cells in a relatively short period of time, and a method for selectively separating samples using the same.

A plurality of micro channels 1 and a sample inlet 2 communicating with the microchannel 1 and a sample outlet 3 are connected to the microchannel 1, A multistage microfluidic chip comprising at least one microfluidic chip, the microfluidic chip comprising a magnetic body (11) for collecting magnetized particles in an upper portion, a lower portion, A microfluidic chip (10); And a central channel (21) in which particles in the sample are aligned with the center of the channel, and at least one or more of the at least one or more channels communicating with the center channel (21) A focusing area F provided with a bypass channel 22; And a separation region S in which an inner wall of the channel is coated with a binding substance a2 'having affinity specific to the specific particle a2 in the sample, and a secondary microfluidic chip 20 formed in this order The present invention provides a multi-stage microfluidic chip for selective separation of a sample.

It is preferable that the magnetic body 11 is a neodymium (Nd) magnet provided in parallel in the upper and lower portions of the outer wall of the channel and the center channel 21 of the focusing region F has a ratio (h / w) is less than 1, that is, the width of the channel is preferably larger than the height.

The separation region S is formed of a channel formed in a herringbone pattern, and the herringbone pattern is a V-shaped or S-shaped horizontal band having a plurality of vertices arranged in parallel, S, the sample is moved in a direction perpendicular to the formation direction of the herringbone pattern so that the sample can be easily coupled to the inner wall of the channel.

According to another aspect of the present invention, there is provided a microchannel comprising a plurality of microchannels, a sample inlet and a sample outlet communicating with the microchannel, A method for selectively separating a sample using a multi-stage microfluidic chip comprising at least one microfluidic chip, comprising the steps of: (a1 ') having a specific affinity for a specific particle (a1) a mixed sample in which the specific particles a1 and the magnetic nanoparticles m are combined in the mixed sample is injected into the first microfluidic chip 10, A primary separating step (S10) of collecting the magnetic material (11) provided in the lower part or the upper part and the lower part; And a specimen discharged from the primary microfluidic chip 10 is injected into the secondary microfluidic chip 20 to form a bonded body a2 'coated on the channel inner wall of the secondary microfluidic chip 20, And a secondary separation step (S20) of collecting the specific particles (a2) in the injected sample having affinity by an antigen-antibody reaction (step S20).

The secondary separation step S20 preferably includes a concentration step S20a passing through the focusing area F and a collecting step S20b passing through the separation area S, It is preferable that the width of the central channel 21 is larger than the height of the channel as described above.

According to a preferred embodiment of the present invention, when the present invention is applied to the separation of circulating rare cells in a sample containing blood, red blood cells that remove red blood cells in the blood through hemolysis and centrifugation prior to the primary separation step (S10) (A1 ') coated on the surface of the magnetic nanoparticles (m) injected into the sample from which erythrocytes have been removed can be preceded by the removal step (S1), and specific binding to the human leukocyte common antigen (CD45) CD45 < / RTI > In the secondary separation step (S20), EpCAM protein antibody (a2 ') having affinity specific to the circulating tumor cells is coated on the inner wall of the channel of the secondary microfluidic chip (20).

In addition, it is preferable to control the flow rate of the in-channel sample of the first microfluidic chip 10 to 300 to 400 / / min in the primary separation step S10.

As described above, the multi-stage microfluidic chip of the present invention and the method of selectively separating samples using the multi-stage microfluidic chip include a primary microfluidic chip employing a Magnetic-Activated Cell Sorting (MACS) technique, a focusing area for concentrating particles in the sample, The microfluidic chip of the secondary microfluidic chip of the secondary microfluidic chip in which the antibody-coated separation region is sequentially formed is used, and thus the processing ability and the cell recovery rate of the device are excellent and the circulating rare cells of various characteristics It is possible to separate a plurality of target cells and improve separation efficiency even at a small flow rate in a channel.

