Microfluidic device and method for manufacturing same

Abstract

Provided is a microfluidic device characterized by comprising a tunnel-shaped separation flow path, a columnar obstacle which is provided inside the separation flow path, and a ligand which is supported on the surface of the obstacle, wherein the ligand is an optically active polymer.

Description

Microfluidic device and method for manufacturing the same

[0001] The present disclosure relates to microfluidic devices and methods for manufacturing the same.

[0002] Conventionally, methods for separating specific components from a sample containing multiple components have been known, using separation devices such as liquid chromatography, which utilize a separation column as a stationary phase. Typical separation columns used in liquid chromatography include particle-packed columns, in which particles such as particulate silica gel are packed into a cylinder, and monolithic columns, in which silica gel with a three-dimensional network structure is packed into a cylinder. Recently, liquid chromatography using columns with a pillar array structure, in which multiple columnar obstacles (pillars) are arranged in a tunnel-shaped separation channel, as a stationary phase has been developed and is currently being studied. While particle-packed columns and monolithic columns have the drawback of being difficult to precisely control the size and arrangement of the particles and the size and arrangement of the mesh that constitutes the three-dimensional network structure, pillar array columns have the advantage of being able to precisely fabricate the structure according to the design, thereby achieving high separation efficiency, thanks to the ability to manufacture and arrange the pillars using techniques such as semiconductor microfabrication.

[0003] As a technology related to a pillar array structure, Patent Document 1 discloses a technology related to a mixer that has a separation channel in which the shape and size of the pillars and the distance between each pillar are designed within a specific numerical range, thereby achieving high mixing characteristics while maintaining the separation state of the separated components. Non-Patent Document 1 also discloses that in a pillar array column having a turn structure, high separation efficiency can be achieved by making the channel shape a low-dispersion shape. Non-Patent Document 2 also discloses that in a pillar array column having a turn structure, high separation efficiency can be achieved by controlling the arrangement of pillars in the channel.

[0004] Japanese Patent Application Laid-Open No. 2018-122295

[0005] Chiaki Aoyama, et al., Analytical Chemistry, 82, 1420-1426 (2010)Muneki Isokawa, et al., Analytical Chemistry, 88, 6485-6491 (2016)

[0006] To impart separation capabilities to a device, Patent Document 1 forms multiple pillars in the separation channel, while Non-Patent Documents 1 and 2 form multiple pillars in the separation channel and then chemically bond octadecylsilyl groups to the pillar surfaces. However, no method for separating optical isomers is disclosed. Optical isomers are used in a wide range of technical fields, particularly in fields such as pharmaceuticals and pesticides, where many optically active compounds are used, and research and development into optical isomers has been actively conducted. Therefore, in separation columns used in devices such as liquid chromatography, the ability to separate optical isomers from samples containing multiple components would be a significant advantage. In other words, the above-mentioned conventional devices, particularly microfluidic devices, which are unable to separate optical isomers, still have room for improvement.

[0007] Therefore, an object of the present disclosure is to provide a microfluidic device capable of separating optical isomers.

[0008] After extensive research, the inventors discovered that the above problem can be solved by supporting an optically active polymer as a ligand on the surface of a separator provided in the separation channel, and thus arrived at the present disclosure.

[0009] [1] A microfluidic device comprising a tunnel-shaped separation channel, pillar-shaped obstacles provided in the separation channel, and a ligand supported on the surface of the obstacle, wherein the ligand is an optically active polymer. [2] The microfluidic device according to [1], wherein the optically active polymer is a polysaccharide or a derivative thereof. [3] The microfluidic device according to [1] or [2], wherein the separation channel has a turn structure, and the turn structure has an inner wall surface shaped like a tapered structure that widens toward the outer periphery when viewed from above. [4] The microfluidic device according to [3], wherein the turn structure has the same outer and inner peripheries when viewed from above. [5] The microfluidic device according to [1] or [2], wherein the separation channel has a turn structure, and the turn structure has a structure in which the density of the pillars decreases gradually from the inner periphery to the outer periphery. [6] The microfluidic device according to any of [1] to [5], further comprising a sample channel for introducing a sample. [7] The microfluidic device according to [6], wherein the separation channel and the sample channel are provided on the same substrate. [8] The microfluidic device according to [7], wherein the substrate is in the form of a chip. [9] A method for manufacturing a microfluidic device, comprising: a substrate preparation step of preparing a substrate having a tunnel-shaped separation channel and columnar obstacles provided in the separation channel; and a supporting step of supporting a ligand on the surface of the obstacles by passing a solution containing the ligand through the separation channel, wherein the ligand is an optically active polymer.

[10] The method for manufacturing a microfluidic device according to [9], wherein the substrate is in the form of a chip.

[11] The method for manufacturing a microfluidic device according to [9] or

[10] , wherein the substrate further comprises a sample channel for introducing a sample.

[0010] According to the present disclosure, a microfluidic device capable of separating optical isomers can be provided.

