Fluid device and method for separating nucleic acids

Abstract

To provide a technique capable of separating nucleic acids of a high molecular weight without damaging them at a low cost.SOLUTION: A fluid device 100 is equipped with a flow channel 120 for electrophoresing nucleic acids. A polymer brush layer 122 is provided on an inner surface of the flow channel 120. The polymer brush layer 122 includes hydrophilic polymers.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a fluidic device and a method for separating nucleic acids. [Background technology]

[0002] Size separation of nucleic acids is an essential process in gene analysis and purification of artificial genes, including nucleic acid medicines. In recent years, improvements in DNA synthesis technology have made it possible to synthesize long-chain DNA with a larger number of base pairs, which has led to a demand for size separation technology for large-molecular-weight DNA.

[0003] Gel electrophoresis is commonly used to separate DNA by size, but when DNA molecules have large molecular weights, they become elongated as they pass through the mesh of the gel, making size separation difficult.

[0004] In contrast to gel electrophoresis, many electrophoresis microchips (also called microfluidic devices) are known, in which numerous nanostructures that replace gels are arranged in microchannels (see, for example, Non-Patent Documents 1 to 3). Because electrophoresis microchips use artificial microstructures instead of gels, it is possible to freely set the size and gap of the structures to design separation performance, and they can also handle large molecular weight DNA fragments. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Anal. Chem., 2004, 76, p. 15-22 [Non-patent document 2] Anal. Chem., 2011, 83, p. 6635-6640 [Non-patent document 3] Jpn. J. Appl. Phys, 2016, 55, 06GN01 Summary of the Invention [Problem to be solved by the invention]

[0006] Although separation is faster than conventional gel electrophoresis, the high cost of using advanced microfabrication technology to fabricate the devices is an issue. Furthermore, the mechanism for separating DNA fragments using these microchips involves randomly coiled DNA fragments being caught on nanostructures and stretched as they are separated, so the larger the molecular weight of the DNA, the more likely it is to be caught on the structure. This means that DNA molecules are subjected to mechanical external forces, increasing the risk of breakage or damage.

[0007] An object of the present disclosure is to provide a technology that can separate large molecular weight nucleic acids at low cost without damaging them. [Means for solving the problem]

[0008] One aspect of the present disclosure is a fluidic device, which includes a channel for electrophoresis of nucleic acids, and a polymer brush layer provided on the inner surface of the channel, the polymer brush layer including a hydrophilic polymer. [Effects of the Invention]

[0009] According to the present disclosure, a technology can be provided that is capable of separating large molecular weight nucleic acids at low cost without damaging them. [Brief explanation of the drawings]

[0010] [Figure 1] 1(a) and 1(b) are diagrams illustrating a fluidic device according to a first embodiment. [Figure 2] 2 is a diagram for explaining the configuration of the inner surface of the flow channel shown in FIG. 1. FIG. [Figure 3] 3(a) and 3(b) are diagrams for explaining the separation of a plurality of nucleic acids in the nucleic acid separation method according to the third embodiment. [Figure 4] 4(a) and 4(b) are diagrams for explaining the separation of a plurality of nucleic acids in the nucleic acid separation method according to the fourth embodiment. [Figure 5] 5(a) to 5(c) are diagrams for explaining the separation of a plurality of nucleic acids in the nucleic acid separation method according to the fifth embodiment. [Figure 6] FIG. 1 is a diagram showing the results of film thickness control by adjusting the monomer concentration in Example 1. [Figure 7] FIG. 10 is a diagram showing the results of film thickness control by adjusting the UV light intensity in Example 1. [Figure 8] FIG. 1 is a diagram showing an experimental apparatus used in Example 2. [Figure 9] 9(a) and 9(b) are diagrams showing schematic diagrams of the apparatus used in the experimental method (1) of Example 2. [Figure 10] FIG. 10 is a schematic diagram showing a jig produced in Example 2. [Figure 11] FIG. 10 is a diagram showing the results of comparing migration speeds in Example 2. [Figure 12] FIG. 10 is a diagram showing the results of comparing the mobility in Example 2. [Figure 13] FIG. 10 is a diagram showing the results of comparing circularity in Example 2. [Figure 14] FIG. 1 shows the film thickness dependence of the migration speed for λ-DNA. [Figure 15] FIG. 1 shows the film thickness dependence of mobility for λ-DNA. [Figure 16] FIG. 10 shows the film thickness dependence of circularity on λ-DNA. [Figure 17] FIG. 1 shows the film thickness dependence of the migration speed for T4-DNA. [Figure 18] FIG. 1 shows the film thickness dependence of mobility for T4-DNA. [Figure 19] FIG. 1 shows the film thickness dependence of circularity for T4-DNA. [Figure 20] FIG. 1 shows the time-series change in the circularity of a 108 nm T4-DNA brush film. [Figure 21] FIG. 1 shows the results of comparing λ-DNA and T4-DNA migrating on an MPC polymer brush film with a brush film thickness of approximately 10 nm. [Figure 22]FIG. 1 shows the results of comparing λ-DNA and T4-DNA migrating on an MPC polymer brush film with a brush film thickness of approximately 40 nm. DETAILED DESCRIPTION OF THE INVENTION

[0011] A fluidic device according to a first aspect of the present disclosure includes a channel for electrophoresis of nucleic acids, and a polymer brush layer is provided on the inner surface of the channel, the polymer brush layer including a hydrophilic polymer. This aspect enables separation of large molecular weight nucleic acids without damaging them.

[0012] In the fluidic device of the second aspect, the hydrophilic polymer is biocompatible, which can prevent nucleic acids from adhering to the inner surface of the flow channel.

[0013] The fluidic device of the third embodiment is the same as that of either the first or second embodiment, except that the thickness of the polymer brush layer is set so as to separate nucleic acids based on the difference in entanglement between the nucleic acids and the surface of the polymer brush layer when the nucleic acids are electrophoresed. This embodiment allows multiple nucleic acids to be separated by their respective sizes. Here, the thickness of the polymer brush layer refers to the thickness of the polymer brush layer in a dry state.