1 is a schematic diagram of a first microfluidic chip 10 according to a preferred embodiment of the present invention.
FIG. 2 is a schematic view illustrating a separation process in a channel of the first microfluidic chip 10 according to a preferred embodiment of the present invention.
3 is a schematic diagram of a secondary microfluidic chip 20 according to a preferred embodiment of the present invention.
FIG. 4 is a schematic view illustrating a separation process performed inside a channel of a secondary microfluidic chip 20 according to a preferred embodiment of the present invention.
5 is a schematic diagram of a secondary microfluidic chip 20 including a focusing area F and a separation area S according to a preferred embodiment of the present invention.
6 is a schematic view showing a channel in a separation region S according to a preferred embodiment of the present invention.
7 and 8 are schematic views showing a pattern of a herringbone pattern formed in a channel in a separation region S according to a preferred embodiment of the present invention.
FIG. 9 is a photograph sequentially showing steps (a) to (g) of manufacturing a first microfluidic chip 10 according to a preferred embodiment of the present invention.
10 is a graph showing the leukocyte removal efficiency according to leukocyte concentration and flow rate as a result of an experiment using the primary microfluidic chip 10. Fig.
11 is a graph showing the cell recovery efficiency according to the sample flow rate as a result of an experiment using the primary microfluidic chip 10. FIG.
12 is an immunostained photograph of a sample cell separated and discharged from the first microfluidic chip 10.
FIG. 13 is a graph showing the number of MCF-7 cells in the sample separated and discharged from the primary microfluidic chip 10. FIG.
FIG. 14 is a photograph showing the expected behavior of the cells in the channel of the focusing area F of the secondary microfluidic chip 20 through the green fluorescent beads.
15 is a graph showing the focusing efficiency of leukocytes and MCF-7 cells in the focusing region F of the secondary microfluidic chip 20.
16 is a graph showing the results of selective separation of sample cells according to whether EpCAM is tested using the secondary microfluidic chip 20. FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. Prior to the description, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary meanings and should be construed in accordance with the technical concept of the present invention.

Throughout this specification, when a member is " on " another member, this includes not only when the member is in contact with another member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as "including" an element, it is understood that it may include other elements as well, without departing from the other elements unless specifically stated otherwise.

The terms "primary "," secondary ", and the like are used to distinguish one element from another, and the scope of the right should not be limited by these terms. For example, the first component may be referred to as a second component, and similarly, the second component may also be referred to as a first component.

In each step, the identification code is used for convenience of explanation, and the identification code does not describe the order of the steps, and each step may be performed differently from the stated order unless clearly specified in the context. have. That is, each of the steps may be performed in the same order as described, or may be performed substantially concurrently or in the reverse order.

The present invention includes at least one microfluidic chip provided with a plurality of microchannels 1 and a sample inlet 2 communicating with the microchannel 1 and a sample outlet 3 according to a preferred embodiment of the present invention A multi-stage microfluidic chip comprising: a primary microfluidic chip (10) having a magnetic body (11) at an upper portion or a lower portion of a channel; a secondary microfluidic chip (10) having a channel in which a focusing region (F) And a multi-stage microfluidic chip for selective separation of a sample including the microfluidic chip (20).

According to another preferred embodiment of the present invention, a primary separation step (S10) of injecting a mixed sample of magnetic nanoparticles (m) coated with a binding substance and a sample into a primary microfluidic chip (10) And a secondary separation step (S20) of injecting the sample discharged from the primary microfluidic chip (10) into the secondary microfluidic chip (20) and collecting the specific particles by the combined body coated on the inner wall of the channel. To provide a method for selectively separating a sample.

As used herein, the term "conjugate" may be DNA, peptide, antibody, antibody or the like that binds to target cells or non-target cells, and may be used to simultaneously capture different target cells or non- ≪ / RTI >

1 and 2, the primary microfluidic chip 10 is a chip-implemented Manic-activated cell Sorting (MACS) technique. The magnetic nanoparticle (m) And a method of separating them into magnetic bodies 11 is proposed. Whereby the magnetic nanoparticles (m1) coated with the binding substance (a1 ') having specific affinity for the specific particles (a1) in the sample are combined with the specific particles (a1) to form the complex (M) (M) is collected by the magnetic force toward the magnetic body (11) provided in the upper, lower or upper and lower parts of the channel.

This may be used as a positive enrichment method for separating only target cells from non-target cells using an antibody specific for the target cell, or may be used as a non-target And can be used as a negative enrichment method in which target cells are finally released by specifically removing the cells.

