JP6244589B2 - Micro-channel chip for separating fine particles, advection integrated unit, system for separating fine particles, and method for separating fine particles - Google Patents

Description

  The present invention relates to a microfluidic chip for separating fine particles, a convection collecting unit, a system for separating fine particles, and a method for separating fine particles for separating fine particles of different sizes mixed in a liquid, and in particular, circulating tumor cells in blood (Circulating tumor cell, which may be abbreviated as “CTC” hereinafter.) CTC separation microchannel chip capable of selectively capturing, CTC separation system using the chip, advection integration used in the CTC separation system The present invention relates to a unit and a CTC separation method.

  CTCs are defined as tumor cells that circulate in the peripheral bloodstream of cancer patients and are tumor cells that have infiltrated into blood vessels from primary or metastatic tumors. The detection of this CTC has recently attracted attention as one of the methods for early detection of metastatic malignant tumors. The reason is that metastatic malignant tumors can be diagnosed more accurately and less invasively than X-ray photographs and tumor marker detection in serum, and can be used as an indicator of patient prognosis and therapeutic effects.

CTC is a very rare cell, and it is known that only about 1 cell is present among 10 ⁸ to 10 ⁹ blood cells contained in the blood of patients with metastatic cancer. For this reason, much effort has been put into technology development for accurately detecting rare CTCs from peripheral blood. Major detection methods that have been developed so far include immunohistochemistry, PCR, flow cytometry, and the like. However, as described above, since CTC is a very rare cell, blood cannot be directly used for these detection methods. Therefore, it is usually necessary to concentrate CTC as a pretreatment. It is necessary to concentrate the CTC abundance ratio to the compliant level.

  Among various techniques that have been developed as CTC enrichment methods, the most widely used is the enrichment of tumor cells targeting specific antigens on the cell surface. Many of them employ a method of concentrating tumor cells using a magnet after mixing magnetic particles on which a monoclonal antibody against an epithelial cell adhesion molecule (EpCAM) is immobilized with blood (for example, non-cells). Patent Document 1). However, it is known that the expression level of EpCAM varies greatly depending on the type of tumor.

  As another concentration method, there is a method of concentrating on the basis of a form such as a cell size. A method of selecting epithelial tumor cells having a size larger than that of leukocytes by filtration is called an ISET method (Isolation by Size of Experimental Tumor cells). ISET is a simple method of filtering blood using a polycarbonate membrane filter having a pore diameter of 8 μm, and is an inexpensive and user-friendly method. The polycarbonate membrane filter used here has holes formed by a technique called track etching in which etching is performed after irradiation with heavy ions. However, since the holes have a relatively low density and there is a problem that two or more holes overlap, the trapping efficiency is 50-60% when used for trapping CTC. A method that is simple and efficient is not yet developed.

  In order for CTC detection to be efficient and accurate, techniques such as enrichment and detection need to be performed consistently. Multi-stage handling operations such as cell staining, washing, separation, and dispensing cause CTC loss. Therefore, avoid these operations as much as possible, and perform analysis in an integrated detector. Is preferred. Cellsearch (Veridex ™, Warren, PA) is the only CTC detector that has received FDA approval. In this apparatus, CTC is concentrated on whole blood using anti-EpCAM antibody-immobilized magnetic microparticles, and tumor cells are immunostained, and then tumor cells are counted using an automated fluorescent microscope (for example, Non-patent document 2). However, when using the apparatus, it is generally necessary to introduce a large apparatus and secure a trained operator, and it is difficult to perform an inspection at a bedside in a short time and accurately.

  On the other hand, small microfluidic devices for CTC detection are also known. For example, a microfluidic device for CTC detection developed by Toner et al. Is called CTC-chip, and 78000 cylindrical structures (microposts) are formed in a silicon flow path formed by photolithography. . The micropost is coated with an anti-EpCAM antibody, and when blood is fed into the channel, CTC in the blood is captured on the micropost. The captured CTC is subjected to fluorescent immunostaining targeting an epithelial cell marker (cytokeratin), and tumor cells are counted using a fluorescence microscope. Although this device is a small device placed on the palm, it has a great advantage that it is possible to use 5 mL or more of blood as it is for analysis. Actually, CTC is detected from the blood of a metastatic cancer patient, and a mutation that produces resistance to a tyrosine kinase inhibitor can be detected from the collected CTC. However, CTC detection using Cellsearch or CTC-chip has been performed vigorously through experiments using actual samples such as blood from metastatic cancer patients, but these methods are anti-EpCAM antibodies and CTCs have been used. The principle is to concentrate. Therefore, there is a problem that EpCAM negative or weak positive tumor cells cannot be detected.

  As another method, a microfluidic device that detects CTC using tumor cell size and morphology as an index has been developed. In these devices, a membrane microfilter, a crescent-shaped cell capture well (see Non-Patent Document 3), and a four-stage channel (see Non-Patent Document 4) are arranged in the channel structure, and blood The blood cells and tumor cells are sorted according to size, and the tumor cells are selectively enriched. In addition, by using the flow channel, operations such as lysis can be continuously performed on the concentrated cells. In the evaluation experiment of the recovery efficiency of model tumor cells using these devices, a CTC recovery efficiency of 80% or more is obtained. However, this evaluation is only conducted in an experiment using model cells, and elemental technical items such as cell staining and washing operations that are actually required for CTC detection have not been studied, and blood of cancer patients, etc. Experiments using real samples were not conducted, and it was not clarified whether they could actually be used for CTC detection.

  Furthermore, as a small device that does not use an anti-EpCAM antibody, there is known a microfluidic device that can capture a CTC by providing a microcavity array (fine through-hole) in a microchannel (see Patent Document 1). ). However, since the microfluidic device is a type that captures CTC in a fine through-hole, there is a problem that the work efficiency is lowered due to clogging of the CTC, and further, it is difficult to collect the separated CTC.

  In order to solve the above problems, the present applicants have (1) a microchannel chip for separating fine particles in which a trapping site larger than the width of the main channel and the main channel is formed, or (2) the main channel, Using a branch channel that branches from the main channel and connects to the main channel again, and a microchannel chip for fine particle separation in which a trapping part larger than the width of the branch channel is formed in the branch channel, the gas-liquid interface The force generated in the meniscus can be used to settle the microparticles, and only the target microparticles can be captured at the capture site. In particular, even if whole blood that has not been pretreated such as concentrated is used as a sample, only CTC Have been newly found that they can be separated and recovered continuously (see Patent Document 2).

  In the method of separating fine particles described in Patent Document 2, a sample is injected between a microchannel chip for microparticle separation and a thin plate for sample, and a sheath is interposed between the microchannel chip for microparticle separation and a thin plate for sheath liquid. The target microparticles are captured on the microchannel chip for particle separation by a meniscus generated by injecting the liquid and moving the microchannel chip for microparticle separation, the thin plate for sample, and the thin plate for sheath liquid relative to each other. It is trapped in the site. By the way, the amount of sample that can be injected between the microchannel chip for fine particle separation described in the above patent application and the thin plate for sample is about 100 μl. However, it is possible to accurately determine whether or not CTC is contained from a sample of a patient having very few CTC cells in blood cells, such as a follow-up of a patient with early cancer metastasis or a patient after cancer treatment. Therefore, it is preferable to analyze a blood sample of about 5 ml, and the method described in Patent Document 2 requires reintroduction of the sample, which makes the separation operation of microparticles very complicated and time consuming. There's a problem.

  Furthermore, in order to analyze individual separated CTC cells, it is necessary to amplify nucleic acids by PCR. However, a system that can separate and recover CTC from a blood sample and perform automatic analysis is not known.