[0014] In the fluidic device of the fourth aspect, in either the first or second aspect, the thickness of the polymer brush layer is set so that nucleic acids smaller than the size of the target nucleic acid are removed by the polymer brush layer when the nucleic acids are electrophoresed. According to this aspect, the target nucleic acid can be separated from multiple nucleic acids.

[0015] A fifth aspect of the fluidic device is the same as either the first or second aspect, in which a polymer brush layer is provided on the inner surface of the flow channel so as to generate a difference in electrophoretic speed of nucleic acids within the flow channel. According to this aspect, the target nucleic acid can be purified or concentrated by separating the target nucleic acid from multiple nucleic acids.

[0016] A sixth aspect of the present disclosure is a nucleic acid separation method using the fluidic device of any one of the first to fifth aspects. In this method, a solution containing at least one nucleic acid is filled into the electrophoresis starting point of the channel, and the nucleic acid is electrophoresed. According to this aspect, large molecular weight nucleic acids can be separated without being damaged.

[0017] A seventh aspect of the present disclosure is a nucleic acid separation method using the fluidic device of the third aspect. In this method, a solution containing multiple nucleic acids is filled into the electrophoresis starting point of the flow channel, the multiple nucleic acids are electrophoresed, and the multiple nucleic acids are separated by size based on the difference in entanglement between each of the multiple nucleic acids and the surface of the polymer brush layer. According to this aspect, the multiple nucleic acids can be separated by size.

[0018] An eighth aspect of the present disclosure is a nucleic acid separation method using the fluidic device of the fourth aspect. This method separates the target nucleic acid by filling a solution containing multiple nucleic acids into the electrophoresis starting point of a flow channel, electrophoresing the multiple nucleic acids, and removing nucleic acids of the multiple nucleic acids that are smaller than or equal to the size of the target nucleic acid using a polymer brush layer. According to this aspect, the target nucleic acid of a predetermined size or larger can be separated from the multiple nucleic acids.

[0019] A ninth aspect of the present disclosure is a nucleic acid separation method using the fluidic device of the fifth aspect. This method involves filling a channel with a solution containing multiple nucleic acids at the electrophoresis starting point, applying a first electrophoresis voltage to the channel to electrophorese nucleic acids that are smaller than a predetermined size among the multiple nucleic acids, and applying a second electrophoresis voltage higher than the first electrophoresis voltage to the channel to electrophorese nucleic acids that exceed the predetermined size. According to this aspect, a target nucleic acid can be separated from the multiple nucleic acids.

[0020] Hereinafter, embodiments for carrying out the present disclosure will be described in detail with reference to the drawings and the like. In the embodiments and modified examples, identical or equivalent components and members are designated by the same reference numerals, and redundant explanations will be omitted as appropriate. The dimensions of the members in each drawing are enlarged or reduced as appropriate to facilitate understanding. In addition, some members that are not important for explaining the embodiments will be omitted from each drawing.

[0021] (First embodiment) A fluidic device according to the first embodiment will now be described. FIGS. 1(a) and 1(b) are diagrams illustrating a fluidic device 100 according to the first embodiment. FIG. 1(a) is a plan view of the fluidic device 100, and FIG. 1(b) is a cross-sectional view of the fluidic device 100 taken along line AA of FIG. 1(a). As shown in FIGS. 1(a) and 1(b), the fluidic device 100 mainly comprises a substrate 110, a flow channel 120, a cover member 130, a first electrode 140, and a second electrode 142.

[0022] A groove-shaped channel 120 is formed on the upper surface of the substrate 110. A cover member 130 is placed on the upper surface of the substrate 110 to seal this channel and use it as a channel. A first electrode 140 and a second electrode 142 are placed on both ends of the channel 120. By applying a voltage to the first electrode 140 and the second electrode 142, nucleic acids can be electrophoresed in the channel 120.

[0023] The substrate 110 may be made of any material that allows electrophoresis of nucleic acids, such as inorganic materials such as silicon and glass, and synthetic resins such as cycloolefin polymer, polyethylene terephthalate, and polydimethylsiloxane (PDMS).

[0024] FIG. 2 is a diagram illustrating the configuration of the inner surface of the channel 120. A polymer brush layer 122 is formed on the inner surface of the channel 120 facing the substrate, i.e., on the upper surface of the substrate 110. The polymer brush layer 122 is composed of multiple brush-like polymer chains. Here, "brush-like" refers to a state in which one end of a linear polymer chain is fixed to the substrate and the other end is not fixed. The brush-like polymer chains are relatively free to deform, so they do not interfere with the movement of biopolymers within the channel.

[0025] Unlike a gel network, the polymer brush layer 122 does not have a finite pore size, and therefore, in principle, there is no upper limit to the size of molecules that can interact with the surface, making it possible to separate large molecular weight nucleic acids by size.

[0026] In the fluidic device 100 according to this embodiment, the interaction with the target nucleic acid, i.e., the resistance to electrophoretic force, can be adjusted by adjusting the length, density, charge, and chemical composition of the polymer chains constituting the polymer brush layer 122 according to the properties of the target nucleic acid. Such adjustments allow the electrophoretic speed of the target nucleic acid to be controlled, enabling efficient separation of the target nucleic acid.

[0027] The smaller the cross-sectional area of ​​the channel 120, such as a nanochannel, the greater the migration speed of the nucleic acid, which may be subjected to excessive shear stress and result in destruction of the nucleic acid. In the fluidic device 100 according to this embodiment, the polymer brush layer 122 interacts with the nucleic acid to be electrophoresed, thereby adjusting the migration speed of the nucleic acid so as to prevent destruction of the nucleic acid.

[0028] Compared to microfluidic devices with nanostructures, the polymer brush layer 122 can be fabricated easily and at low cost. Furthermore, since polymer brushes can be formed on a large surface area, the device can be easily scaled up.