More preferably, the magnetic substance 11 may be a neodymium (Nd) magnet provided in a sandwich manner in parallel with the upper and lower portions of the outer wall of the channel of the first microfluidic chip 10. By using neodymium magnets which have the strongest magnetic force (25 ~ 50MGOe) among the magnets currently used on the earth and which are relatively good in processability and relatively inexpensive, and are installed at the upper and lower portions of the channel, Can be collected downward.

3 and 4, the secondary microfluidic chip 20 focuses particles in a sample to remove substances other than intracellular particles, that is, water and a buffer solution A focusing region F and a separation region S for sequentially coating the antibody on the inner wall of the channel to immobilize specific particles in the sample in the channel.

The focusing region F aligns the particles in the sample to the center of the center channel 21 and removes the remaining non-corresponding substances to the outer periphery of the channel through the separately provided bypass channel 22, Concentrate the particles. In particular, in the sample containing blood, the circulating rare cell has a very small content in the blood as described above, and therefore, the process of concentrating the sample as described above should be preceded before separation and detection. Whereby the particle components in the sample of various sizes are concentrated.

Hereinafter, the principle of the concentration of the particle components in the sample in the focusing area F will be described in more detail with reference to FIG. This is also in line with the principle of the concentration step S20a in the secondary separation step S20. The channel of the secondary microfluidic chip 20 constituting the focusing region F is a channel through which the sample is injected through the sample injection port 2 and the concentrated channel is moved to the separation channel F, And a bypass channel 22 provided at an outer portion of the center channel 21 and communicating with the center channel 21 to be connected to a discharge port of the material except for the particles in the sample.

The center channel 21 preferably has a ratio (h / w) of a width w of the channel to a height h of less than 1. In other words, the width (w) of the channel must be larger than the height (h). In this case, the particle component in the sample is aligned with the center of the channel due to the inertial force applied to the fluid sample in the channel . If the ratio (h / w) is 1 or more, the opposite phenomenon occurs and the concentration of the sample becomes difficult. Depending on the ratio (h / w) There is no particular limitation on the lower limit value, so that the size of the microfluidic chip and the manufacturing process can be adjusted as needed.

By the above-described principle, the particle components in the sample are aligned with the central part of the central channel 21, so that the substances other than the particle components in the sample are gathered in the outer part of the center channel 21. [ Therefore, in the focusing region F where the center channel 21 and the bypass channel 22 intersect, materials other than the particles in the sample, typically water and buffer solution, are discharged to the outside of the microfluidic chip through the bypass channel 22 do. As a result, the sample can be concentrated, effectively separating a small amount of target particles in the sample, and improving the separation performance of the device.

The separation area S on the secondary microfluidic chip 20 used in the collection step S20b in the secondary separation step S20 is a region into which the concentrated sample is injected through the focusing area F , And a complex (a2 ') having affinity specific to the specific particle (a2) in the sample is coated on the inner wall of the channel.

As in the case of the first microfluidic chip 10 described above, the inner wall of the channel may be coated with a binding substance specific to the target cell and used in a positive enrichment method, or a non-target cell-specific binding substance may be coated and used in a negative enrichment method .

The channel provided in the isolation region S may be formed in a herringbone pattern. The inventors of the present invention have focused on a channel in which a pattern in which chaos mixing can occur in a large area is referred to by referring to a herringbone pattern known to be capable of chaotic mixing.

More specifically, the channel in the isolation region S is characterized in that a void formed therein is formed in a herringbone pattern. That is, the channel can be a fine void, pore, or space formed inside the microfluidic chip structure, which forms a thin tube-like channel. (See FIG. 6)

In the present specification, the term " herringbone pattern "means a V-shaped or S-shaped horizontal band shape closely arranged like an interference pattern occasionally seen on a screen of a television receiver. Among them, a V- And S-shaped horizontal strips may be arranged in parallel (refer to Figs. 7 and 8).

The present invention is characterized in that a shape of a channel is formed by a herringbone pattern having a plurality of vertices, and a desired cell can be intensively detected and / or separated by a channel having such a shape in the vicinity of a vertex. In this regard, the herringbone pattern is a geometrically activated surface interaction pattern (GASI pattern) that induces geometrically activated surface interactions.