[0011] FIG. 1 is a schematic perspective view showing one embodiment of a pillar array column. FIG. 2 is a cross-sectional view showing one embodiment of a turn structure. FIG. 3 is a diagram for explaining a method for producing a ligand-unsupported substrate. FIG. 4 is a diagram showing one embodiment of a microfluidic device. FIG. 5 is a diagram showing one embodiment of a microfluidic device. FIG. 6 is a diagram showing one embodiment of a microfluidic device. FIG. 7 is a diagram showing a chromatograph according to the results of separation evaluation in the examples. FIG. 8 is a diagram showing a chromatograph according to the results of separation evaluation in the examples. FIG. 9 is a diagram showing a chromatograph according to the results of separation evaluation in the examples.

[0012] Embodiments of the present disclosure are described in detail below. However, each configuration and combination thereof in each embodiment is merely an example, and additions, omissions, substitutions, and other modifications of configurations are possible as appropriate within the scope of the gist of the present disclosure. The present disclosure is not limited by the embodiments, but only by the scope of the claims. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits, and "A to B" means A or greater and B or less. Also, in this specification, "plurality" means "two or more." Note that the schematic diagrams in the drawings depict various components as appropriately enlarged or reduced for the purpose of explanation, and do not represent the actual size or proportions of the embodiments of the present disclosure.

[0013] <Configuration and Characteristics of Microfluidic Device> A microfluidic device (also simply referred to as a "microfluidic device") according to one embodiment of the present disclosure is a microfluidic device comprising a tunnel-shaped separation channel, columnar obstacles (also referred to as "pillars") provided within the separation channel, and ligands supported on the surfaces of the obstacles, wherein the ligands are optically active polymers. In the microfluidic device according to this embodiment, because the optically active polymer is supported on the surfaces of the pillars, optical isomers can be separated from a fluid passed through the tunnel-shaped separation channel. In the present disclosure, an "element comprising a tunnel-shaped separation channel, columnar obstacles provided within the separation channel, and ligands supported on the surfaces of the obstacles" provided in the microfluidic device is also referred to as a pillar array column, and this pillar array column will be described below.

[0014] FIG. 1 shows an example of a pillar array column included in the microfluidic device according to this embodiment. The pillar array column 10 shown in FIG. 1 includes a tunnel-shaped separation channel formed by separation channel walls (not shown) and columnar obstacles (pillars) 12 disposed within the separation channel. Arrow F(i) in FIG. 1 indicates the direction of fluid flow into the channel, and arrow F(o) indicates the direction of fluid flow out of the separation channel. In this disclosure, unless otherwise specified, numerical conditions in which multiple targets may exist represent their average values. Specifically, for example, the height of a pillar is the average of the heights of multiple pillars. Each component of the pillar array column 10 is described in detail below.

[0015] [Tunnel-Shaped Separation Channel] The pillar array column 10 is equipped with a tunnel-shaped separation channel. The form of the tunnel-shaped separation channel is not particularly limited, and the cross-sectional shape of the separation channel (cross-sectional shape of the separation channel) perpendicular to the flow direction (direction of arrow F(i) in FIG. 1 ) may be, for example, a polygonal shape such as a rectangle, a circle, a semicircle, or an ellipse, and is preferably a rectangle. The cross-sectional area of ​​the separation channel perpendicular to the flow direction is not particularly limited, but is usually 0.01 μm from the viewpoint of manufacturing stability. ２ or more, and ２It is preferable that the thickness is 10 μm or more. ２ More preferably, it is 100 μm or more. ２ More preferably, it is 1,000 μm or more. ２ It is particularly preferable that the thickness is 5,000 μm or more. ２ It is particularly preferable that the thickness is 1,000,000 μm or more, and from the viewpoint of usability as a microfluidic device, it is usually 1,000,000 μm ２ less than 500,000 μm ２ Preferably, it is 100,000 μm or less. ２ More preferably, it is 50,000 μm or less. ２ More preferably, it is 30,000 μm or less. ２The height of the separation channel (particularly, when the cross section of the separation channel perpendicular to the flow direction is rectangular, the height of the rectangle in the height direction of the pillar) is not particularly limited, but from the viewpoint of production stability, it is usually 0.1 μm or more, preferably 1 μm or more, more preferably 5 μm or more, and even more preferably 10 μm or more, and from the viewpoint of usability as a microfluidic device, it is usually 1,000 μm or less, preferably 500 μm or less, more preferably 100 μm or less, and even more preferably 50 μm or less. Furthermore, the width of the separation channel (particularly the width of the rectangle when the cross section of the separation channel perpendicular to the flow direction is rectangular) is not particularly limited, but from the viewpoint of production stability, it is usually 0.1 μm or more, preferably 1 μm or more, more preferably 5 μm or more, even more preferably 10 μm or more, and particularly preferably 100 μm or more, and from the viewpoint of usability as a microfluidic device, it is usually 10,000 μm or less, preferably 5,000 μm or less, more preferably 1,000 μm or less, and even more preferably 500 μm or less. The length of the separation channel in the flow direction (separation channel length) is not particularly limited, but from the viewpoint of facilitating the reduction in device size and the time required for separation, and also of suppressing production costs, it is usually 1,000 mm or less, preferably 750 mm or less, more preferably 500 mm or less, even more preferably 300 mm or less, and particularly preferably 200 mm or less, and there is no need to set a lower limit, and it may be 1 mm or more, 10 mm or more, or 50 mm or more.