[0029] The polymer brush layer 122 is formed from a hydrophilic polymer. A hydrophilic polymer is a polymer that has a high affinity for water. Since nucleic acids are dissolved in water during electrophoresis, the polymer brush layer 122 formed from a hydrophilic polymer is able to interact with the nucleic acids dissolved in water due to its properties. From the perspective of enhancing the interaction with nucleic acids, it is preferable that the hydrophilic polymer have high hydration properties.

[0030] Examples of hydrophilic polymers include polyacrylic acid, polyethyleneimine, polyalkylene glycol, 2-methacryloyloxyethyl phosphorylcholine (MPC), polyvinyl alcohol, and polyvinylpyrrolidone. The hydrophilic polymer is preferably biocompatible because it can prevent nucleic acids from adhering to the channel. Examples of biocompatible hydrophilic polymers include polyalkylene glycol, 2-methacryloyloxyethyl phosphorylcholine (MPC), polyvinyl alcohol, and polyvinylpyrrolidone.

[0031] Methods for forming the polymer brush layer 122 on the substrate 110 are widely known. For example, the polymer brush layer 122 can be formed by a surface-initiated graft polymerization method. The surface-initiated graft polymerization method generally includes a step of introducing a polymerization initiator group onto the surface of the substrate and a step of graft polymerizing the polymerization initiator group with a monomer.

[0032] The step of introducing polymerization initiator groups into the surface of substrate 110, which will become the inner surface of flow channel 120, can be performed, for example, by preparing a solution of a polymerization initiator and applying this solution to the surface of substrate 110. If the material of substrate 110 is one into which polymerization initiator groups are difficult to introduce, the polymerization initiator groups can be introduced after forming a coating onto the surface of substrate 110 into which polymerization initiator groups can be introduced. The type of polymerization initiator group can be selected appropriately depending on the type of polymer that constitutes polymer brush layer 122.

[0033] The step of graft polymerizing the polymerization initiator groups and the monomers can be carried out, for example, by preparing a monomer solution, applying this solution to the surface of substrate 110 to which the polymerization initiator groups have been introduced, and graft polymerizing the polymerization initiator groups and the monomers. The reaction conditions for the graft polymerization can be adjusted as appropriate depending on the desired properties of the polymer brush layer, such as the thickness and density.

[0034] In the fluidic device 100 according to this embodiment, the thickness of the polymer brush layer 122, i.e., the length of the polymer chains constituting the polymer brush layer 122, is set to adjust the migration speed of the nucleic acids to be electrophoresed and to separate them without damaging them. The thickness of the polymer brush layer 122 can be set to this value by adjusting the reaction conditions for graft polymerization.

[0035] The width, length, and height of the channel 120 can be appropriately set according to the thickness of the polymer brush layer 122 so as to separate the nucleic acids to be electrophoresed. For example, in this embodiment in which the polymer brush layer 122 is formed on the lower surface of the channel 120, the height of the channel 120 is 100 nm to 10 μm.

[0036] The cover member 130 is formed of a transparent material that allows electrophoresis of nucleic acids and allows the inside of the channel 120 to be viewed, allowing the state of electrophoresis of nucleic acids to be observed. Examples of materials for the cover member 130 include glass and resin.

[0037] The first electrode 140 and the second electrode 142 can be formed from a material (such as platinum) that is generally used for electrodes in electrophoresis.

[0038] The following describes the modified examples. In the description of the modified examples, the same components and members as those in the embodiment are denoted by the same reference numerals. Descriptions that overlap with the embodiment will be omitted as appropriate, and the description will focus on the configurations that differ from the embodiment.

[0039] (Variation 1) In the first embodiment, an example was shown in which the polymer brush layer 122 was formed on the lower surface of the flow channel 120, i.e., on the upper surface of the substrate 110, but the present disclosure is not limited to this. In the fluidic device 100 according to the present disclosure, the polymer brush layer 122 may be formed on the upper surface of the flow channel 120, i.e., on the lower surface of the cover member 130. This modification provides the same effects as the fluidic device 100 of the first embodiment.

[0040] (Variation 2) In the fluidic device 100 according to the present disclosure, polymer brush layers 122 may be formed on both the upper and lower surfaces of the channel 120. This modification can better prevent nucleic acids from being damaged in the channel during electrophoresis. The height of the channel 120 in this modification is, for example, 1 to 10 μm.

[0041] (Variation 3) In the first embodiment, an example was shown in which the thickness of the polymer brush layer 122 was set to adjust the migration speed of the nucleic acids to be electrophoresed and separate them without damaging them, but the present disclosure is not limited to this. In the fluidic device 100 according to the present disclosure, the thickness of the polymer brush layer 122 may be set to separate nucleic acids based on the difference in entanglement between the nucleic acids and the surface of the polymer brush layer 122 when the nucleic acids are electrophoresed. In this modification, multiple nucleic acids can be separated according to their respective sizes.

[0042] (Variation 4) In the fluidic device 100 according to the present disclosure, the thickness of the polymer brush layer 122 may be set so that nucleic acids smaller than the size of the target nucleic acid are removed by the polymer brush layer by electrophoresis. In this modification, the target nucleic acid can be separated from multiple nucleic acids.

[0043] (Variation 5) In the first embodiment, the polymer brush layer 122 is formed on the lower surface of the flow channel 120, but the present disclosure is not limited thereto. In the fluidic device 100 according to the present disclosure, at least one polymer brush layer may be provided on the inner surface of the flow channel to generate differences in electrophoretic speed of nucleic acids within the flow channel. For example, a polymer brush layer may be provided on a portion of the inner surface of the flow channel 120, or multiple polymer brush layers of different thicknesses may be provided on the inner surface of the flow channel 120. Specifically, for example, the thickness of the polymer brush layer at the electrophoresis start point of the flow channel 120 may be increased to align the migration start points of multiple nucleic acids. Alternatively, multiple polymer brush layers may be patterned on the inner surface of the flow channel 120. In this variation, the target nucleic acid can be purified or concentrated by separating it from multiple nucleic acids.