The method of forming the herringbone pattern as described above in the channel of the separation region S is not particularly limited, and various methods known in the art can be employed. For example, it is possible to make a mold by using machining, then to make a plastic chip by injection, or to fabricate a pattern by etching the silicon (Si) or glass wafer.

On the other hand, the direction of movement of the sample in the separation region S is not particularly limited, but it is preferable to move in the direction perpendicular to the formation direction of the herringbone pattern as shown in FIGS. It is possible to maximize the contact area or frequency with the vertex when the sample moves in a direction perpendicular to the formation direction of the herringbone pattern because the purpose is to adhere more target cells in the vicinity of the vertex of the herringbone pattern, Do.

By providing the secondary microfluidic chip 20 of the present invention with the separation region S including the channels having the internal structure of the herringbone pattern as described above, the target cells can be attached to the channel inner wall in a larger amount , The position of the vertex array of the herringbone pattern and the longitudinal or transverse length of the pattern or channel can be controlled to greatly increase the cell separation efficiency and both the positive and negative concentration methods can be used to obtain a general and economical effect .

However, in order to effectively fix the specific particle (a2) in the sample to the coupled substance (a2 ') coated on the inner wall of the channel in this channel structure, a very slow flow rate of 20 to 30 ㎕ / min must be maintained, There are disadvantages. However, the present invention is not limited to this, but a focusing area F may be placed so as to precede the separation area S to compensate for the disadvantages of such a separation area S channel according to the preferred embodiment, Thereby maximizing the treatment efficiency of the sample.

By using the first and second microfluidic chips 10 and 20 as described above, various target cells can be separated or extracted from a biological sample in which various cells are mixed by performing the first and second separation steps (S10 and S20) . In addition, by using such a multi-stage microfluidic chip, separation techniques can be implemented by various separation mechanisms as follows.

First, the first and second microfluidic chips 10 and 20 are operated in a positive enrichment mode. For example, a cell that specifically binds to an antibody A can be isolated in a first step, and then a cell that specifically binds to an antibody B can be isolated in two steps to extract various cells. Alternatively, if the cells specifically binding to the antibody A in both steps 1 and 2 are separated, the cells that have not yet been separated in step 1 are separated again in step 2, thereby improving the recovery rate of the target cells.

Second, the primary microfluidic chip 10 is operated in a negative enrichment mode, the secondary microfluidic chip 20 is operated in a positive enrichment mode, or vice versa. For example, by removing antibodies in a first step using an antibody specific to a non-target cell, and separating cells specific to the antibody A in step 2, many cells are removed in step 1, Of the target cell can be separated.

Third, all of the first and second microfluidic chips 10 and 20 are operated in a negative enrichment mode. Target cells are removed using an antibody specific to the non-target cells in step 1, and non-target cells that have not yet been removed in the second step are removed once again, thereby finally separating the target cells with high purity, There is an advantage that a target cell recovery process is not required.

Hereinafter, embodiments of a multi-stage microfluidic chip of the present invention and a method of selectively separating samples using the multi-stage microfluidic chip will be described. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents. shall.

≪ Method of producing microfluid chip >

A manufacturing process of the first microfluidic chip 10 will be described with reference to FIG.

A double-sided tape (a double-sided tape, which is a conventional double-sided tape, has a structure in which acrylic adhesive is provided on both sides of a PET film (13 μm) and a paper release paper is attached to one side) is laminated on both sides of a PMMA sheet. And cut into the shape of the desired microfluidic channel. (Fig. 9 (a), hereinafter referred to as " material 1 &

Prepare one 3M hydrophilic polyester film that has one hydrophilic polyester film and one laser injection port (2) and one sample outlet (3), which are commonly used. (Fig. 9 (b), hereinafter referred to as 'material 2a' and 'material 2b'),

After removing the paper release sheet on one side of the material 1 and attaching the material 2a from which the protective film has been removed, the paper release sheet on the other side of the material 1 is removed and the material 2b from which the protective film has been removed is inserted into the sample inlet 2, And the sample outlet (3).