[0016] The separation channel may be composed of only a linear structure or may have a curved structure, and the curved structure preferably has a turn structure from the viewpoint of realizing a reduction in the size of the pillar array column 10. The turn structure refers to a structure in which the direction of the separation channel is changed, for example, by 10° or more, 45° or more, 90° or more, 110° or more, 130° or more, 150° or more, 160° or more, 170° or more, or by 200° or less, or by 190° or less, preferably by at least 180°.

[0017] The shape of the turn structure is not particularly limited. However, from the viewpoint of suppressing fluid diffusion at the turn portion and flow resistance of the separation flow path, it is preferable that the inner wall surface in plan view be formed in a tapered shape that widens toward the outer periphery, as disclosed in documents such as "Chiaki Aoyama, et al., Analytical Chemistry, 82, 1420-1426 (2010)" (Non-Patent Document 1). Furthermore, it is preferable that the tapered structure have the same outer and inner periphery lengths in plan view. However, "the two lengths being the same" includes not only cases where the two lengths are almost the same, but also cases where there is an error (e.g., a difference of less than ±1% in the outer periphery length) that can be recognized as substantially the same (for example, within the range in which the effects of the present disclosure can be obtained). The tapered structure can be appropriately designed according to the conditions disclosed in known documents such as "Chiaki Aoyama, et al., Analytical Chemistry, 82, 1420-1426 (2010)." The tapered structure can be appropriately designed according to the application. A structure in which the shape of the inner peripheral wall surface in plan view is formed in a tapered shape that widens toward the outer peripheral side means a structure in which a channel length with a constant width is bent into a U-shape (for example, a structure as shown in FIG. 3 ), where U is the length of the inner peripheral side in plan view, and the length of the inner peripheral side of this U-shape is greater than U (more specifically, the shape of the inner peripheral wall surface is widened and tapered toward the inner peripheral wall surface) (while maintaining the shape of the outer peripheral wall surface). An example of a tapered structure is shown in FIG. 2 (pillars in the separation channel are not shown). The directions of arrows T(a) and T(b) in FIG. 2 indicate the flow direction of the fluid in the separation channel, and the length of T(a) is the length of the outer peripheral side, and T(b) is the length of the inner peripheral side.

[0018] Furthermore, from the viewpoint of suppressing fluid diffusion and flow path resistance at the turn portion, it is preferable that the flow path has a turn structure disclosed in documents such as "Muneki Isokawa, et al., Analytical Chemistry, 88, 6485-6491 (2016)" (Non-Patent Document 2), and that the turn structure has a gradient such that the density of the number of pillars decreases from the inner periphery to the outer periphery. Note that, for this structure with a gradient, the conditions disclosed in known documents such as Non-Patent Document 2 can be applied as appropriate, but the structure is not limited thereto and can be designed as appropriate depending on the application.

[0019] The number of turn structures (number of turns) of the pillar array column is not particularly limited and can be designed appropriately depending on the application. It may be 1, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, or 1000 or less, or 100 or less, and an even number is preferable from the viewpoint of suppressing fluid diffusion (diffusion is offset by the flow from the inside to the outside and the flow from the outside to the inside).

[0020] The shape of the components constituting the separation channel is not particularly limited and may be any shape, but for example, it may be a shape produced by the method for producing a ligand-unloaded substrate described below, or it may be a shape that is the same as the shape of the separation channel, that is, a shape that is composed of walls of a certain thickness that surround the separation channel. As a method for forming the separation channel, for example, when a shape is formed of walls of a certain thickness that surround the separation channel, a method can be used to form the separation channel by combining sheet members that will become the separation channel walls. When an arbitrary shape is to be formed, a method can be used in which through-holes that will become the separation channel are provided in a component of an arbitrary shape. The material of the components constituting the separation channel, i.e., the material of the channel walls, is not particularly limited, but silicon is particularly preferred from the viewpoint of ease of semiconductor processing.

[0021] [Columnar Obstacles (Pillars)] The pillar array column 10 includes columnar obstacles (pillars) 12. The shape of the pillars 12 is not particularly limited, and the shape of the bottom of the pillars 12 may be a polygonal shape such as a triangular shape, a quadrangular shape (particularly a rectangular shape), a pentagonal shape, or a hexagonal shape, or a cylindrical shape. However, from the viewpoint of ease of semiconductor processing, a quadrangular shape is preferable, and a rectangular shape is particularly preferable. The shape of the bottom of the pillars 12 in the pillar array column 10 of FIG. 1 is rectangular. The arrangement of the pillars is not particularly limited, but from the viewpoint of preventing peak broadening and an increase in column pressure, it is preferable that they are uniformly arranged. In addition, the members constituting the separation channel and the pillars may be manufactured separately and then joined, or may be manufactured as an integrally molded body.