[0044] Methods for patterning polymer brush layers are well known, and techniques for patterning polymer brush layers on a micrometer-scale have been established. For example, photolithography can be used to form a pattern of regions containing polymerization initiator groups, followed by graft polymerization.

[0045] (Variation 6) In the first embodiment, an example was shown in which the fluidic device 100 includes one channel 120, but the present disclosure is not limited to this. The fluidic device 100 according to the present disclosure may include multiple channels. In this modification, multiple samples can be simultaneously passed through the multiple channels to separate the nucleic acids contained in each sample.

[0046] (Second embodiment) A nucleic acid separation method according to the second embodiment will be described. The nucleic acid separation method according to the second embodiment uses the fluidic device 100 of the first embodiment. In the second embodiment, the channel 120 of the fluidic device 100 is pre-filled with a buffer solution. The first electrode 140 and the second electrode 142 are connected to a power source via conductors. A solution (buffer solution) containing at least one nucleic acid is filled into the channel 120 at the starting point of electrophoresis. A voltage is applied between the first electrode 140 and the second electrode 142 by the power source, causing the nucleic acid to electrophorese within the channel 120.

[0047] As described above in the first embodiment, during electrophoresis, the migration speed of nucleic acids can be adjusted by interaction between the polymer brush layer 122 on the inner surface of the channel 120 and the nucleic acids, allowing large molecular weight nucleic acids to be separated without being damaged.

[0048] (Third embodiment) A nucleic acid separation method according to the third embodiment will be described. The nucleic acid separation method according to the third embodiment is a nucleic acid separation method using a fluidic device according to Modification 3 of the first embodiment. As in the second embodiment, in the third embodiment, the channel 120 of the fluidic device 100 is pre-filled with a buffer solution, and the first electrode 140 and the second electrode 142 are connected to a power source via conductors. Unlike the second embodiment, in the third embodiment, a solution containing multiple nucleic acids is filled into the channel at the electrophoresis start point, and a voltage is applied between the first electrode 140 and the second electrode 142 by the power source, causing the multiple nucleic acids to electrophorese.

[0049] 3(a) and 3(b) are diagrams illustrating the separation of multiple nucleic acids by a nucleic acid separation method according to a third embodiment. FIG. 3(a) is a schematic diagram showing the interior of a channel before electrophoresis. FIG. 3(b) is a schematic diagram showing the interior of a channel after electrophoresis. The polymer brush layer 122 is composed of polymer chains 122a. The nucleic acids 10, 12, and 14 have different sizes, with nucleic acid 10 being the largest and nucleic acid 14 being the smallest. In the third modification of the first embodiment, the thickness of the polymer brush layer 122 of the fluidic device 100, i.e., the length of the polymer chains 122a, is set to separate the nucleic acids based on the difference in entanglement between the nucleic acids and the surface of the polymer brush layer 122 during electrophoresis. Therefore, when the nucleic acids 10, 12, and 14 are electrophoresed in the direction of arrow D, the nucleic acids 10, 12, and 14 migrate at different distances depending on their sizes, as shown in FIG. 3(b). At the same electric field strength, the migration distance of the smallest nucleic acid 14 is the longest, and the migration distance of the large nucleic acid 10 is the shortest. In this way, the nucleic acids 10, 12, and 14 can be separated according to their respective sizes.

[0050] (Fourth embodiment) A nucleic acid separation method according to the fourth embodiment will be described. The nucleic acid separation method according to the fourth embodiment is a nucleic acid separation method using a fluidic device according to Modification 4 of the first embodiment. As in the second and third embodiments, in the fourth embodiment, the channel 120 of the fluidic device 100 is pre-filled with a buffer solution, and the first electrode 140 and the second electrode 142 are connected to a power source via conductors. As in the third embodiment, in the fourth embodiment, a solution containing multiple nucleic acids is filled at the electrophoresis start point of the channel, and a voltage is applied between the first electrode 140 and the second electrode 142 by a power source, causing the multiple nucleic acids to electrophorese.

[0051] 4(a) and 4(b) are diagrams illustrating the separation of multiple nucleic acids by a nucleic acid separation method according to the fourth embodiment. FIG. 4(a) is a schematic diagram showing the interior of a channel before electrophoresis. FIG. 4(b) is a schematic diagram showing the interior of a channel after electrophoresis. The polymer brush layer 122 is composed of polymer chains 122a. As in the third embodiment, the nucleic acids 10, 12, and 14 have different sizes. In the fourth modification of the first embodiment, the thickness of the polymer brush layer 122 of the fluidic device 100, i.e., the length of the polymer chains 122a, is set so that nucleic acids smaller than the size of the target nucleic acid are removed by the polymer brush layer during electrophoresis. In the fourth embodiment, the target nucleic acid is the nucleic acid 10. When the nucleic acids 10, 12, and 14 are electrophoresed in the direction of arrow D, the nucleic acids 12 and 14 migrate within the polymer brush layer 122, while the nucleic acid 10 migrates on the surface of the polymer brush layer 122, as shown in FIG. 4(b). At the same electric field strength, the nucleic acid 10 that has a small interaction with the polymer chain 122a has the highest migration speed. This allows the target nucleic acid 10 to be separated from the nucleic acids 12 and 14.

[0052] (Fifth embodiment) A nucleic acid separation method according to the fifth embodiment will be described. The nucleic acid separation method according to the fifth embodiment is a nucleic acid separation method using a fluidic device according to the fifth modification of the first embodiment. As in the second, third, and fourth embodiments, in the fifth embodiment, the channel 120 of the fluidic device 100 is pre-filled with a buffer solution, and the first electrode 140 and the second electrode 142 are connected to a power source via conductors. As in the third and fourth embodiments, in the fifth embodiment, a solution containing multiple nucleic acids is filled at the electrophoresis start point of the channel, and a voltage is applied between the first electrode 140 and the second electrode 142 by a power source.