A Tygon tube (10 cm) is placed on the sample inlet (2) and the sample outlet (3) and bonded using an epoxy adhesive. A neodymium (Nd) magnet is sandwiched between the top and bottom of the channel.

On the other hand, in the case of the secondary microfluidic chip 20, general PDMS soft lithography is used.

≪ Secondary Microfluidic Chip (20) Fixing Method in Channel >

Neutravidin is dissolved in 10 mM tris tris buffer at a concentration of 0.2 mg / ml (hereinafter, referred to as 'material 1'). Specific antibody to which biotin is bound is dissolved in phosphate buffer (Hereinafter referred to as "material 2") was dissolved in phosphate buffer saline (PBS) (hereinafter referred to as "material 2"). A phosphate buffer solution containing 10 mM Tris buffer (hereinafter referred to as "material 3") and 1% bovine serum albumin Prepare a saline solution (hereinafter referred to as "material 4").

0.3 ml of the sample 1 is injected into the secondary microfluidic chip 20 and allowed to stand at room temperature for 5 minutes so that the neptavidin in the channel is coated by the protein adsorption. Thereafter, 0.5 ml of the sample 3 is injected to wash off the non-adsorbed neutravidin on the chip. After injecting the sample 2 into 0.3 ml of the secondary microfluidic chip 20, the neutravidin coated in the channel forms a biotin-avidin bond between the sample 2 and the antibody is immobilized on the inner wall of the channel. do. To prevent nonspecific binding of the cells, 0.5 ml of the sample 4 is injected to wash the unbound antibody.

<Application to circulating rare cell separation>

By using the multistage microfluidic chip of the present invention, it is possible to isolate and detect circulating rare cells (CRC), particularly circulating tumor cells (CTC), in a sample containing blood. Hereinafter, this will be described in detail.

First, a red blood cell hemolytic agent is added to a sample containing blood to hemolyze red blood cells contained therein, and the red blood cell removing step (S1) is performed to collect only white blood cells and circulating rare cells by centrifuging them.

Thereafter, in order to remove leukocytes from the injected sample, the magnetic nanoparticles (m) coated with an antibody binding to an antigen specifically present in leukocytes were mixed with a sample from which erythrocytes were removed, and this was mixed with a syringe pump And injected into the primary microfluidic chip 10. Preferably, the antigen specifically present in leukocytes is a human leukocyte common antigen (CD45), and the antibody binding thereto is a CD45 antibody. Among the injected samples, leukocytes bind with the magnetic nanoparticles (m) and move in the direction of the magnetic body (11) provided at the upper and lower parts of the channel to be fixed. After the first separation step (S10), red blood cells and white blood cells are removed, and a sample containing various circulating rare cells is discharged along the channel to the sample outlet (3) of the first microfluidic chip (10).

Thereafter, the discharged sample is injected into the secondary microfluidic chip 20 by using a syringe pump. An antibody specifically binding to the circulating rare cell to be a target is present on the inner wall of the channel forming the separation region S of the secondary microfluidic chip 20 to fix the target cell on the inner wall of the channel through antigen-antibody binding . After the secondary separation step (S20), the sample containing the circulating rare cells other than the target cell comes out of the secondary microfluidic chip (20) through the sample outlet (3).

When the target cell is set as CTC, a secondary separation step (S20) is performed by coating EpCAM protein having specific affinity with the inner wall of the channel of the secondary microfluidic chip (20).

<Application to Separation of Bacterial Cell Samples>

On the other hand, the present invention can be applied to cell sample separation such as bacteria, similar to the method of separating the circulating rare cells as described above.

For example, in the case of bacteria, the target bacteria is concentrated by removing the non-target cells from the primary microfluidic chip 10 and mixed in the buffer such as a large amount of water through the focusing area F in the secondary microfluidic chip 20 The target bacteria that have been extracted are concentrated, and then only the target bacteria are separated through the separation region S to use the extracted target bacteria for the subsequent analysis.

<Performance Test of Primary Microfluidic Chip 10>

The primary microfluidic chip 10 is used to remove white blood cells to which magnetic nanoparticles (m) coated with a CD45 antibody are bound according to an embodiment of the present invention. Therefore, leukocyte removal rate was tested using normal blood.