[0022] The area of ​​the bottom surface of the pillar 12 is not particularly limited, but from the viewpoint of manufacturing stability, it is usually 0.01 μm ２ or more, and 0.1 μm ２ It is preferable that the thickness is 1 μm or more. ２ More preferably, it is 5 μm or more. ２ More preferably, it is 1,000,000 μm or more. From the viewpoint of usability as a microfluidic device, it is usually 1,000,000 μm ２ less than 100,000 μm ２ Preferably, it is 10,000 μm or less. ２ More preferably, it is 1,000 μm or less. ２ More preferably, it is 100 μm or less. ２ More preferably, it is 50 μm or less. ２It is particularly preferable that the maximum width of the bottom surface of the pillar 12 (the length of the longest line segment that can be taken on the bottom surface of the pillar) is not particularly limited, but from the viewpoint of manufacturing stability, it is usually 0.1 μm or more, preferably 0.5 μm or more, more preferably 1 μm or more, and even more preferably 2 μm or more. Also, from the viewpoint of usability as a microfluidic device, it is usually 1,000 μm or less, preferably 100 μm or less, more preferably 50 μm or less, and even more preferably 10 μm or less. When the shape of the bottom surface of the pillar 12 is rectangular, the length of each side is not particularly limited, but from the viewpoint of manufacturing stability, it is usually 0.1 μm or more, preferably 0.5 μm or more, more preferably 1 μm or more, and even more preferably 2 μm or more. Also, from the viewpoint of usability as a microfluidic device, it is usually 1,000 μm or less, preferably 100 μm or less, more preferably 50 μm or less, and even more preferably 10 μm or less. The height of the pillars 12 is not particularly limited, but from the viewpoint of manufacturing stability, it is usually 0.1 μm or more, preferably 1 μm or more, more preferably 5 μm or more, and even more preferably 10 μm or more, and from the viewpoint of usability as a microfluidic device, it is usually 1,000 μm or less, preferably 500 μm or less, more preferably 100 μm or less, and even more preferably 50 μm or less.

[0023] The minimum distance between each pillar (the minimum distance between the side surfaces of each pillar) is not particularly limited, but from the viewpoint of manufacturing stability, it is usually 0.1 μm or more, preferably 0.5 μm or more, more preferably 1 μm or more, and even more preferably 1.5 μm or more. Also, from the viewpoint of usability as a microfluidic device, it is usually 1,000 μm or less, preferably 100 μm or less, more preferably 50 μm or less, and even more preferably 10 μm or less. The pillar pitch distance (the minimum distance between the central axes of each pillar) is not particularly limited, but from the viewpoint of manufacturing stability, it is usually 0.1 μm or more, preferably 0.5 μm or more, more preferably 1 μm or more, and even more preferably 3 μm or more. Also, from the viewpoint of usability as a microfluidic device, it is usually 1,000 μm or less, preferably 100 μm or less, more preferably 50 μm or less, and even more preferably 10 μm or less. The number of pillars in the separation channel is not particularly limited, and can be designed appropriately according to the amount of sample to be separated and the size of the device.

[0024] The pillars carry optically active polymers as ligands on their surfaces. This makes it possible to separate optical isomers from a fluid passed through a tunnel-shaped separation channel. The type of optically active polymer is not particularly limited, and examples thereof include polysaccharides or derivatives thereof, poly(meth)acrylic acid amide, polyamino acids, polyamides, etc., but polysaccharides or derivatives thereof are preferred from the viewpoint of high separation performance. The type of polysaccharide or derivative thereof is not particularly limited, and examples of polysaccharides include β-1,4-glucan (cellulose), α-1,4-glucan (amylose, amylopectin), α-1,6-glucan (dextran), β-1,6-glucan (pustulan), β-1,3-glucan (curdlan, schizophyllan), α-1,3-glucan, β-1,2-glucan (Crown Gall polysaccharide), β-1,4-galactan, β-1,4-mannan, α-1,6-mannan, β-1,2-fructan (inulin), β-2,6-fructan (levan), β-1,4-xylan, β-1,3-xylan, β-1,4-chitosan, β-1,4-N-acetylchitosan (chitin), pullulan, agarose, alginic acid, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, nigeran, or amylose. Among these, from the viewpoint of easily obtaining a high-purity polysaccharide, cellulose, amylose, β-1,4-chitosan, chitin, β-1,4-mannan, β-1,4-xylan, inulin, curdlan, pullulan, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or nigeran are preferred, and cellulose, amylose, pullulan, or nigeran are more preferred. The optically active polymers can be used alone or in any combination of two or more kinds.

[0025] The number-average degree of polymerization of the polysaccharide (the average number of pyranose or furanose rings contained in one molecule) is preferably 5 or more, more preferably 10 or more, and although there is no particular upper limit, it is preferably 1,000 or less from the viewpoint of ease of handling, more preferably 5 or more and 1,000 or less, even more preferably 10 or more and 1,000 or less, and particularly preferably 10 or more and 500 or less.

[0026] For these polysaccharides, for example, ester derivatives and carbamate derivatives obtained by chemically modifying cellulose or amylose can be used as ligands. Such polysaccharide derivatives are known to have high optical resolution ability as chiral stationary phases. Specific examples of ester derivatives and carbamate derivatives include, for example, JP-B 4-42371 discloses a cellulose derivative in which the hydroxyl groups of cellulose are modified with a substituent in which some of the hydrogen atoms on the aromatic ring of phenyl carbamate are substituted with halogen (fluorine or chlorine), and JP-A 2005-315668 discloses a cellulose derivative and amylose derivative in which the hydroxyl groups of cellulose or amylose are modified with a substituent in which some of the hydrogen atoms on the aromatic ring of phenyl carbamate are substituted with fluorine, an alkyl group, or an alkoxy group. These derivatives can also be used as ligands in the present disclosure.