JP 2011-163830 A Japanese Patent Application No. 2012-227717

Allard WJ, Matera J, Miller MC, Repolet M, Connelly MC, Rao C, Tibbe AG, Uhr JW, Terstrappen LW. 2004. Tumor cells circulate in the peripheral block of all major carcinomas but not in health subjects with valents diligents. Clin Cancer Res 10 (20): 6897-904. Riethdorf S, Fritsch H, Muller V, Rau T, Schindlbeck C, Rack B, Janni W, Coith C, Beck K, Janice F and others. 2007. Detection of circling tutor cells in peripheral blood of patents with metallic breast cancer: avalidation study of the CellSearch. Clin Cancer Res 13 (3): 920-8. Tan SJ, Yobas L, Lee GY, Ong CN, Lim CT. 2009. Microdevice for the isolation and enumeration of cancer cells from blood. Biomed Microdevices 11 (4): 883-92. Mohamed H, Murray M, Turner JN, Cagana M. et al. 2009. Isolation of tutor cells using size and deformation. J Chromatogr A 1216 (47): 8289-95.

  The present invention has been made in order to solve the above-described conventional problems, and as a result of extensive research, (1) a substrate, a main flow channel formed on the substrate, a branch from the main flow channel, and a main flow channel again. A microchannel chip for separating fine particles in which the main channel is formed radially from the center of the substrate, including a branch channel connected to the channel, and a capture site formed in the branch channel and larger than the width of the branch channel, or (2) A fine particle including a substrate, a main channel formed on the substrate, and a trapping portion that is larger than the width of the main channel and formed on the main channel, and the main channel is formed radially from the center of the substrate A separation microchannel chip is placed on a rotating means for rotating the microparticle separation microchannel chip, and a sheath liquid inlet, a sample inlet, and a sheath liquid are placed on the surface of the microchannel chip for microparticle separation. Flat for advection accumulation And an advection and accumulation unit including at least a flat part for sample advection and accumulation, and while rotating the rotating means, the sheath liquid is injected from the sheath liquid inlet, and the sample is injected from the sample inlet, A microfluidic chip for separating fine particles, a sheath liquid advection and accumulation plane and a sample advection and accumulation plane are moved relative to each other to generate a meniscus, and the sheath liquid is aspirated by a suction means, so that the intended fine particles The sample is continuously captured by the microfluidic chip for particle separation by capturing the captured microparticles in the microfluidic chip for separation and removing the removed microparticles together with the sheath liquid from the microfluidic chip for separating microparticles. It was newly found that target fine particles can be efficiently captured and separated. Furthermore, when capturing biomaterials containing nucleic acids such as CTC, the biomaterial captured at the capture site is taken out by the fine particle extraction means and transferred to the PCR means for amplifying the nucleic acid, thereby extracting and amplifying the biomaterial in the sample. The present invention has been completed.

  That is, the present invention is to provide a microchannel chip for particle separation, an advection accumulation unit, a particle separation system, and a particle separation method.

  The present invention relates to a microfluidic chip for separating fine particles, an advection and accumulation unit, a system for separating fine particles, and a method for separating fine particles described below.

(1) Substrate, main flow channel formed on the substrate, a branch flow channel branched from the main flow channel and connected to the main flow channel again, and a particulate trapping portion formed in the branch flow channel and larger than the width of the branch flow channel A microchannel chip for separating fine particles, wherein the main channel is formed radially from the center of the substrate.
(2) When the size of the fine particles captured at the capture site is X and the size of the fine particles to be separated / removed is Y, the width F of the main channel and the branch channel is Y <F <X, The fine particle according to (1), wherein the capture region has a width G of 1X <G <10X, and the main channel, the branch channel, and the depth H of the capture site are 1X <H <10X. Microchannel chip for separation.
(3) The microchannel chip for microparticle separation according to (2), wherein the width G is 1X <G <2X and the depth H is 1X <H <2X.
(4) The microchannel chip for separating fine particles according to (2) or (3) above, wherein the width F is Y <F <0.8X.
(5) The above-mentioned (1) to (1), wherein a flow path having a width F and a depth J Y <J is further formed below the main flow path, the branch flow path, and the capture site. The microchannel chip for fine particle separation according to any one of 4).
(6) It includes a substrate, a main channel formed on the substrate, and a capture portion that is larger than the width of the main channel and formed on the main channel, and the main channel is formed radially from the center of the substrate. A microchannel chip for fine particle separation characterized by the above.
(7) When the size of the fine particles captured at the capture site is X and the size of the fine particles to be separated / removed is Y, the width A of the main channel is Y <A <X, and the width of the capture site B is 1X <B <10X, the depth C of the capture site is 1X <C <10X, the depth D of the main channel at the capture site is Y <D, and the depth of the main channel other than the capture site The microfluidic chip for separating fine particles according to (6) above, wherein E is E = C + D.
(8) The microchannel chip for separating fine particles according to (7), wherein the width B is 1X <B <2X and the depth C is 1X <C <2X.
(9) The microchannel chip for microparticle separation according to (7) or (8) above, wherein the width A is Y <A <0.8X.
(10) The circular groove part which connects the front-end | tip part of the said main flow path extended radially is formed on the said board | substrate, The said any one of said (1)-(9) characterized by the above-mentioned. Microchannel chip for fine particle separation.
(11) The microchannel for microparticle separation according to any one of the above (1) to (10), wherein the microparticles captured at the capture site are CTC, and the microparticles to be removed are blood cells. Chip.
(12) An advection and accumulation unit including at least a sheath liquid inlet, a sample inlet, a sheath liquid advection and accumulation plane and a sample advection and accumulation plane.
(13) The advection accumulation unit according to (12), further including a hole for mounting the sheath liquid suction pad and a sheath liquid suction port.
(14) The advection accumulation unit according to (12), further including a hole for sucking the sheath liquid by capillary force and a sheath liquid suction port.
(15) The microchannel chip for separating fine particles according to any one of (1) to (11) above,
The advection accumulation unit according to any one of the above (12) to (14),
Rotating means for rotating the micro-channel chip for separating fine particles, and sheath liquid suction means,
A system for separating fine particles.
(16) The method as described in (15) above, further comprising a fine particle extraction means for taking out the fine particles captured at a capture site formed in the microchannel chip for fine particle separation and a detection means for detecting the fine particles. Fine particle separation system.
(17) The system for separating fine particles according to (16) above, further comprising PCR means for amplifying the nucleic acid.
(18) Any one of the above (15) to (17), further including a magnetic field generator for generating a magnetic field and / or an electric field generator for generating an electric field at a capturing part of the micro-channel chip for microparticle separation The system for separating fine particles according to claim 1.
(19) The microchannel chip for microparticle separation described in any one of (1) to (11) above,
The microparticle chip for microparticle separation is placed on a rotating means for rotating, and a sheath liquid inlet, a sample inlet, a flat section for sheath liquid advection and accumulation, and a sample advection and accumulation are placed on the microchannel chip for microparticle separation. Arranging an advection accumulating unit including at least a plane part for
The microfluidic chip for fine particle separation, the flat portion for sheath liquid advection and accumulation, and the sample advection by injecting a sheath liquid from the sheath liquid inlet and injecting a sample from the sample inlet while rotating the rotating means Relative movement of the flat part for accumulation, capture the target fine particles by the meniscus generated by the relative movement to the capture site formed in the micro-channel chip for fine particle separation,
A fine particle separation method comprising removing fine particles to be removed together with a sheath liquid from a fine particle separation microchannel chip by sucking a sheath liquid with a sheath liquid suction means.
(20) The fine particle separation method according to (19), wherein the fine particles captured at the capture site are CTCs, and the removed fine particles are blood cells.

  The particle separation system according to the present invention rotates a particle separation microchannel chip in which a main channel having a capture site is provided radially from the center of a substrate, and a sample and a sheath liquid from a sample inlet formed in an advection accumulation unit The sheath liquid can be continuously introduced into the microparticle chip for separating fine particles from the injection port. Therefore, for example, only a CTC with a low content can be separated with high accuracy in a short time from whole blood in which red blood cells, white blood cells, and the like are mixed, without pretreatment. Therefore, CTC can be efficiently separated even in samples of patients with very few CTC cells in blood cells, such as follow-up of patients with early cancer metastasis or after cancer treatment. Thus, bedside cancer diagnosis can be performed by simple operation.