[0053] 5(a) to 5(c) are diagrams illustrating the separation of multiple nucleic acids in a nucleic acid separation method according to a fifth embodiment. FIG. 5(a) is a schematic diagram showing the interior of a channel 120 before electrophoresis. FIG. 5(b) is a schematic diagram showing the interior of the channel 120 when a first electrophoresis voltage is applied to the channel 120. FIG. 5(c) is a schematic diagram showing the interior of the channel 120 when a second electrophoresis voltage higher than the first electrophoresis voltage is applied to the channel 120. In the fifth modification of the first embodiment, a polymer brush layer 122 is provided on the inner surface of the channel 120 to generate a difference in electrophoresis speed of nucleic acids within the channel 120. As shown in FIG. 5(a), the polymer brush layer 122 aligns the electrophoresis starting points of nucleic acid 16 and nucleic acid 18. Nucleic acid 16 is a target nucleic acid and is larger in size than nucleic acid 18. By applying a first electrophoresis voltage to the flow channel, as shown in Figure 5(b), small nucleic acids 18 electrophores within the polymer brush layer 122 while interacting with the polymer chains 122a, while large nucleic acids 16 remain at the starting point of electrophoresis. Then, by applying a second electrophoresis voltage higher than the first electrophoresis voltage to the flow channel, the nucleic acids 18 electrophores. This allows the target nucleic acid 18 to be separated from a mixture of nucleic acids 16 and 18, and purified or concentrated.

[0054] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to these examples. [Example]

[0055] Example 1: Preparation of polymer brush film In this example, a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer brush film was fabricated on a silicon substrate by surface-initiated graft polymerization. The fabrication process consisted of four steps: (1) deposition of a parylene film, (2) application of a photopolymerization initiator, (3) preparation of an MPC monomer aqueous solution, and (4) photopolymerization by UV irradiation. Each of these steps is described below.

[0056] (1) Deposition of parylene film Parylene is known as a coating agent with excellent waterproofing, insulating properties, chemical resistance, biosafety, and surface modification properties. Its basic structure is a benzene ring with methylene groups attached to both ends, and upon polymerization it becomes a very stable, colorless, transparent polymer. By modifying the benzene ring with various functional groups, it can exhibit a wide variety of properties. Its role in this example is to create a polymerizable surface on a silicon substrate. This is because silicon substrates do not have double bonds, making polymerization with MPC polymers impossible.

[0057] A substrate is placed in the chamber (evaporation chamber) of the parylene coating device and evaporated. A solid granular raw material called dimer is heated in the vacuum and vaporized to form a dimer gas. This gas is thermally decomposed, cleaving the dimer and converting it to a monomer form. In the evaporation chamber at room temperature, this monomer gas polymerizes on all surfaces, forming a thin, transparent polymer film. The amount of dimer used is 0.03 g, and the parylene film thickness is approximately 30 nm. The experimental equipment used was a parylene coating device (manufactured by KICSO, DACS-LAB).

[0058] (2) Application of photopolymerization initiator Benzophenone was used as the photopolymerization initiator. Benzophenone is an organic compound that has the property of absorbing ultraviolet light. In this example, it was used as a photopolymerization initiator, taking advantage of its ability to absorb ultraviolet light and exhibit photosensitization. Photosensitization is the ability to transfer the energy absorbed from light to other substances, thereby aiding in a reaction.

[0059] Benzophenone is soluble in acetone, so it was dissolved in acetone and applied uniformly using a spin coater. The concentration of the benzophenone acetone solution was 10 wt%, and 100 μL of the benzophenone acetone solution was dropped onto the substrate. The spin coater was used at a rotation speed of 2000 rpm for 30 seconds. The applied solution was then degassed to evaporate only the acetone. It was then heated at 50°C for 10 minutes to achieve a more uniform coating.

[0060] (3) Preparation of MPC monomer aqueous solution An MPC monomer aqueous solution is added dropwise and photopolymerized by UV irradiation. The concentration of the MPC monomer aqueous solution was adjusted by placing the MPC monomer in a vial and adding pure water. The concentration was adjusted according to the desired film thickness, based on 10 wt% (0.1 g of MPC monomer and 0.9 g of pure water). The relationship between film thickness and the concentration of the MPC monomer aqueous solution will be described later. The amount of liquid to be used is placed in a test tube and degassed by bubbling. The degassing time was 10 minutes per 10 mL of solution.

[0061] (4) Photopolymerization by UV irradiation The substrate coated with the photopolymerization initiator was placed on the irradiation table. A UV irradiation device (manufactured by CCS Corporation, model HLDL-100U6-NWPSC) was used for UV irradiation. The irradiation intensity ranged from 80 to 663 mW / cm. 2 Since UV irradiation causes the substrate temperature to rise, a water-cooled heat sink was introduced to suppress the temperature increase. The irradiation stage is a copper plate with a flow path, connected to a water-cooled heat sink. The water temperature of the water-cooled heat sink was set to 20°C. An aqueous solution of MPC monomer was dropped onto the substrate, which was then covered with a cover glass and irradiated with UV. When irradiated with UV, a photopolymerization reaction occurs starting from the photopolymerization initiator.

[0062] After the MPC polymer brush film was formed, the substrate was adsorbed onto the cover glass. When pure water was poured into the side of the cover glass, it became easier to slide off the cover glass. After that, the free polymer that had not grafted onto the substrate was removed by washing with pure water and ethanol.

[0063] An ellipsometer was used to measure the film thickness. To measure the film thickness of the prepared MPC polymer brush film, an automatic ellipsometer (MARY-102, manufactured by Five Labs) and a multispectral ellipsometer (FS-1, manufactured by Film Sense) were used.

[0064] To control the degree of entanglement between the nucleic acid and the MPC polymer brush, we controlled the brush length (film thickness) of the MPC polymer brush. The film thickness was controlled by adjusting the concentration of the MPC monomer aqueous solution and the UV irradiation intensity. These two parameters are shown in Table 1. [Table 1]

[0065] Figure 6 shows the results of film thickness control by adjusting the monomer concentration. UV irradiation was performed at an irradiation intensity of 200 mW / cm. 2 It was confirmed that the MPC polymer brush film thickness increased with increasing monomer concentration. The standard deviation within the same substrate also increased with increasing film thickness. Electrophoresis experiments were performed on substrates with a monomer concentration of 15 wt% or less, where the standard deviation within the same substrate was less than 10 nm.