[0027] Among the polysaccharides or derivatives thereof, the polysaccharide derivatives are particularly preferred from the viewpoint of the separation performance of the optical isomers to be separated and the ease of loading onto the pillars. The polysaccharide derivatives are not limited to those mentioned above, and any desired type can be used. The method for loading the polysaccharide or its derivative onto the pillars is not particularly limited, and the polysaccharide or its derivative may be loaded by physical adsorption or chemical bonding. Examples of physical adsorption methods include coating the pillars with an optically active polymer. Examples of chemical bonding methods include chemical bonding between the pillars and the polysaccharide derivative, chemical bonding using a third component, or a reaction induced by irradiation of the polysaccharide derivative on the support with light, radiation such as gamma rays, or electromagnetic waves such as microwaves, or a radical reaction using a radical initiator to generate a chemical bond.

[0028] The amount of optically active polymer supported in the separation channel (the amount of optically active polymer supported on the pillars, particularly the amount of optically active polymer supported on the pillars and separation channel walls) is not particularly limited as long as it is within a range in which the effects of the present disclosure can be obtained. However, from the viewpoint of being able to stably separate optical isomers, the average amount per unit area of ​​the separation channel when viewed in plan is usually 0.001 μmol / m ２ or more, and ２ It is preferable that the concentration is 0.1 μmol / m or more. ２ More preferably, it is 2.0 μmol / m or more, and usually, it is 2.0 μmol / m or more. ２ is 1.0 μmol / m or less ２ It is preferable that the concentration is 0.9 μmol / m or less. ２ More preferably, it is:

[0029] The pillars may be supported with a compound other than the above-mentioned optically active polymers, and examples thereof include optically inactive polyesters such as polyethylene terephthalate, polybutylene terephthalate, polyethylene-polybutylene terephthalate, polytrimethylene terephthalate, polyethylene naphthalate, polybutylene naphthalate, polylactic acid, polyglycolic acid, polyε-caprolactone, or poly(oxycarbonyloxy-1,4-phenylene-2,2-isopropylidene-1,4-phenylene) {polycarbonate of bisphenol A); proteins such as protein A, protein G, protein L, or albumin, and functional variants thereof; nucleic acids such as DNA, RNA, oligonucleotides, or modified oligonucleotides; or other low molecular weight compounds or oligomers.

[0030] <Method for Manufacturing a Microfluidic Device> Another embodiment of the present disclosure, a method for manufacturing a microfluidic device (hereinafter also referred to simply as a "method for manufacturing a microfluidic device"), includes a substrate preparation step of preparing a substrate having a tunnel-shaped separation channel and pillar-shaped obstacles provided in the separation channel, and a supporting step of supporting a ligand on the surface of the obstacles by passing a ligand-containing solution through the separation channel, wherein the ligand is an optically active polymer. The terms and conditions used in the above-described description of the microfluidic device can also be applied to this embodiment of the manufacturing method, unless otherwise specified. In the present disclosure, a substrate having a pillar array column in which no ligand is supported on the pillars is also referred to as a ligand-unsupported substrate.

[0031] [Substrate Preparation Step] The manufacturing method for a microfluidic device according to this embodiment includes a substrate preparation step of preparing a substrate (ligand-unloaded substrate) having a tunnel-shaped separation channel and columnar obstacles disposed within the separation channel. The method for manufacturing the substrate is not particularly limited and can be performed by a known method or a combination of known methods. However, from the viewpoint of being able to manufacture according to the design, a method including a step of using semiconductor processing technology, specifically, a step of patterning the substrate using semiconductor processing technology, is preferred. The shape of the substrate is not particularly limited, but a chip shape is preferred from the viewpoints of ease of handling and ease of manufacturing. In the present disclosure, a chip-shaped substrate (sometimes simply referred to as a "chip") refers to a single plate-like member. The form of the substrate is not particularly limited as long as it can be provided with a pillar array column, and commercially available products can be used. When using a chip-shaped substrate, for example, it may be formed by cutting it from a bulk material such as silicon, or by curing a composition containing a material such as silicon, or a commercially available product may be used as is. An example of a specific method for manufacturing a ligand-unloaded substrate is described with reference to FIG. 3.

[0032] A photosensitive resin 23 is applied to one side of a silicon substrate 21 having oxide films 22 on both sides (FIG. 3(a)). This surface is exposed to light through a mask according to the design of the separation channels, and then photolithography is performed to form a resin pattern on the substrate (FIG. 3(b)). The substrate is then immersed in acid, and the silicon oxide film in the areas not covered by the resin is dug down (wet etching) (FIG. 3(c)). The remaining resin is then removed, and deep-reactive ion etching (deep-RIE) is performed using the oxide film as a resist to dig down the separation channels (FIG. 3(d)). After that, wet etching is performed to remove the remaining oxide film (FIG. 3(e)). Dust generated by etching can also be removed at the same time. O ２ The surface of the silicon substrate is reoxidized using plasma (for the subsequent surface modification reaction), and finally, this substrate is bonded to a glass substrate 24 by anodic bonding to form a separation channel 25 (FIG. 3(f)), completing the production of the ligand-unloaded substrate.

[0033] The material of the substrate is not limited to silicon, and may be any of the materials used for the flow path walls. The exposure, development, pattern formation, digging, anodic bonding, and other processes may be performed by known methods.