  Since the microfluidic chip for particle separation used in the particle separation system of the present invention does not use an anti-EpCAM antibody, even CTC-negative or weakly positive tumor cells can be reliably detected. In addition, the microchannel chip for particle separation of the present invention allows small cells such as red blood cells and white blood cells to flow out of the chip with a sheath liquid, and large cells such as CTCs are captured at a capture site provided in the channel. Therefore, unlike a conventional filter type device, the device is not clogged and can be processed continuously.

  When the microparticles to be separated are biomaterials containing nucleic acids such as CTC, the biomaterial captured at the capture site is extracted by the microparticle extraction means and transferred to the PCR means for amplifying the nucleic acid, thereby extracting and amplifying the biomaterial in the sample. Can be automated.

  Further, a rotating means is attached to a known cell picking system in which cells on the plate are automatically extracted by the extracting means and transplanted to the PCR means for amplifying the nucleic acid. By using a channel chip, a conventional apparatus can be used as a system for separating fine particles.

  Furthermore, since the microchannel chip for particle separation used in the system for particle separation of the present invention can be mass-produced using a semiconductor formation process, the cost of CTC inspection can be greatly reduced.

FIG. 1 is a schematic view showing an example of a microchannel chip for separating fine particles of the present invention. FIG. 2 is an enlarged view of the main flow path 12, the branch flow path 13 branched from the main flow path 12 and connected again to the main flow path 12, and the capture site 14 formed in the branch flow path 13. FIG. 3 is a diagram showing another embodiment of the microchannel chip for separating fine particles of the present invention. FIG. 4 is a view showing another embodiment of the microchannel chip for separating fine particles of the present invention. FIG. 5 is a diagram showing another form of the main channel 20 and the capture site 21 of the microchannel chip for particle separation of the present invention. FIG. 6 is a flowchart showing a procedure for producing the microchannel chip for separating fine particles of the present invention. FIG. 7 is a diagram showing an outline of the fine particle separation system of the present invention. FIG. 8 is a diagram illustrating the principle of meniscus generation. FIG. 9 is a drawing-substituting photograph showing the configuration of the advection accumulation unit 50 and the positional relationship when the advection accumulation unit 50 and the chip 1 are set in the system 30. FIG. 10 is a drawing-substituting photograph that is an enlarged view of the advection stacking unit 50 of the system 30 from above. FIG. 11 is a drawing-substituting photograph from the side of the advection stacking unit 50. FIG. 12 is a drawing-substituting photograph showing the entire particulate separation system of the present invention. FIG. 13 is a drawing-substituting photograph showing a state in which fine particles captured at a capture site are taken out after the fine particles are separated using the system of the present invention. FIG. 14 is a drawing-substituting photograph showing the appearance of the chip obtained in Example 1. FIG. 15 is a drawing-substituting photograph showing the appearance of the advection accumulation unit obtained in Example 3. FIG. 16 is a photograph substituted for a drawing, and is an enlarged photograph of a hole portion for sucking the sheath liquid of the advection accumulation unit obtained in Example 4 by capillary force.

  Hereinafter, the micro-channel chip for separating fine particles, the advection collecting unit, the system for separating fine particles, and the method for separating fine particles will be described in detail.

  FIG. 1 shows an example of a micro-channel chip for separating fine particles according to the present invention (hereinafter sometimes simply referred to as “chip”). The chip 1 is formed from a substrate 10 and a center 11 of the substrate 10. It includes at least a main channel 12 formed radially, a branch channel 13 that branches from the main channel 12 and connects to the main channel 12 again, and a capture site 14 that is formed in the branch channel 13 and captures target particles. It is out. In addition, a circular groove portion 15 that connects the tip portions of the main flow channels 12 may be formed at the tip portions of the main flow channels 12, and removed from the sheath liquid and the sample injected from the advection accumulation unit described later. The fine particles to be guided may be guided to the groove portion 15 so that they can be sucked and removed from the groove portion 15 via the portion 18 in contact with the sheath liquid of the sheath liquid suction means.

  In the present invention, the term “fine particles” means particles that can be dispersed in a liquid, and the form of the particles may be either single or aggregated. The size of the fine particles is not particularly limited as long as the meniscus principle can be applied, and may be about 1 mm or less. Any non-biological material or biomaterial can be used as long as it satisfies the above conditions. FIG. 1 shows an example in which the fine particles to be captured are CTC and the fine particles to be removed are blood cells.

  The chip 1 is not particularly limited in shape and the like as long as it can be mounted and rotated on a rotating means of a particle separation system described later, but a circular shape is preferable from the viewpoint of space and convenience of handling. Further, it is preferable that the chip 1 is not displaced after being placed on the rotating means, and the substrate 10 may be formed of a material having high adhesion to the material forming the rotating means. A convex portion may be formed on the substrate 10 and a concave portion may be formed on the substrate 10. Examples of the material of the substrate 10 include, but are not limited to, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), plastic made of hard polyethylene, glass, and the like.

  When the fine particles are separated using the chip 1 of the present invention, an area corresponding to a sheath liquid advection and accumulation plane portion (hereinafter also referred to as “sheath liquid plane portion”) of an advection and accumulation unit described later. Thus, a meniscus is generated in the sheath liquid, and the outer peripheral side of the area where the meniscus is generated in the sheath liquid corresponds to a sample advection and accumulation plane part (hereinafter also referred to as “sample plane part”). A meniscus is generated in the sample in the area to be used. Therefore, the main flow path 12 may be formed by the portion 16 where the capture site 14 is not formed and the portion 17 where the capture site 14 is formed. The capture site 14 may be formed over the entire length of 12.

  FIG. 2 is an enlarged view of the main flow path 12, the branch flow path 13 branched from the main flow path 12 and connected again to the main flow path 12, and the capture site 14 formed in the branch flow path 13. The capture site 14 formed in the branch channel 13 may be formed in the middle of the branch channel 13, or as shown in (1) to (4) of FIG. The part may be in contact with the main flow path 12. FIG. 2 (1) shows an example in which the capture part 14 is circular, a part of the circular capture part 14 is in contact with the main flow path 12, and (2) is the flow direction of the main flow path 12. An example in which a substantially square capturing part 14 having a smooth corner is formed in the vertical direction, one side of the substantially square capturing part 14 is in contact with the main flow path 12, and (3) is the main flow path 12. An example is shown in which a substantially square capturing part 14 having a smoothed corner is formed in the flow direction and the flow direction of the main flow path 12 is changed by about 90 degrees, and one side of the substantially square capturing part 14 is the main stream. (4) shows an example in which the capture part 14 formed in the branch flow path 13 is substantially elliptical, and a part of the elliptical capture part 14 is in contact with the main flow path 12. doing. In addition, the shape of the capture | acquisition site | part 14 described in said (1)-(4) is only a mere illustration, For example, as long as microparticles | fine-particles can be capture | acquired, such as a hexagon or an octagon polygon, it is another shape. Good. In addition, one capture site 14 may be formed in one branch channel 13, or one capture site 14 may be formed in a plurality of branch channels. Further, the main flow path 12 and the branch flow path 13 may be straight lines or curved lines, or may be broken lines that change directions in the middle of the straight lines.