[0066] Figure 7 shows the results of film thickness control by adjusting the UV light intensity. The MPC monomer concentrations were 22.2 wt% and 29.6 wt%. It was confirmed that the MPC polymer brush film thickness increased with increasing UV light intensity. The standard deviation within the same substrate decreased with increasing UV irradiation intensity.

[0067] Example 2 In this example, the film thickness dependency of the electrophoretic velocity of nucleic acids electrophoresing on the surface of a hydrated polymer brush film was investigated.

[0068] (Experimental sample) In this example, 48 kbp λ-DNA and 166 kbp T4-DNA were used as nucleic acid samples. bp is a unit representing the number of base pairs. The length of the fully extended state is 0.34 nm per bp, which is 16.3 μm for λ-DNA (48 kbp) and 56.4 μm for T4-DNA. The difference in length between the two different sizes of DNA is approximately 3.5 times.

[0069] To observe DNA molecules, DiYO™-1 (manufactured by AAT bioquest; excitation wavelength: 491 nm, emission wavelength: 508 nm, quantum yield: 0.52) was used as a fluorescent staining sample.

[0070] The concentration of DiYO-1 was adjusted so that one molecule of DiYO-1 intercalates for every five base pairs of the DNA molecule. Specifically, the DNA sample and DiYO-1 were mixed at a ratio of 1 ng to 0.1 μM / μL, resulting in a total volume of 3 μL. The procedure was as follows: 500 ng / μL λ-DNA was diluted to 10 ng / μL, and 5 mM / μL DiYO-1 was diluted to 0.1 μM / μL, and then mixed. After each dilution, the mixture was left for 1 hour to allow for natural diffusion to allow for mixing.

[0071] Tris-borate-EDTA (TBE) buffer was added for electrophoresis. The buffer was diluted with pure water to a concentration of 1 / 10. The nucleic acid solution and TBE buffer were mixed on the substrate.

[0072] The MPC polymer brush film used in this example had a dry thickness of 10 to 160 nm. The MPC polymer brush film used was hydrated with pure water. It is known that the brush film thickness increases several times when hydrated. The hydration time was approximately one day.

[0073] (Experimental equipment) Figure 8 shows the experimental setup used in Example 2. The experimental setup consisted of a silicon substrate with an MPC polymer brush film, a voltage / current source, and a fluorescence detection system. Platinum electrodes were fixed to the substrate and a voltage was applied. A multifunction generator (NF Circuit Design Block, WF1948) and a power amplifier (MESS-TEK, M-2617) were used as the voltage / current source. An arbitrary waveform was set using the function generator, and the voltage value was amplified using the power amplifier. In this example, the voltage of the function generator was set in the range of 0.1 V to 4.0 V, and the voltage was amplified 10 times using the power amplifier before being applied. The electrodes were made by cutting approximately 3 cm of 0.3 mm diameter platinum wire (As One, Φ0.3 mm x 1 m) and soldering it.

[0074] The fluorescence detection system used to detect DNA stained with DiYO-1 was comprised of an epifluorescence microscope (OLYMPUS, BX51N-33FL-2-SP), an EMCCD camera (Andor, iXonEM+897), and a PC. Fluorescence observation was performed using the epifluorescence microscope with an LED light source (LUMEN DYNAMICS, XLED1). Objective lenses were available with magnifications of 4x, 10x, 20x, 50x, and 100x, allowing observation from 40x to 1000x. Because the absorption spectrum of DiYO-1 used as a fluorescent sample is 491 nm and the emission spectrum is 508 nm, a 460 nm light source was used, and the MMNIBA3 fluorescence mirror unit was selected as the filter. Because high light source output caused fragmentation of nucleic acid molecules, the light source output was reduced and the EM gain was increased for imaging. Fluorescently labeled nucleic acid molecules were observed using an EMCCD camera (Andor, iXonEM+897). Data acquired by the camera was sent to a PC, imported into the imaging software Micro Manager 2.0 and Image J, and output as images and videos. In this example, the exposure time was set to 100 msec and the EM gain was set to 500.

[0075] (Experimental Method) The experimental method was to fix a silicon substrate with an MPC polymer brush film onto which a nucleic acid sample had been dropped onto a stage, and then fix an electrode onto the substrate. While maintaining a constant room temperature of 22°C, a voltage was applied and the migration of the nucleic acid sample was observed using a fluorescence microscope and a camera. The following two methods were used to add the nucleic acid sample to the silicon substrate with an MPC polymer brush film and to fix the electrode:

[0076] (1) A fluorescently labeled nucleic acid sample was dropped onto the center of a substrate coated with an MPC polymer brush film. The drop volumes were 0.3 μL for the nucleic acid sample and 2.7 μL for the buffer. A cover glass (18 mm × 18 mm) was placed over the sample. The nucleic acid solution spreads across the cover glass. The depth between the cover glass and the substrate was approximately 10 μm. This was determined by adjusting the height adjustment screw on the microscope stage when the focus was on the cover glass surface and the substrate surface. Platinum electrodes were placed next to the cover glass so that the distance between them was 2 cm. A Bemcot® (Ozu Sangyo Co., Ltd.) was placed next to the electrodes to secure the electrodes and retain the buffer. 10 μL of buffer was dropped onto the Bemcot and the experiment was performed. As the experiment continued, the buffer evaporated, sometimes preventing voltage application. In such cases, additional buffer was added. If images were taken immediately after the buffer was dropped, a flow would occur due to differences in the flow rate inside the cover glass, and this flow would carry away the nucleic acid molecules. Therefore, after dripping the buffer, the sample was left for 5 minutes and then photographed using a fluorescence microscope and an EMCCD camera. The voltage application was confirmed at an electric field strength of 10 V / cm. Figures 9(a) and 9(b) show schematic diagrams of the apparatus used in this experimental method (1). Figure 9(a) is a schematic diagram of the apparatus seen from above, and Figure 9(b) is a schematic diagram of the apparatus seen from the side. The voltage was gradually increased, and the migration of the DNA molecules was photographed.