[0034] [Loading Step] The method for manufacturing a microfluidic device according to this embodiment includes a loading step of loading a ligand onto the surface of the obstacles (pillars) by passing a solution containing the ligand through the separation channel. The method for passing the ligand solution through the channel is not particularly limited and can be performed by a known method. For example, the ligand-containing solution can be passed from F(i) to F(o) in FIG. 1. The flow rate at which the solution is passed is not particularly limited and can be, for example, 0.1 cm ３ / s or more, 10.0cm ３ / s or less, and ３ / s or more, 5.0cm ３ / s or less. After passing the liquid through the pillar array column, the ligand solution present (applied) on the pillars in the separation flow path is dried by natural drying, vacuum drying, heat treatment, or the like, thereby allowing the pillars to be supported (coated) with the ligand. When heat treatment is performed, the heating temperature is not particularly limited and can be set depending on the solution used. For example, it may be 10°C or higher and 100°C or lower, or 30°C or higher and 80°C or lower.

[0035] The solution containing the ligand is not particularly limited as long as it contains a ligand. The ligand is not particularly limited as long as it is an optically active polymer, and the optically active polymers described above can be used. The content of the ligand (particularly the optically active polymer) in the solution passed through to attach the optically active polymer to the pillars is not particularly limited as long as the effects of the present disclosure can be obtained. However, from the viewpoint of stable separation of optical isomers, the content is usually 0.01 mmol / L or more, preferably 0.1 mmol / L or more, and more preferably 0.5 mmol / L or more, and usually 2.0 mmol / L or less, preferably 1.5 mmol / L or less, and more preferably 1.0 mmol / L or less.

[0036] The type of solvent in the solution containing the ligand is not particularly limited as long as it can dissolve the ligand, and may be, for example, acetone, tetrahydrofuran, dimethylformamide, dimethylacetamide, etc. Among these, acetone is preferred from the viewpoint of achieving good separation performance. The solvent may be used alone or in any combination of two or more kinds.

[0037] The solution containing the ligand may contain components other than the ligand and the solvent (other components).

[0038] [Other Steps] The method for manufacturing a microfluidic device according to this embodiment may include steps (other steps) other than the above-described substrate preparation step and supporting step, such as a synthesis step of synthesizing a ligand (optically active polymer), a solution preparation step of dissolving the ligand in a solvent to produce a solution containing the ligand, etc. Furthermore, when a mixer or the like that can be provided in the microfluidic device described below is provided, the method may also include a mixer formation step, a step of forming an inlet for introducing a derivatization reagent, and a step of forming a derivatization reagent flow path.

[0039] The method for synthesizing the ligand in the synthesis step is not particularly limited, and the ligand can be synthesized by a known method or a combination of known methods depending on the type of ligand.

[0040] In the solution preparation step, the method for dissolving is not particularly limited and can be performed by a known method. For example, the ligand can be added to a solvent and dissolved while stirring and, if necessary, heating.

[0041] The methods for forming each component in the step of forming the mixer, the step of forming the inlet for introducing the derivatization reagent, and the step of forming the derivatization reagent flow path can be similar to the method for forming the separation flow path described above.

[0042] A microfluidic device including a pillar array column according to the present disclosure can be used as a device for liquid chromatography or the like for separating a specific component from a sample containing a mixture of multiple components. Note that, in the present disclosure, a microfluidic device refers to a device having flow channels or structures on the order of μm, and the overall size of the device does not necessarily have to be on the order of μm.

[0043] In another embodiment of the microfluidic device of the present disclosure, the microfluidic device (preferably, the substrate of the microfluidic device) according to the above embodiment further comprises a sample channel for introducing a sample. The mode in which the microfluidic device comprises a sample channel is not particularly limited, and the sample channel may be formed on the microfluidic device itself. For example, the sample channel may be obtained by patterning a microfluidic device having a pillar array column formed thereon using a method similar to the above-described method for providing a pillar array column on a substrate using patterning, thereby forming a sample channel. Alternatively, the sample channel may be obtained by connecting a separate component having a sample channel to the microfluidic device. Of these modes, from the viewpoints of ease of manufacturing, manufacturing stability, and miniaturization, the mode in which the sample channel is formed on the microfluidic device itself is preferred. In particular, the mode in which the separation channel and the sample channel are provided on the same substrate is preferred. The mode in which the separation channel and the sample channel are provided on the same substrate will be specifically described below.

[0044] In the microfluidic device according to the present embodiment, the sample channel and the separation channel are formed on the same substrate, which makes it easier to reduce the size of the device and the time required for separation, and also reduces manufacturing costs, compared to devices in which the sample channel and the separation channel exist as separate components and are manufactured by joining them. There are no particular limitations on the method for forming the sample channel in the microfluidic device, but from the perspective of being able to manufacture the device according to the design, it is preferable to use a method that uses semiconductor processing technology to form the separation channel in the substrate, specifically, a method that includes a step of patterning a substrate using semiconductor processing technology.