  The width and depth of the main flow path 12, the branch flow path 13, and the capture site 14 may be set as appropriate according to the size of the fine particles to be captured. When the size of the fine particles to be removed is Y, the width F of the main channel 12 and the branch channel 13 is preferably Y <F <X. Width G of the capture site 14 (Note that the width G of the capture site 14 is a diameter when the shape of the capture site 14 is circular, and a polygon or ellipse such as one side, 6 or octagon when it is square) Means the shortest length passing through the center of the polygon or ellipse.) Is preferably 1X <G <10X. Moreover, it is preferable that the depth H of the main flow path 12, the branch flow path 13, and the capture part 14 be the same depth in the range of 1X <H <10X. If G and H are set to 10 or more, the width of the branch flow path 13 with respect to the size of the capture site 14 becomes too small, and the processing capacity for separating and removing fine particles is not preferable. In the case where a plurality of branch flow paths 13 connected to the capture site 14 are provided, G and H may be 10X or more because the processing capacity for separating fine particles is improved. The above numerical value is a range in the case where a plurality of fine particles are captured at each capture site 14 such as concentration of captured fine particles, but is captured at each capture site 14 in order to analyze the captured fine particles. When the number of fine particles is one, it is preferable that 1X <G <2X and 1X <H <2X. Further, when the shape of the microparticles captured at the capturing site 14 is likely to change, such as a living cell, the shape of the living cell captured at the capturing site 14 is changed by the fluid force so that it does not flow out to the branch channel 13. Therefore, the width F of the branch channel 13 may be appropriately selected according to the change rate of the shape of the living cell. For example, in the case of CTC, it is preferable to satisfy Y <F <0.8X. The width F of the main channel and the branch channel may be the same or different as long as they are within the range of F. Moreover, the depth H of the main flow path 12, the branch flow path 13, and the capture | acquisition site | part 14 may be the same or different, if it is in the range of said H. Furthermore, in the present invention, a flow path may be formed below the capture site 14. In that case, a flow path having a width F and a depth J of Y <J may be further provided below the main flow path 12, the branch flow path 13 and the capture site 14, and FIG. Sectional drawing of the capture | acquisition part 14 part at the time of providing is represented.

  For example, when CTC is captured from whole blood and cells such as red blood cells and white blood cells other than CTC are removed, the width F of the main flow path 12 and the branch flow path 13 is smaller than the diameter (15 to 30 μm) of the CTC. What is necessary is just to make it larger than a cell (about 7 micrometers), and 8-12 micrometers is preferable. On the other hand, since the capture site 14 needs to capture CTC, the width of the capture site 14 needs to be larger than the diameter of the CTC. For example, when the capture site 14 shown in FIG. 2 (1) is circular, the diameter G is preferably 16 to 36 μm, and when the capture site 14 shown in FIGS. 2 (2) and (3) is substantially square. The side G is preferably 16 to 36 [mu] m, and the short axis G is preferably 16 to 36 [mu] m when the capture site 14 shown in FIG. In addition, when the shape of the capture | acquisition site | part 14 is a polygon, the length of the shortest line | wire which passes along a center should just be 16-36 micrometers as above-mentioned.

  In the case of the chip 1 including the capture site shown in FIGS. 2 (1) to 2 (4), CTC is trapped in the capture site 14, but the sheath liquid described later flows through the main flow path 12, and therefore CTC is the flow of the sheath liquid. No physical strength. Furthermore, many blood cells smaller than CTC flow through the main flow path 12 together with the sheath liquid described later, and the blood cells that have flowed into the capture site 14 pass again through the branch flow channel 13 extending further from the capture site 14. It can be returned to the main flow path 12. Therefore, in the chip 1 shown in FIG. 2, since the main flows of cells other than CTC and CTC are different, it is not essential to form a flow path below the capture site 14. In the case where no flow path is provided below, the depth of the main flow path 12, the branch flow path 13 and the capture portion 14 is preferably 16 to 36 μm. When a flow path is provided below the capture site 14, the depth of the flow path below the capture site 14 is preferably 8 to 20 μm. For the main flow channel 12 and the branch flow channel 13 other than the capture site 14, the capture site 14 is used. And the total depth of the flow paths.

  The above example is the size when separating CTC from whole blood, for example, when separating gastric cancer cell mass (25-50 μm) from blood cells or mesothelial cells (about 7-15 μm) in the peritoneal lavage fluid The width F of the main flow path 12 and the branch flow path 13 may be 8 to 24 μm, and the width G and the depth H of the main flow path 12, the branch flow path 13 and the capture site 14 may be 26 μm to 60 μm.

  The width and depth of the groove 15 in FIG. 1 are not particularly limited as long as the sheath liquid can flow from the main flow path 12, but the width of the groove 15 may be larger than the portion 18 that contacts the sheath liquid of the sheath liquid suction means. desirable. The depth of the groove 15 is preferably the same as that of the main channel 12 so that the sheath liquid flows smoothly.

  FIG. 3 is a view showing another embodiment of the micro-channel chip for separating fine particles according to the present invention. The groove 15 is not limited to the shape shown in FIG. 1, and for example, the tip portion of each main channel 12 extending radially. The width of the circular shape connecting the two is made smaller than the portion 18 of the sheath liquid suction means that contacts the sheath liquid, and the part of the portion 18 of the sheath liquid suction means that contacts the sheath liquid overlaps the groove 15. Good. In addition, it is not essential to form the groove portion 15. For example, as shown in FIG. 4, a portion 18 that contacts the sheath liquid of the sheath liquid suction unit may be in contact with the distal end portion of the main flow path 12. .

  FIG. 5 (1) shows another form of the main channel 20 and the capture site 21. In this embodiment, the capture site 21 is arranged on the main channel 20 without forming a branch channel. The shape of the capture site 21 is not particularly limited as long as it can capture the target fine particles, such as a circle, a substantially square, a polygon such as a hexagon or an octagon, and an ellipse, like the capture site 14. The definition of the width of the capture site is the same as that of the capture site 14.

  5 (2) is a cross-sectional view of the position aa where the capture site 21 is not formed on the main channel 20 of FIG. 5 (1), and FIG. 5 (3) is the capture site 21 formed on the main channel 20. It is sectional drawing of the position bb made. The width and depth of the main channel 20 and the width and depth of the trapping part 21 may be set as appropriate according to the size of the particulates to be trapped. In this embodiment, the flow of the lower part shown in FIG. Since the fine particles to be removed in the passage are discharged using the sheath liquid and the target fine particles are captured at the upper capture site 21, the size of the fine particles captured at the capture site 21 is separated and removed. When the size of the fine particles is Y, the width A of the main flow path 20 is Y <A <X, the width B of the capture site 21 is 1X <B <10X, and the depth C of the capture site 21 is 1X < It is preferable that C <10X, and the depth D of the flow path in the capture part 21 is Y <D, and the depth E of the flow path other than the capture part 21 is preferably E = C + D. If B and C are 10 or more, the width A of the main flow path 20 with respect to the size of the capture site 21 becomes too small, and the processing capacity for separation / removal of fine particles is not preferable. The above-mentioned 1X <B <10X and 1X <C <10X are examples in which the lower main flow path 20 of the capture site 21 is one example, but a plurality of main flow paths 20 are provided at the lower level of the capture site 21. B and C may be 10X or more as long as the processing capacity for fine particle separation is increased.

  Similarly to the trapping site 14, when one particle is captured at each trapping site 21, 1X <B <2X, 1X <C <2X, and when the target particle is CTC, The width A of the main channel 20 is preferably Y <A <0.8X.

  When capturing CTC from whole blood and removing blood cells such as red blood cells and white blood cells other than CTC, the width A of the main flow path 20 is preferably 8 to 12 μm, and the width B and depth of the capture site 21 are the same as described above. The length C is preferably 16 to 36 μm.

  Moreover, in the capture part 21, since CTC is captured by the upper stage part shown by FIG. 5 (3), and a blood cell is removed by a sheath liquid in a lower part part, the main flow path of the part in which the capture part 21 is formed The depth D of the lower portion 20 needs to be at least larger than the diameter of blood cells, and it is preferable that one or more blood cells can be removed at the same time. Therefore, the depth D is preferably 8 to 20 μm. The depth E of the main channel 20 in which the capture site 21 is not formed may be C + D.