[0077] (2) A jig was used to secure the electrodes. Figure 10 is a schematic diagram of the jig we fabricated. The electrodes were secured by placing the substrate, fitting the fasteners, and tightening the screws. The distance between the electrodes was 5.0 cm, and the height between the substrate and the cover glass was approximately 30 μm. This was determined by turning the height adjustment screw on the microscope stage when the focus was on the cover glass surface and the substrate surface. This method secures the electrodes more securely than experimental method (1), but the buffer had to fill the area without the substrate, so 200 μL of buffer was dripped all over. As a result, the height between the substrate and the cover glass was higher than in method (1).

[0078] (Analysis method) ImageJ was used to analyze electrophoresed DNA molecules. ImageJ is image analysis software, and a plugin called QuimP was used to track DNA molecules (Piotr Baniukiewicz, Richard Tyson and Till Bretschneider, QuimP Guide, https: / / pilip.lnx.warwick.ac.uk / docs / master / QuimP_Guide.html#x1-140008). QuimP is software for analyzing cell morphology. A function called BOA was used. Nucleic acid molecules can be tracked by adjusting the segmentation parameters. Image tracking provides information about the contour line. Specifically, the central coordinates, central velocity, perimeter of the contour line, elongation of the fitted ellipse (elongation = major axis / minor axis; a value of 1 indicates that a circle has been fitted, not a perfect circle), and circularity (circularity = 4πS / l 2 A value of 1 represents a perfect circle.) The area can be obtained. The segmentation parameters were Image F = 0.2, Node Spacing = 2, and Blowup = 10.

[0079] (Experimental conditions) The experimental conditions for Experimental Method (2) are summarized in Table 2. In Experimental Method (2), we compared the presence and absence of entanglement with the brush membrane, and investigated the electrophoresis of two types of nucleic acids with different sizes. The distance from the substrate to the cover glass was 20 to 30 μm. Images were taken with a 40x objective lens. [Table 2]

[0080] Next, the experimental conditions for experimental method (1) are summarized in Table 3. In experimental method (1), the distance from the substrate to the cover glass was 10 to 20 μm. This was the distance measured by turning the height adjustment screw on the microscope stage when the focus was on the cover glass surface and the substrate surface. The film thickness dependency of the MPC polymer brush film was investigated in the range of 10 to 130 nm. Images were taken with a 100x objective lens. [Table 3]

[0081] Figures 11 to 13 show a comparison of the results using the jig for experimental method (2). Figure 11 shows a comparison of the migration speed. Figure 12 shows a comparison of the mobility. Figure 13 shows a comparison of the circularity. Nucleic acids that are not entangled with the MPC polymer brush membrane were observed to migrate in a random coiled state, and in low electric fields, they exhibited significant Brownian motion. On the other hand, nucleic acids that are entangled with the MPC polymer brush membrane were observed to migrate while repeatedly expanding and contracting on the brush membrane. The presence or absence of entanglement is thought to be influenced by the distance from the substrate of the migrating nucleic acid.

[0082] Figures 11 to 13 show that the migration speed of nucleic acids decreases due to entanglement with the MPC polymer brush membrane. When there is no entanglement, the migration speed is proportional to the electric field strength. On the other hand, when there is entanglement, the change in migration speed is small. Looking at the mobility, which is the migration speed divided by the electric field strength, we can see that the mobility decreases as the electric field strength increases. The mobility of λ-DNA decreases linearly, while that of T4-DNA decreases exponentially. It is thought that the resistance of the brush increases with an increase in electric field strength, and this is thought to be a migration model different from conventional gel electrophoresis.

[0083] Experiments were conducted using MPC polymer brush membranes with different brush thicknesses in the experimental method (1). Figure 14 shows the film thickness dependence of migration velocity for λ-DNA. Figure 15 shows the film thickness dependence of mobility for λ-DNA. Figure 16 shows the film thickness dependence of circularity for λ-DNA.

[0084] Comparing the migration speeds, we found that the migration speeds tended to slow as the brush film thickness increased. This is thought to indicate that the entanglement between the nucleic acid and the MPC polymer brush film increases with increasing brush film thickness, resulting in an increased resistance force from the MPC polymer brush acting on the nucleic acid. The increase in speed differs between brush film thicknesses of 12 nm and above. For brush film thicknesses of 12 nm, the increase is seen to be linear with the electric field strength. On the other hand, for brush film thicknesses above this, the change in speed is small from an electric field strength of 4 V / cm to 12 V / cm, but becomes significant above 12 V / cm. The difference in mobility is striking, with mobility increasing at a brush film thickness of 12 nm. The increase in mobility represents a decrease in resistance. This is thought to be due to the dissociation of nucleic acid from the brush at higher electric fields. On the other hand, for film thicknesses of 29 nm and above, the mobility switches from decreasing to increasing at an electric field strength of 12 to 14 V / cm.

[0085] Next, experiments were performed using MPC polymer brush membranes with different brush thicknesses using experimental method (1). Figure 17 shows the membrane thickness dependence of migration velocity for T4-DNA. Figure 18 shows the membrane thickness dependence of mobility for T4-DNA. Figure 19 shows the membrane thickness dependence of circularity for T4-DNA. As with λ-DNA, when the brush membrane thickness was 15, 27, or 46 nm, the velocity increased linearly. When the brush membrane thickness was 62 or 108 nm, the velocity change became significant at the boundary of 12 to 14 V / cm.