[0045] An example of a microfluidic device according to this embodiment is shown in FIG. 4 . The microfluidic device 30 shown in FIG. 4 includes a sample channel 31 for introducing a sample and a separation channel 32 for separating components in the sample by flowing them together with a mobile phase, both of which are formed on the same substrate (herein also referred to as a “chip” (chip-like substrate)) 33. In FIG. 4 , for convenience, all components are depicted in black, and the separation channel 32 (the portion of the channel where pillars are present) is depicted surrounded by a dotted line (the dotted line includes a sample injection section 36, but the portion where the pillars are present is depicted by the dotted line, and pillars can be disposed in the sample injection section 36). The meaning of this dotted line is the same in FIGS. 5 and 6 , which will be described later. In the present disclosure, the region where pillars are present among the portions through which the mobile phase and sample are passed is referred to as the separation channel. The configuration of the mobile phase and sample channels before and after the separation channel 32 in FIG. 4 is not particularly limited, and may be any configuration that allows the passage of the mobile phase and sample. For example, the separation channel may be a tunnel-shaped cavity excluding the pillars from the separation channel. Furthermore, the mobile phase flow path through which only the mobile phase flows (the flow path through which the mobile phase flows before the mobile phase and sample are mixed) is not particularly limited as long as it is capable of passing the mobile phase. In FIG. 4 , the microfluidic device includes a mobile phase inlet 34 through which a mobile phase such as water, an organic solvent, or a buffer solution is introduced into the device, and a sample inlet 35 through which a sample is introduced into the device. The sample introduced through the sample inlet 35 is mixed with the mobile phase introduced through the mobile phase inlet 34 in the sample injection section 36 and introduced into the separation flow path 32 together with the mobile phase. The components contained in the sample introduced together with the mobile phase are separated in the flow direction in the separation flow path 32. The sample and mobile phase introduced into the separation flow path 32 are discharged through the outlet 37. In this case, the sample and mobile phase flow into the separation flow path along F(i) shown in FIG. 1 . The sample introduced into the sample processing device through the sample inlet 35 and not mixed with the mobile phase in the sample injection section 36 is discharged through the sample outlet 38. The sample inlet 35 and the sample outlet 38 are connected by a sample flow channel 31 which is a groove formed in the chip 33 .The separation channel 32, mobile phase inlet 34, sample inlet 35, outlet 37, and the like that constitute the microfluidic device 30 are formed as grooves on a substrate such as silicon, and a mixer or the like may also be formed as needed, with the upper part of the groove (opening) covered with glass as shown in Figure 4.

[0046] There are no particular limitations on the method of feeding the liquid to the mobile phase inlet 34, and for example, the liquid can be fed using a micro liquid chromatography pump, and to the sample inlet 35 by pressure feeding.

[0047] The shape of the substrate is not particularly limited, and for example, the shape of the surface of the substrate on which the separation flow path is provided may be a polygonal shape such as a rectangle, a circle, a semicircle, an ellipse, etc., preferably a rectangle. The shape of the substrate may be a polygonal plate such as a rectangular plate, a circular plate, a semicircular plate, an ellipse, etc., preferably a rectangular plate.

[0048] The parameters relating to the shape of the substrate will be explained below, but these parameters can also be treated as parameters of the shape of the microfluidic device within the applicable range. The area of ​​the surface of the substrate on which the pillar array column is provided is not particularly limited, but from the viewpoint of ease of handling, it is usually 100 mm ２ or more, and 400 mm ２ From the viewpoint of cost reduction, it is usually 10,000 mm ２ is less than or equal to 5,000 mm ２ The thickness of the substrate is not particularly limited, but from the viewpoint of ease of handling, it is usually 100 μm or more, and preferably 150 μm or more, and from the viewpoint of cost reduction, it is usually 1,000 μm or less, and preferably 500 μm or less. Furthermore, when the shape of the surface of the substrate on which the pillar array column is provided is rectangular, the length of each side is not particularly limited, but from the viewpoint of ease of handling, it is usually 10 mm or more, and preferably 20 mm or more, and from the viewpoint of cost reduction, it is usually 100 mm or less, and preferably 70 mm or less.

[0049] With the above-described configuration, optical isomers can be separated by causing a mobile phase and a sample to flow into the separation flow path in the microfluidic device.

[0050] The microfluidic device according to this embodiment has a configuration as shown in Fig. 4 when the separation channel is configured only in a linear shape, but can have a configuration as shown in Fig. 5 when the above-described turn structure is included. There is no limit to the number of turn structures, and when multiple turn structures are used, the device can have a configuration as shown in Fig. 6. Note that the turn structures of the separation channel shown in Figs. 5 and 6 have inner wall surfaces that are tapered and diverge toward the outer periphery when viewed from above, but are not limited to this shape and can have any turn structure.

[0051] 4 may be provided on the same substrate 33. For example, a mixer for derivatizing the separated substances may be provided between the separation channel 32 and the outlet 37. When a mixer is provided, an inlet for introducing a derivatization reagent and a derivatization reagent channel may also be provided.

[0052] The present disclosure will be described in more detail below with reference to examples, but the present disclosure should not be construed as being limited to the following examples.