The erythrocyte hemolytic agent is added to the normal blood at a volume ratio of 1:10, and the mixture is allowed to stand at room temperature for 10 minutes. After centrifugation at 600 G for 10 min, the hemolyzed red cells were removed and the collected cells were suspended in phosphate buffered saline (PBS). Thereafter, 20 μl of the magnetic nanoparticles (m) coated with the CD45 antibody are added per 10 6 cells and allowed to stand at room temperature for 15 minutes.

The sample prepared as described above is injected into the primary microfluidic chip 10 by using a syringe pump and the number of cells in the sample that exits to the sample outlet 3 is counted using a hemocytometer .

FIG. 10 is a graph showing an experimental result of leukocyte removal efficiency according to leukocyte concentration and flow rate. Samples were prepared by suspending the leukocytes recovered from 3 ml, 5 ml and 7.5 ml of blood in 3 ml of phosphate buffered saline, respectively. When the sample was injected into the first microfluidic chip 10 at a flow rate of 100-400 μl / min, the maximum flow rate was 400 μl / min. In the sample with the highest leukocyte concentration of 7.5 ml, leukocyte was 99.99% .

11 is a graph showing how well cancer cells are recovered when MCF-7 cells, which are breast cancer cell model cell lines, are injected at a concentration of 10 6 / ml without mounting the magnetic substance 11 on the primary microfluidic chip 10 . At a flow rate of 300 to 400 μL / min, it was confirmed that the cells were evacuated to the sample outlet (3) by 90% or more. Since the flow rate was slow in the flow rate range of 100 to 200 μL / min, (3) and settled in the channel. On the other hand, if the flow rate exceeds 400 μL / min, the cost due to the maintenance of the flow rate is excessive and the economical efficiency is deteriorated.

In summary, the present inventors optimized the experimental conditions at a flow rate in the range of 300-400 μL / min, which removes more than 99.99% of white blood cells and effectively separates cancer cells by 90% or more.

In order to verify the performance of the primary microfluidic chip 10, MCF-7 cells were spiked into 5 ml of normal blood at 50, 100, 500 and 1000, respectively, The separation test of the first microfluidic chip 10 was performed. Finally, the separated samples were stained with a cytospin centrifuge on a slide glass, and the cells were counted by immunostaining. Immunostaining was performed using DAPI staining to identify the nuclei of cells, CD45 (green) conjugated with FITC fluorescent antibody (fluorescent green) to identify leukocytes, PE fluorescence antibody (fluorescence red) to identify MCF-7 cells ) (Cytokeratin) (red). Leukocytes were counted for DAPI positive, CD45 positive, and Cytokeratin negative, and MCF-7 cells were counted for DAPI positive, CD45 negative, and Cytokeratin positive cells. 12 is an immunostained photograph of a separated sample.

Figure 13 is a graph showing the number of MCF-7 cells counted after separation according to the degree of spiking of the initial MCF-7 cells. When MCF-7 cells were spiked with 50, 100, 500, and 1000 cells, 99.99% of leukocytes were removed, and 48, 96, 483, and 913 MCF-7 cells were isolated, respectively.

<Performance Test of Secondary Microfluidic Chip 20>

The secondary microfluidic chip 20 is a chip for injecting a sample into a channel in which an antibody specific to the inner wall is immobilized to selectively immobilize specific cells inside the chip through an antigen-antibody reaction. Therefore, it is important to keep the flow rate of the fluid sample in the channel low, because the bond is broken and the specific cell is not fixed to the channel inner wall when the shear force of the fluid is larger than the binding force of the antigen-antibody.

In addition, since the fixing efficiency of the cell varies depending on the shape of the channel, the fixing efficiency of specific particles in the channel can be further improved by designing the channel of the separation region S with a herringbone structure according to an embodiment of the present invention.

However, in order to immobilize the cells in the channel of the separation region S, it is necessary to maintain a very low flow rate of 20 to 30 μL / min. Accordingly, in order to solve this problem, the present invention allows the sample to concentrate the sample through the focusing region F before the separation region S to improve the throughput.