  The above example is the size when separating CTC from whole blood, for example, when separating gastric cancer cell mass (25-50 μm) from blood cells or mesothelial cells (about 7-15 μm) in the peritoneal lavage fluid The depth D of the main flow path 20 may be 8 to 24 μm, and the width B and the depth C of the capture site 21 may be 26 μm to 60 μm.

  The number of the main flow paths 12 and 20 formed in the chip 1 is at least 2 or more, and the number of main flow paths to be formed is particularly limited as long as it does not interfere with the capturing portions 14 and 21 formed in the adjacent main flow paths. There is no. Moreover, the number of the capture | acquisition parts 14 and 21 formed in each main flow path 12 and 20 is at least 1 or more, What is necessary is just to adjust suitably in consideration of the length of the main flow paths 12 and 20, the width | variety of the capture | acquisition part to form. Moreover, the shape of the main flow paths 12 and 20 may be a straight line, or may be a curve as shown in FIG. In the present invention, the meniscus is generated by relatively moving the tip 1 and the advection integrated unit. However, since the tip 1 rotates around the center 11 of the substrate 10, the sheath liquid plane portion and the sample plane The line where the meniscus occurs in the part is not a straight line but a curved line. Therefore, it is preferable that the line where the meniscus is generated coincides with the lines of the main flow paths 12 and 20, and the main flow paths 12 and 20 are preferably curved so as to secure a large number of capture sites. What is necessary is just to adjust the curve | curvature degree of a curve suitably, considering the rotational speed of the chip | tip 1, etc. FIG.

  The chip 1 can be manufactured using a photolithography technique. FIG. 6 is a flowchart showing an example of a manufacturing procedure. As shown in FIG. 5, a procedure using a two-stage exposure technique in the case of manufacturing a two-stage chip in which a flow path is provided below the capturing portion 21. Is shown.

First, the silicon substrate is organically cleaned with an ultrasonic cleaner and baked. Next, it is manufactured by the following procedure shown in FIG.
1. A negative photoresist (SU-8) is spin-coated on a Si substrate and prebaked on a hot plate.
2. The exposure is performed using a photomask such as a chrome mask for forming the capture site, the main flow path portion other than the capture site and the groove.
3. After performing post-exposure baking on a hot plate and developing with a developer (PM thinner or the like), rinse with ultrapure water, and then dry with a spin dryer or the like.
4. Spin coat the second stage SU-8 negative photoresist and pre-bake.
5). The exposure is performed using a lower main channel of the capture site, a main flow channel portion other than the capture site, and a chrome mask for forming a groove.
6). Post-exposure baking, development and rinsing are performed to form a pattern.
7). The formed pattern is transferred to polydimethylsiloxane (PDMS).
8). PDMS is separated from the formed pattern.
9. PDMS surface is hydrophilized.

  The organic cleaning is not particularly limited as long as it is a cleaning agent generally used in the semiconductor manufacturing field, such as acetone and ethanol. In the above procedure, an example in which Si is used as a substrate has been shown. However, the material of the substrate is not particularly limited as long as it is a material generally used in the photolithography technical field. For example, silicon carbide, Examples include sapphire, gallium phosphide, gallium arsenide, gallium phosphide, and gallium nitride. The negative photoresist is not limited to SU-8. For example, a commonly used resist such as PMER or AZ can be used as long as it is a positive photoresist.

  The above procedure is a procedure in the case of forming the main channel 20 below the capture site 21, but the main channel 12, the branch channel 13, the capture site 14 and the groove 15 as shown in FIG. In this case, it can be produced in the same procedure as described above except that the masks of those shapes are used and the procedure of providing the second resist layer in the procedure “4.-6.” Is omitted.

  Since the surface of the chip 1 is hydrophilized, it is possible to prevent bubbles from entering the groove when a liquid is injected into the microchip. Examples of the hydrophilization treatment method include plasma treatment, surfactant treatment, PVP (polyvinylpyrrolidone) treatment, photocatalyst and the like. For example, by subjecting the chip surface to plasma treatment for 10 to 30 seconds, hydroxyl groups are formed on the chip surface. Can be introduced.

  FIG. 7 is a diagram showing an outline of a fine particle separation system (hereinafter sometimes simply referred to as “system”) 30 according to the present invention. Rotating means 40 for mounting and rotating the chip 1 and an advection accumulation unit 50, which includes at least a sheath liquid suction means (not shown), and may include a fine particle extraction means 70 when taking out the fine particles captured at the capture site. Furthermore, when the captured microparticles are biomaterials containing nucleic acids and PCR is performed after separation, PCR means 80 may be included, and FIG. 7 shows an example in which wells used for PCR are arranged. Further, a sheath liquid injection 61 for feeding a sheath liquid to the sheath liquid inlet of the advection accumulation unit described later and a sample injection 62 for feeding a sample to the sample inlet may be provided. The sheath liquid injection 61 and the sample injection 62 are not particularly limited as long as the liquid can be fed, and may be manually fed using a syringe or a commercially available constant flow pump or the like. Moreover, you may drop by weight from a bottle etc., without using motive power for liquid feeding, In that case, you may provide the clamp used for the flow volume adjustment of drip, and may adjust a flow volume.

  FIG. 8 is a diagram for explaining the principle of meniscus generated when the chip 1 and the advection accumulating unit 50 are relatively moved by the rotating means 40. Capillary force between fine particles existing at the gas-liquid interface, called advection accumulation method. A technique is used in which fine particles are arranged in a closely packed structure by utilizing (especially called lateral capillary force). When the tip 1 and the sheath liquid plane portion 53 and the sample plane portion 54 of the advection and accumulation unit 50 are relatively moved, the meniscus of the suspension in which the fine particles are dispersed in the solution is interposed between the tip 1 and the sheath liquid plane portion 53 and the sample plane portion 54. In the tip of the meniscus, as shown in FIG. 8, a portion where the fine particles protrude from the solution is formed. At the point where the head protrudes, the force pressed down by the interfacial tension and gravity is generated along with the meniscus while being generated in the fine particles, the fine particles are pressed against the chip 1, and the target fine particles are formed in the main flow path. Captured at the capture site.

  The rotating means 40 is not particularly limited as long as the chip 1 can be placed and rotated. For example, a driving means such as a motor is provided under the rotatable disk on which the chip 1 can be placed to rotate the disk. You just have to do it. In addition, since it is preferable to make the magnitude | size of the meniscus to generate | occur | produce constant, it is preferable that a drive means is what can carry out rotation control of the disk at a fixed speed.

  FIG. 9 is a photograph showing the configuration of the advection accumulation unit 50 and the positional relationship when the advection accumulation unit 50 and the chip 1 are set in the system 30. The advection accumulation unit 50 includes at least a sheath liquid inlet 51, a sample inlet 52, a sheath liquid flat portion 53, and a sample flat portion 54. Further, the advection accumulation unit 50 of the present invention is equipped with a sheath liquid suction pad or a hole 55 for sucking the sheath liquid by capillary force, and a sheath liquid suction for connecting to the hole 55 and connecting to a sheath liquid suction device (not shown). A mouth 56 may be provided. Since the sheath liquid plane portion 53 and the sample plane portion 54 generate a meniscus by moving relative to the tip 1, the surface facing the tip 1 is preferably a planar shape. Further, a meniscus is generated between the sheath liquid plane part 53 and the sample plane part 54 and the chip 1, and it is not necessary to generate a meniscus other than the sheath liquid plane part 53 and the sample plane part 54. The sheath liquid flat portion 53 and the sample flat portion 54 need to be thicker than other portions and provided with a step. On the other hand, with respect to the hole 55, when a sheath liquid suction pad is attached to the hole 55, the amount of protrusion from the hole 55 where the sheath liquid suction pad is attached can be adjusted so that the sheath liquid suction pad contacts the sheath liquid. Therefore, the hole 55 may be formed on the same surface as the other portions, or a flat surface portion in which the hole 55 is provided at the same height as the sheath liquid flat surface portion 53 and the sample flat surface portion 54 is formed. The hole 55 may be formed in the part, or the hole 55 is provided corresponding to the groove part 15, and therefore the flat part provided with the hole 55 may be thicker than the sheath liquid flat part 53 and the sample flat part 54. Further, when the hole 55 is used as a hole for sucking the sheath liquid by capillary force, the hole 55 is preferably disposed so as to substantially contact with the tip 1 without causing friction. As will be described later, since the tip 1, the sheath liquid plane part 53, and the sample plane part 54 are set at positions 500 to 1000 μm apart, the plane part forming the hole 55 for sucking the sheath liquid by capillary force is the sheath part. It is preferable to form it thicker by about 500 to 1000 μm than the liquid plane portion 53 and the sample plane portion 54.