[0053] Example 1 Production of Ligand-Unsupported Substrates A microfluidic device was prepared using the configuration shown in FIG. 6 (with the exception that the separation channel had 12 turn structures). A 60-μm-deep groove was formed as the sample channel 31 on a square microchip with a thickness of 500 μm and sides of 20 mm. Other channels through which the mobile phase and sample flowed were formed as 30-μm-deep grooves. The separation channel 32 had a width of 400 μm (the narrowest part of the curved portion of the turn structure was 110 μm). The separation channel had a width (length perpendicular to the flow direction) of 400 μm and a length in the flow direction of 110 mm. The pillars in the separation channel 32 had a side length of 3 μm and were aligned with a 2-μm spacing between faces. The above microfluidic devices were manufactured by forming each structure on a silicon substrate using conventional photolithography and deep reactive ion etching, and then bonding the substrate to glass using conventional anodic bonding. The ligand-unsupported substrates were then manufactured.

[0054] [Ligand Loading] 500 mg of a cellulose derivative (Cellulose tris (3,5-dimethylphenylcarbamate)) was dissolved in 1 L of acetone. Subsequently, this solution was passed through the above-mentioned ligand-unloaded substrate using a syringe pump (YSP-101 "standard type", manufactured by YMC Co., Ltd.) at room temperature (25°C) at a flow rate of 2 μL / min for 60 minutes. Thereafter, the chip was vacuum-dried (vacuum pressure <10 mmHg) (rectangular vacuum constant temperature dryer DP300, manufactured by Yamato Scientific Co., Ltd.) at a temperature of 60°C for 6 hours to load the ligand, thereby producing a microfluidic device.

[0055] <Separation Evaluation> For the above-described microfluidic device, water / acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) = 40 / 60 (v / v) was delivered from the mobile phase inlet 34, and flurbiprofen (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) fluorescently derivatized with 4-bromomethyl-7-methoxycoumarin (Br-Mmc) represented by the following formula (1) was delivered from the reagent inlet 35 at a flow rate of 0.5 μL / min, a sample concentration of 100 μM, and a detection position near the outlet (column length approximately 110 mm). During sample separation, a video was captured using a fluorescence microscope with a fixed field of view. From this video, the time change in fluorescence intensity of a portion of the flow path was plotted using Andor SOLIS (ver. 4.28.30001.0: Andor Technologies, South Windsor, CT, USA) to obtain a chromatogram. The back pressure during delivery was recorded according to the pressure value displayed on the micropump monitor. The evaluation results are shown in Figure 7.

[0056]

[0057] Example 2 [Production of Ligand-Unsupported Substrate] A ligand-unsupported substrate was produced in the same manner as in Example 1.

[0058] [Ligand Loading] A microfluidic device was produced by loading a ligand in the same manner as in Example 1, except that the amount of cellulose derivative (Cellulose tris(3,5-dimethylphenylcarbamate)) was changed from 500 mg to 10 mg.

[0059] <Separation Evaluation> Separation evaluation was carried out in the same manner as in Example 1. The evaluation results are shown in FIG.

[0060] Example 3 [Production of Ligand-Unsupported Substrate] A ligand-unsupported substrate was produced in the same manner as in Example 1.

[0061] [Ligand Loading] A microfluidic device was produced by loading a ligand in the same manner as in Example 1, except that the amount of cellulose derivative (Cellulose tris(3,5-dimethylphenylcarbamate)) was changed from 500 mg to 100 mg.

[0062] <Separation Evaluation> Separation evaluation was performed in the same manner as in Example 1, except that the ratio of water / acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) supplied from the mobile phase inlet 34 was changed from 40 / 60 (v / v) to 50 / 50 (v / v). The evaluation results are shown in Figure 9.

[0063] REFERENCE SIGNS LIST 10 pillar array column 12 pillar 21 silicon substrate 22 oxide film 23 photosensitive resin 24 glass substrate 25 separation channel 30 microfluidic device 31 sample channel 32 separation channel 33 chip 34 mobile phase inlet 35 sample inlet 36 sample injection section 37 outlet 38 sample outlet

Claims

1. a tunnel-shaped separation channel; and a columnar obstacle provided in the separation channel; and a ligand supported on the surface of the obstacle, A microfluidic device, wherein the ligand is an optically active polymer.

2. The microfluidic device according to claim 1 , wherein the optically active polymer is a polysaccharide or a derivative thereof.

3. 3. The microfluidic device according to claim 1, wherein the separation channel has a turn structure, and the turn structure has an inner wall surface that is tapered and diverges toward the outer periphery when viewed from above.

4. The microfluidic device according to claim 3 , wherein the turn structure has an outer periphery and an inner periphery that are the same in plan view.

5. 3. The microfluidic device according to claim 1, wherein the separation channel has a turn structure, and the turn structure has a gradient such that the density of the number of pillars decreases from the inner periphery to the outer periphery.

6. The microfluidic device according to claim 1 or 2, further comprising a sample flow channel for introducing a sample.

7. The microfluidic device according to claim 6 , wherein the separation channel and the sample channel are provided on the same substrate.

8. The microfluidic device according to claim 7 , wherein the substrate is in the form of a chip.

9. a substrate preparation step of preparing a substrate having a tunnel-shaped separation channel and a columnar obstacle provided in the separation channel; a supporting step of supporting a ligand on the surface of the obstacle by passing a solution containing the ligand through the separation channel; wherein the ligand is an optically active polymer.

10. The method for producing a microfluidic device according to claim 9 , wherein the substrate has a chip shape.

11. The method for manufacturing a microfluidic device according to claim 9 or 10, wherein the substrate further comprises a sample flow channel for introducing a sample.