[0086] The results for λ-DNA and T4-DNA showed two patterns of speed change in response to electric field strength: a pattern in which the speed increases at a constant rate (linear), and a pattern in which the migration speed increases sharply at a certain electric field strength (nonlinear). The migration speed increases linearly with electric field strength at 12 nm for λ-DNA and at 15 nm, 27 nm, and 46 nm for T4-DNA. At film thicknesses greater than this, the migration speed increases sharply at a certain electric field strength.

[0087] For λ-DNA, the migration speed increased sharply at a certain electric field strength when the brush film thickness was 29, 41, 72, and 125 nm, and for T4-DNA, it increased sharply at a certain electric field strength when the film thickness was 62 and 108 nm. Looking at the mobility, this is easier to understand, as the graph is U-shaped, centered around 12-14 V / cm. The resistance force from the brush film increases, and then decreases. This can be divided into two regions: one where mobility decreases, and one where mobility increases.

[0088] For both λ-DNA and T4-DNA, the circularity decreased with increasing electric field strength, indicating that the nucleic acid molecules were elongated.

[0089] Figure 20 shows the time series change in circularity of a 108 nm brush film of T4-DNA. The higher the electric field strength, the greater the fluctuation in circularity. This suggests that at high electric field strengths, the mobility increases due to the stochastic disentanglement of the DNA with the polymer brush. On the other hand, when the electric field strength is low, the circularity remains roughly constant, and the DNA does not disentangle from the polymer brush film, migrating while maintaining an elongated state, resulting in slower mobility.

[0090] Figure 21 shows the results of comparing λ-DNA and T4-DNA migrating on an MPC polymer brush membrane with a brush thickness of approximately 10 nm. The larger T4-DNA became more entangled with the brush membrane, resulting in a difference in migration speed. A maximum speed difference of 1.9 times was achieved at an electric field strength of 16 V / cm. Size separation using electrophoresis on this brush membrane surface is thought to enable size separation of multiple nucleic acids, as the magnitude of the resistance due to entanglement is thought to depend on molecular size.

[0091] Figure 22 shows the results of comparing λ-DNA and T4-DNA migration on an MPC polymer brush membrane with a brush film thickness of approximately 40 nm. As shown in Figure 22, for λ-DNA (48 kbp) and T4-DNA, the migration patterns differ depending on their size at film thicknesses of 30 to 40 nm. At an electric field strength of 12 V / cm, a maximum speed difference of 3.1 times was achieved. Because larger nucleic acids have higher mobility, this method may be applicable to applications such as the purification of nucleic acid drugs, where nucleic acids shorter than a certain size are problematic.

[0092] The results obtained in this example are described below. We observed that the migration speed of electrophoretic nucleic acid molecules slows down due to entanglement with the MPC polymer brush membrane. The electrophoresis of nucleic acid molecules was divided into two types depending on the thickness of the MPC polymer brush film: a linear type, where the dependence of the electrophoretic velocity on the electric field strength increases linearly from low electric fields, and a nonlinear type, where the dependence increases at an electric field strength of 15 V / cm. Furthermore, the film thickness at which the electrophoretic pattern switched differed between λ-DNA and T4-DNA. At a brush thickness of approximately 10 nm, a maximum speed difference of 1.9 times was achieved for λ-DNA and T4-DNA at an electric field strength of 16 V / cm due to differences in resistance caused by entanglement on the brush surface. Furthermore, at a brush thickness of approximately 40 nm, where the dependence of different migration speeds on electric field strength was observed, a maximum speed difference of 3.1 times was achieved.

[0093] The present disclosure has been described above based on the embodiments. These embodiments are merely examples, and it will be understood by those skilled in the art that various modifications are possible in the combination of the respective components and the respective treatment processes, and that such modifications are also within the scope of the present disclosure. [Explanation of symbols]

[0094] 100 fluidic device, 120 flow channel, 122 polymer brush layer.

Claims

1. a channel for electrophoresis of nucleic acids; A fluidic device, wherein a polymer brush layer is provided on the inner surface of the flow channel, the polymer brush layer including a hydrophilic polymer.

2. The fluidic device according to claim 1 , wherein the hydrophilic polymer is biocompatible.

3. The fluidic device according to claim 1, wherein the thickness of the polymer brush layer is set so as to separate the nucleic acids by utilizing the difference in entanglement between the nucleic acids and the surface of the polymer brush layer when the nucleic acids are electrophoresed.

4. The fluidic device according to claim 1 , wherein the thickness of the polymer brush layer is set so that nucleic acids smaller than the size of the target nucleic acid are removed by the polymer brush layer when the nucleic acid is electrophoresed.

5. The fluidic device according to claim 1 , wherein at least one of the polymer brush layers is provided on an inner surface of the flow channel so as to generate a difference in electrophoretic velocity of the nucleic acid within the flow channel.

6. A nucleic acid separation method using the fluidic device described in any one of claims 1 to 5, comprising filling a solution containing at least one nucleic acid at the electrophoresis starting point in the flow channel and electrophoresing the nucleic acid.

7. A nucleic acid separation method using the fluidic device described in claim 3, comprising filling a solution containing multiple nucleic acids at the electrophoresis starting point in the flow path, electrophoresing the multiple nucleic acids, and separating the multiple nucleic acids according to their respective sizes based on the difference in entanglement of each of the multiple nucleic acids with the surface of the polymer brush layer.

8. A nucleic acid separation method using the fluidic device described in claim 4, comprising filling a solution containing multiple nucleic acids at the electrophoresis starting point in the flow path, electrophoresing the multiple nucleic acids, and removing nucleic acids from the multiple nucleic acids that are smaller than the size of the target nucleic acid using the polymer brush layer, thereby separating the target nucleic acid.

9. 6. A nucleic acid separation method using the fluidic device described in claim 5, comprising filling a solution containing a plurality of nucleic acids at an electrophoresis starting point in the flow channel, applying a first electrophoresis voltage to the flow channel to electrophorese nucleic acids of a predetermined size or less among the plurality of nucleic acids, and applying a second electrophoresis voltage higher than the first electrophoresis voltage to the flow channel to electrophorese nucleic acids exceeding the predetermined size.

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