  Further, as shown in FIG. 9, the sheath liquid inlet 51 may be formed in the sheath liquid plane portion 53, and the sample inlet 52 may be formed in the sample plane portion 54. When moving, the sheath liquid inlet 51 may be formed on the upstream side of the sheath liquid flat portion 53, and the sample inlet 52 may be formed on the upstream side of the sample flat portion 54. When the chip 1 and the advection integration unit 50 are set in the system 30, if there is a portion 16 in which the capture site 14 is not formed on the chip 1, the sheath liquid plane portion 53 is set corresponding to the portion 16, and the sample The flat surface portion 54 is set corresponding to the portion 17 where the capturing site is formed. Therefore, the shape of the sheath liquid plane portion 53 is not particularly limited as long as the portion 16 and the portion 16 correspond to each other. However, the sheath liquid plane portion 53 is preferably formed in a shape including two arcs concentric with the portion 16. More preferably, the two arcs have the same width R1 as that of the portion 16. The length of the arc may be adjusted as appropriate. Similarly, the sample plane part 54 is not particularly limited as long as the part 17 and the part 17 correspond to each other. However, the sample flat part 54 is preferably formed into a shape including two arcs concentric with the part 17. More preferably, the width of the two arcs is the same width R2 as the portion 17. The length of the arc may be adjusted as appropriate. In the present embodiment, the hole 55 for mounting the sheath liquid suction pad is disposed so as to correspond to the groove portion 15. Therefore, if the hole 55 can be mounted with the sheath liquid suction pad and is smaller than the groove portion 15, the width and shape of the hole 55. Although there is no restriction | limiting in particular, It is preferable that it is a circular-arc-shaped hole concentric with the groove part 15. FIG. Further, the number and position of the sheath liquid suction port 56 formed as long as it communicates with the hole 55 is not particularly limited, and may be adjusted as appropriate.

  FIG. 10 is a photograph of an enlarged view of the advection accumulation unit 50 of the system 30 from above, and the advection accumulation unit 50 is attached to a height adjusting means 57 for keeping a distance from the chip 1 mounted on the rotation means 40. It has been. The height adjusting means 57 is not particularly limited as long as the height of the advection stacking unit can be adjusted by a screw or the like. Further, when the advection accumulation unit 50 is used, the sheath liquid injection port 51 is a sheath liquid injection 61, the sample injection port 52 is a sample injection 62, and the sheath liquid suction port 56 is illustrated via a tube 58 such as silicon. What is necessary is just to connect with the sheath liquid suction apparatus which does not.

  The sheath liquid suction means is not particularly limited as long as the sheath liquid can be sucked from the groove portion 15 of the chip 1 or the main flow paths 12 and 20. For example, the sheath liquid may be sucked by contacting a sheath liquid suction pad such as cloth, cotton, sponge, chamois or the like directly or through the hole 55 of the advection and accumulation unit 50 with the groove 15 or the main flow paths 12 and 20.

  Further, instead of inserting the sheath liquid suction pad into the hole 55, the width of the hole 55 is adjusted to a width at which the capillary force is generated, and the hole 55 is brought into contact with the groove portion 15 or the main flow paths 12 and 20 by the capillary force. The sheath liquid may be aspirated. In that case, since it is necessary to pass at least the fine particles removed together with the sheath liquid, the width of the hole 55 should be at least 8 μm or more when the sample is whole blood, and 10 μm or more in order to increase the processing capability. More preferred. On the other hand, the width of the hole 55 is not particularly limited as long as a capillary force is generated, and may be appropriately adjusted in consideration of the amount of sheath liquid to be sucked, the capillary force, etc. For example, a width of about 200 μm may be provided.

  As the sheath liquid suction means, in addition to the above-described examples, a suction device such as a suction pump is used, and the suction port connected to the suction device is brought into contact with the groove portion 15 or the main flow paths 12 and 20 to suck the sheath liquid. Good. In the present invention, the flow rate of the sheath liquid flowing through the main flow paths 12 and 20 is adjusted by the suction force of the sheath liquid suction means. For this reason, the amount of sheath liquid and sample used for separating fine particles increases, and if the sheath liquid suction pad or the hole for generating capillary force is saturated with the sheath liquid, the suction speed of the sheath liquid may not be stable. . Therefore, the sheath liquid suction means may be used in combination so that the flow rate of the sheath liquid can be kept more stable. For example, one end of a sheath liquid suction pad such as cotton is brought into contact with the sheath liquid to absorb the sheath liquid, and is absorbed into the sheath liquid suction pad from the other end of the sheath liquid suction pad using a suction device such as a suction pump. The sheath liquid may be aspirated. Further, the sheath liquid may be sucked by a suction device from the suction port communicating with the hole 55 while the sheath liquid is sucked by the capillary force from one end of the hole 55 that can suck the sheath liquid by the capillary force. Further, the advancing and accumulating unit 50 is not provided with the hole 55, and the suction port connected to a suction device such as a sheath liquid suction pump is inserted into a suction pad of a size or a suction port where capillary force is generated. Alternatively, the main channels 12 and 20 may be contacted.

  FIG. 11 is a photograph taken from the side of the advection and accumulation unit 50, showing a state in which a sheath liquid suction pad 59 is attached to the hole 55 and a tube 58 such as silicon is connected to the sheath liquid suction port 56. . The lower end of the sheath liquid suction pad 59 is in contact with the sheath liquid in the groove portion 15 of the chip 1, and the sheath liquid sucked by the sheath liquid suction pad 59 passes through a tube 58 made of silicon or the like connected to the sheath liquid suction port 56. Are sucked by a suction device such as a suction pump connected to the sheath liquid and removed from the sheath liquid suction pad 59.

  The material constituting the advection stacking unit 50 is not particularly limited as long as it does not react with the sample or the sheath liquid, such as resin such as acrylic, nylon, and Teflon (registered trademark), or glass. The advection accumulation unit 50 can be produced by cutting using a cutting tool such as a drill and an end mill, or by producing a mold having the shape of the advection accumulation unit 50 and injection molding. In addition, the advection accumulation unit 50 in the present invention includes a sheath liquid injection port 51, a sample injection port 52, a sheath liquid flat surface portion 53 and a sample flat surface portion 54, and a hole 55 and a sheath liquid suction port 56 that are formed as necessary. As long as it is included, it may be formed of a single member or may be a combination of separately prepared members.

  The particulate extraction means 70 is not particularly limited as long as it can extract the particulates captured at the capture sites 14 and 21, and examples thereof include a manipulator provided with a cell suction means. The fine particle extraction means 70 may be automatically collected by a manipulator interlocked with a detection means 90 for detecting target fine particles captured by the capture sites 14 and 21. For example, Japanese Patent Application Laid-Open No. 2010-29178. A cell picking system as described in the publication can be used. Further, as the detection means 90, when the captured fine particles are CTC, a fluorescence microscope or the like capable of observing CTC fluorescently stained with a CTC-specific antibody such as an anti-EpCAM antibody labeled with FITC or PE is used. Can do. In addition, when performing bright field observation using an optical microscope, CTC detection can be performed using morphological features such as intracellular nuclei and cytoplasm as an index by performing Papanicolaou staining or Giemsa staining.