When the sample is injected into the sample injection port 2, the particles in the sample in the focusing area F are aligned to the center of the center channel 21 by the inertial force and moved to the channel in the separation area S. Except the particles in the sample, the material is discharged through the bypass channel 22 at a flow rate of 70 to 80 μl / min. The flow rate of the injected sample is 20 μl / min to 20 μl / min, S). 14 is a photograph showing the behavior of expected cells in a channel using green fluorescent beads of 7 占 퐉.

In the central channel 21 of the focusing area F, it is important that all the cells are aligned to the center of the channel and then moved to the channel of the separation area S. Therefore, focusing efficiency was confirmed using leukocytes and MCF-7 cells. Referring to the graph of FIG. 15, leukocyte and MCF-7 cells exhibited high focusing efficiency of 80.40% and 95.39%, respectively.

In order to confirm that selective circulating rare cells can be separated in the separation region S, an antibody specifically binding to EpCAM was immobilized on a channel in the separation region S, and then EpCAM, among the same breast cancer cell lines, And 106 cells / ml of MDA-MB-231 cells not expressing EpCAM were prepared. Referring to the graph of FIG. 16, MCF-7 cells were isolated and fixed on the chip by an average of about 98.81%, and 93.12% of the MDA-MB-231 cells were separated from the chip outlet I confirmed that it came out.

The present invention is not limited to the above-described specific embodiment and description, and various changes and modifications may be made by those skilled in the art without departing from the scope of the present invention as claimed in the claims. And such modifications are within the scope of protection of the present invention.

a1: Specific particles in the first microfluidic chip
a1 ': a bond for the specific particle (a1)
a2: Specific particles in the secondary microfluidic chip
a2 ': a bond to the specific particle (a2)
m: magnetic nanoparticles
M: a composite of the specific particle (a1) and the magnetic nanoparticle (m)
F: focusing area
S: separation area
1: Microchannel
2: Sample inlet
3: Sample outlet
10: Primary microfluidic chip
11: magnetic substance
20: Secondary microfluidic chip
21: Central channel
22: Bypass channel

Claims (11)

delete delete delete delete A method for selectively separating a sample using a multistage microfluidic chip comprising at least one microfluidic chip having a plurality of microchannels (1) and a sample inlet (2) communicating with the microchannel (1) In the method,
To isolate circulating rare cells (CRC) in the sample containing blood,
A red blood cell removing step (S1) for hemolyzing the red blood cells by injecting a hemolyzing agent into the sample and centrifuging the red blood cells;
(M) coated with an antibody having specific affinity for human leukocyte common antigen (CD45), which is specifically present in leukocytes, is mixed with the sample, and the flow rate of the sample in the channel is adjusted to 300 to 400 (M) coupled with the human leukocyte common antigen (CD45), the antibody and the magnetic nanoparticles (m) in the mixed sample is injected into the first microfluidic chip 10 while controlling the concentration of the antibody A first separation step (S10) of collecting the magnetic bodies (11) provided on the upper, lower or upper and lower sides to remove white blood cells from the sample from which red blood cells have been removed; And
A sample discharged from the primary microfluidic chip 10 is injected into the secondary microfluidic chip 20 to collect the circulating rare cell (CRC), and the sample in which the circulating rare cell (CRC) (S20);
/ RTI &gt;
The secondary separation step (S20)
All the cells in the sample injected into the secondary microfluidic chip 20 are aligned at the center while passing through the center channel 21 having the ratio of the width w of the channel to the height h of the channel h / (20a) for discharging the liquid material except the cells in the sample collected at the outer portion of the center channel (21) through at least one bypass channel (22) communicating with the center channel (21); And
A collection step (S20b) of passing a concentrated sample through an inner wall of a channel coated with a complex (a2 ') having specific affinity for circulating rare cells (CRC) in order to collect circulating rare cells (CRC) in the sample;
The method comprising the steps of: delete delete delete delete 6. The method of claim 5,
In the collecting step (S20b), the conjugate (a2 ') coated on the inner wall of the channel of the secondary microfluidic chip 20 is transferred to EpCAM (Epithelial cell) having affinity specific to circulating tumor cells (CTC) wherein the sample is selected from the group consisting of an antibody and an antibody. delete
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