  When the fine particles extracted by the fine particle extraction means 70 are biomaterials containing nucleic acids and PCR is performed after extraction, the extracted biomaterials may be transferred to the PCR means 80. The PCR means may be a known device.

  Next, a fine particle separation method using the system 30 will be described. FIG. 12 is a photograph showing the entire system 30. The chip 1 is placed on the rotating means 40, and the sheath liquid plane portion 53 and the sample plane portion 54 of the advection accumulation unit 50 are located at a position 500 to 1000 μm away from the chip 1. Is arranged to be located. When the distance between the tip 1 and the sheath liquid plane portion 53 and the sample plane portion 54 of the advection stacking unit 50 is 500 μm or less, the amount of sample liquid introduced decreases and the processing capacity decreases, and when it is 1000 μm or more, the meniscus force decreases. However, sufficient separation cannot be obtained.

  The sheath liquid is not particularly limited as long as it does not damage the fine particles to be separated. When whole blood is used as a sample, various buffer solutions such as phosphate buffered saline (PBS), Tris buffer, There is no particular limitation as long as it is a commonly used sheath fluid such as simulated body fluid (SBF) and general cell culture fluid. In the example shown in FIG. 12, the sheath liquid stored in the bottle 61 is dropped by gravity, and the flow rate is adjusted using a clamp at that time. The sample is put in the syringe 62 and dropped by gravity. The sample may be injected as it is in the case of whole blood, or in the case of separating / extracting the desired microparticles from a non-biological material or a mixture of biological materials, the sample is suspended in a solution having the same composition as the sheath liquid. Also good.

  Since the sheath liquid plane part 53 and the sample plane part 54 are different in radial position, the relative speeds of the two when the chip 1 is rotated are different, but both of them have a relative speed of 50 to 1500 μm / s. It is preferable to rotate the chip 1 so as to be in the range of 60 to 1000 μm / s. When it is slower than 50 μm / s, the processing time becomes longer and the processing capacity is lowered, and when it is faster than 1500 μm / s, fine particles are not captured and the separation efficiency is reduced. In the system shown in FIG. 12, the advection accumulation unit 50 is fixed and the chip 1 is rotated, but the chip 1 may be fixed and the advection accumulation unit 50 may be rotated.

  The amount of sample to be injected is preferably 1.0 to 10.0 μl / s. When the amount is less than 1.0 μl, the processing time becomes long and the processing capacity is lowered. The flow rate of the sheath liquid flowing through the main channel 12 is preferably 10 to 1000 μm / s, more preferably 100 to 700 μm / s, although it varies depending on the depth and width of the main channel and the shape and size of the capturing site. If it is slower than 10 μm / s, the separation efficiency is reduced due to a decrease in the ability to wash blood cells, and if it is faster than 1000 μm / s, the captured CTC is sucked and the separation efficiency is reduced. The flow rate of the sheath liquid may be adjusted by the suction force of the sheath liquid suction means. The sheath liquid may be injected so as to be the same as the amount of sheath liquid to be sucked.

  FIG. 13 shows how the fine particles captured at the capture site are taken out after the fine particles are separated using this system. In order to facilitate movement of the fine particle extraction means 70 when taking out the fine particles, the height adjusting means 57 to which the advection accumulating unit 50 is attached can be moved in the X, Y, and Z axis directions. It may be possible to be separated from. The target fine particles detected by the detection means 90 are sucked by the fine particle extraction means 70 and transferred to the well of the PCR means 80.

  The system 30 of the present invention may be provided with a magnetic field generator and / or an electric field generator for increasing the capture efficiency of fine particles. For example, the disk part of the rotating means 40 corresponding to the part 17 where the capturing part of the chip 1 is formed may be formed of a permanent magnet or the like, or the magnetic field generator is provided below the disk part corresponding to the part 17. As an alternative, a permanent magnet or electromagnet may be installed to generate a magnetic field potential field. After magnetizing particles to be captured, such as CTC that specifically adsorbs magnetic particles labeled with EpCAM antibody or the like, or CTC that adsorbs magnetic particles nonspecifically (taken from endocytosis), By using the fine particle separation system of the present invention, it is possible to accurately separate target fine particles from other fine particles not magnetically labeled.

  In addition, an electric field generator is provided on the disk portion corresponding to the capture site or below the disk portion to generate an electric field potential field (in a non-uniform electric field), and the polarization of the CTC and the surrounding medium and the electric field gradient It is also possible to assist the capture of CTC using the electrostatic force (Coulomb force) that is generated.

  The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.

[Preparation of microchannel chip for fine particle separation]
<Example 1>
First, the silicon substrate was subjected to organic cleaning with an ultrasonic cleaner at 45 kHz for 5 minutes in order of acetone, ethanol, and ultrapure water, and baked at 145 ° C. for 20 minutes. Next, SU-8 was spin-coated on a silicon substrate, and prebaked at 95 ° C. for 30 minutes on a hot plate. Next, after exposure using a chromium mask in which the trapping portion 14 has the shape shown in FIG. 2 (4) and the groove 15 has the shape shown in FIG. 1, post exposure baking is performed on a hot plate at 95 ° C. for 2 minutes to obtain PM thinner. And developed. After development, the substrate was rinsed with ultrapure water, dried with a spin dryer or the like. The formed pattern was transferred to polydimethylsiloxane (PDMS), and after transfer, both were separated, and the PDMS surface was hydrophilized by plasma treatment (frequency 50 kHz, output 700 W, 30 seconds).

  FIG. 14 (1) is a photograph showing the appearance of the chip obtained in Example 1. The center 11 of the chip is a hole with a diameter of 5 mm, the width of the portion 16 where the capture site is not formed in the main channel is 10 mm, and the main channel The width of the portion 17 where the capturing site is formed is 10 mm, and the width of the groove 15 is 10 mm. Moreover, the depth of the main flow path, the capture site, and the groove was about 30 μm. FIG. 14 (2) is an enlarged photograph of the main channel, the branch channel, and the capture site. The width F of the main channel and the branch channel is about 8 μm, and the length G of the shortest line passing through the center of the capture site is It was about 30 μm. The distance between the center of the main channel and the center of the adjacent main channel was about 66 μm, the number of formed main channels was 736, and the number of capture sites was 62,651.

<Example 2>
As shown in FIG. 4, a chip was fabricated in the same procedure as in Example 1 except that the main channel was extended without forming a groove.

[Production of advection integrated unit]
<Example 3>
The advection accumulation unit was manufactured by cutting using an acrylic material and a cutting tool (a drill and end mill manufactured by Mitsubishi Materials Corporation). FIG. 15 is a photograph of the advection integrated unit produced in Example 2 taken from the direction facing the chip 1. The sizes of the sheath liquid inlet 51, the sample inlet 52, and the sheath liquid inlet 56 are 2 mm in diameter. there were. The width of the two arcs of the sheath liquid plane portion 53 is 8 mm, the length of the short arc is 4.1 mm, the width of the two arcs of the sample plane portion 54 is 10 mm, and the length of the short arc is 9.2 mm. there were. Further, the width of the hole 55 was 4 mm, and the length of the arc of the hole 55 was 46 mm.
<Example 4>
Advection and accumulation in the same procedure as in Example 3 except that the width of the hole 55 for sucking the sheath liquid by capillary force is 150 μm, and the plane part where the hole 55 is provided is about 500 μm thicker than the sheath liquid plane part and the sample plane part A unit was made. FIG. 16 is an enlarged photograph of the hole 55 portion for sucking the sheath liquid of the advection and accumulation unit produced in Example 4 by capillary force.

[Preparation of blood sample]
10 × 10 ⁵ green fluorescent protein (GFP) -introduced MKN-45 (human stomach cancer) cells were added to 20 μl of collected human blood, and a blood sample simulating the blood of a cancer patient (whole blood including CTC) Was made. The average particle size of the cancer cells was 25 μm.

<Example 5>
[CTC separation experiment from blood sample]
The microfluidic chip for separating fine particles produced in Example 1 is placed on the rotating means 40 of the system 30, and the distance between the advancing and integrating unit produced in Example 2 and the microfluidic chip for separating microparticles is about It set so that it might become 500 micrometers. In addition, cotton was inserted into the holes 55 of the advection accumulation unit so as to be in uniform contact with the grooves 15 of the microchannel chip for particle separation. Using the clamp from the bottle storing the sheath liquid so that the speed of the sheath liquid (phosphate buffered saline (PBS)) flowing through the main channel is 600 μm / s while rotating the rotating means 40 at 0.3 rpm. While supplying the sheath liquid, one end of a silicon tube (manufactured by ASONE) was connected to the sheath liquid suction port 56, and the other end was connected to micro-shilling (manufactured by KD Scientific) to suck the sheath liquid. Moreover, the sample was supplied so that it might become 8.3 microliters / s. The captured CTC was confirmed by a fluorescence microscope.

  The results of Example 5 are shown in Table 1. In Table 1, “number of cells per field of view” means any sample selected on the chip 1 by applying only the sample prepared in the above [Preparation of blood sample] on the chip 1 without generating a meniscus. 4 represents the number of CTC cells contained in 4 places (a to d in Table 1). “The number of captured cells” refers to CTCs captured at any of the four capture sites (a to d) while removing blood cells by generating a meniscus and flowing sheath fluid according to Example 5. Represents the number of As shown in Table 1, in Example 5, about 60% of CTCs in the sample could be captured. In the case of capturing CTC using a conventional anti-EpCAM antibody, it is said that only about 100 to 1/1000 of CTC in blood can be captured, but the microchannel chip for microparticle separation of the present invention is used. It was revealed that CTC can be captured very efficiently.

<Example 6>
[Velocity change experiment of sheath liquid flowing in main channel]
Using the chip produced in Example 2 and the advection accumulation unit produced in Example 4, the distance between the sheath liquid plane part and the sample plane part of the chip and the advection accumulation unit was about 500 μm, and the hole 55 of the chip and the advection accumulation unit was almost The experiment was performed under the same conditions as in Example 5, except that the contact was set. During the experiment, the flow rate of the sheath liquid flowing through the main channel at arbitrary intervals was measured by video shooting. After the sheath liquid started to be sucked by capillary force through the holes of the advection and accumulation unit, the sheath liquid flowed at a substantially constant flow rate. It was confirmed that the gas flowed through the main channel.

  By using the microparticle separation system including the microchannel chip for microparticle separation of the present invention, microparticles having different sizes in a sample can be separated quickly and efficiently without using an antibody or the like. Therefore, it is very effective in clinical settings such as separation of CTC from whole blood, so it can be used as a cancer diagnosis system in medical institutions such as hospitals and emergency centers, research institutions such as university medical departments, and educational institutions. Is possible.

Claims (20)

  A substrate, a main flow path formed on the substrate, a branch flow path branched from the main flow path and connected to the main flow path again, and a trapping part of fine particles formed in the branch flow path and larger than the width of the branch flow path, A microchannel chip for separating fine particles, wherein the main channel is formed radially from the center of the substrate.   When the size of the fine particles captured at the capture site is X and the size of the fine particles to be separated / removed is Y, the width F of the main channel and the branch channel is Y <F <X, and the capture site 2. The micro flow for separating fine particles according to claim 1, wherein the width G is 1X <G <10X, and the depth H of the main flow path, the branch flow path, and the capture site is 1X <H <10X. Road chip.   The microchannel chip for microparticle separation according to claim 2, wherein the width G is 1X <G <2X, and the depth H is 1X <H <2X.   4. The microchannel chip for separating fine particles according to claim 2, wherein the width F is Y <F <0.8X. The flow path according to any one of claims 2 to 4, wherein a flow path having a width F and a depth J <Y <J is further formed below the main flow path, the branch flow path, and the capture portion. The microchannel chip for separating fine particles according to Item. Including a substrate, a main flow path formed on the substrate, and a capture portion that is larger than the width of the main flow path and formed on the main flow path,
The main flow path is provided in a portion where the capture site is provided, with a width smaller than the capture site below the capture site,
The main flow path of the part where the capture part and the capture part are not provided is open on the substrate,
A microchannel chip for separating fine particles, wherein the main channel is formed radially from the center of the substrate.   When the size of the fine particles captured at the capture site is X and the size of the fine particles to be separated / removed is Y, the width A of the main channel is Y <A <X, and the width B of the capture site is 1X. <B <10X, the depth C of the capture site is 1X <C <10X, the depth D of the main channel at the capture site is Y <D, and the depth E of the main channel other than the capture site is The microchannel chip for microparticle separation according to claim 6, wherein E = C + D.   8. The microchannel chip for separating fine particles according to claim 7, wherein the width B is 1X <B <2X, and the depth C is 1X <C <2X.   9. The microchannel chip for fine particle separation according to claim 7, wherein the width A is Y <A <0.8X.   10. The microfluidic for microparticle separation according to claim 1, wherein a circular groove portion that connects the distal end portions of the main flow paths extending radially is formed on the substrate. Road chip.   The microchannel chip for microparticle separation according to any one of claims 1 to 10, wherein the microparticles captured at the capture site are CTC and the microparticles to be removed are blood cells.   An advection and accumulation unit comprising at least a sheath liquid inlet, a sample inlet, a sheath liquid advection and accumulation plane and a sample advection and accumulation plane.   13. The advection accumulation unit according to claim 12, further comprising a hole for mounting the sheath liquid suction pad and a sheath liquid suction port.   The advection accumulation unit according to claim 12, further comprising a hole for sucking the sheath liquid by capillary force and a sheath liquid suction port. A microchannel chip for separating fine particles according to any one of claims 1 to 11,
An advection accumulation unit according to any one of claims 12 to 14,
Rotating means for rotating the micro-channel chip for separating fine particles, and sheath liquid suction means,
A system for separating fine particles.   The system for separating fine particles according to claim 15, further comprising a fine particle extracting means for taking out the fine particles captured at a capture site formed in the fine particle separation microchannel chip and a detecting means for detecting the fine particles. .   The system for separating fine particles according to claim 16, further comprising PCR means for amplifying the nucleic acid.   The magnetic field generator for generating a magnetic field and / or the electric field generator for generating an electric field are further included in the capture part of the microchannel chip for microparticle separation. Fine particle separation system. The microfluidic chip for microparticle separation described in any one of claims 1 to 11 is placed on a rotating means that rotates the microfluidic chip for microparticle separation,
On the microchannel chip for fine particle separation, an advection and accumulation unit including at least a sheath liquid inlet, a sample inlet, a sheath liquid advection and accumulation plane and a sample advection and accumulation plane is disposed,
The microfluidic chip for fine particle separation, the flat portion for sheath liquid advection and accumulation, and the sample advection by injecting a sheath liquid from the sheath liquid inlet and injecting a sample from the sample inlet while rotating the rotating means Relative movement of the flat part for accumulation, capture the target fine particles by the meniscus generated by the relative movement to the capture site formed in the micro-channel chip for fine particle separation,
A fine particle separation method comprising removing fine particles to be removed together with a sheath liquid from a fine particle separation microchannel chip by sucking a sheath liquid with a sheath liquid suction means. The fine particle separation method according to claim 19, wherein the fine particles to be captured at the capture site are CTC, and the fine particles to be removed are blood cells.