Flow path device for separating proteins and separation method using the flow path device for separating proteins

Abstract

To separate normal protein from denatured and inactivated protein only by making them pass through a flow channel device.SOLUTION: A flow channel device separating normal protein from denatured and inactivated protein is provided, having: a hydrophilic coating portion for inducing an aqueous solution including protein; a liquid pool-shaped portion having a bent, curved shape; and a hydrophobic coating portion for binding and trapping denatured and inactivated protein.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a flow path device for separating normal proteins from denatured and inactivated proteins, and a separation method using the flow path device for separating normal proteins from denatured and inactivated proteins. [Background technology]

[0002] In recent years, medical and biomedical applications have revealed that even cells of the same type possess unique properties. This necessitates the need for analysis at the single-cell level. Analysis at the single-cell level is extremely challenging due to the much smaller sample volumes required compared to conventional methods. However, microfluidic devices, which perform various chemical reactions and analyses within microchannels as thin as a human hair (several tens to hundreds of micrometers), have attracted attention since the 2000s, and their applications have been widely adopted in a variety of fields. In recent years, "extended nanofluidic devices" have been proposed to enable more advanced analysis in the "extended nanoregion," a region of several tens to hundreds of nanometers, approximately one-hundredth the size of a microchannel. This technology is expected to revolutionize medical and biomedical researchers. "Extended nanofluidic devices" have extended nanochannels, whose channel volumes are much smaller than the volume of a single cell, enabling efficient sampling of components from a single cell. For this reason, the "extended nanofluidic device" is widely anticipated in fields ranging from research to production technology as a "dream analytical technology" that will enable analysis to distinguish individual differences within a group of cells.

[0003] On the other hand, proteins present in cells are structured by peptide bonds between amino acids, forming a primary structure (sequence of amino acids), a secondary structure (a local, regular three-dimensional structure formed by the main chain of a polypeptide chain with nearby amino acids in the primary sequence), a tertiary structure (a spatial three-dimensional structure formed by polypeptide chains that have formed secondary structures), and a quaternary structure (a spatial arrangement in which multiple polypeptide chains with tertiary structures associate to form multi-subunits). When these proteins are denatured or inactivated by heat or other factors, the protein structure collapses and they can no longer be reacted with enzymes. While it is possible to determine the state of inactivation by actually causing the reaction, this takes time and effort. A method is needed to separate normal proteins from inactivated proteins without causing the reaction.

[0004] An example of using microchannels to separate proteins is a method in which a restricted permeation region is provided in the channel to separate molecules based on their size (Patent Document 1). For example, when separating particles with a particle size of 50 nm or less, such as proteins, high processing precision is required for the shape of the obstacles relative to the size of the particles to be separated. This has required expensive processing technology that can achieve high processing precision. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-354364 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made in view of the above circumstances, and aims to separate normal proteins from denatured and inactivated proteins simply by passing them through a flow path device. [Means for solving the problem]

[0007] In order to solve the above problems, the first aspect of the present invention is to provide a method for treating a normal protein by denaturing the normal protein. Separation of activated protein Dimethylpolysiloxane-based A flow path device for conducting an aqueous solution containing a protein The substrate was grafted with a hydrophilic polymer layer. Hydrophilic coating and , curved, curvaceous whole A liquid pool-shaped portion having a shape to , hydrophobicity to bind and capture denatured and inactivated proteins region The present invention is characterized by having the following.

[0008] In order to solve the above problems, a second aspect of the present invention is a separation method using a flow channel device for separating normal proteins from denatured and inactivated proteins, the method comprising: In parts A step of inducing an aqueous solution containing a protein, a step of generating a vortex in the aqueous solution by a liquid pool-shaped portion having a curved, curved shape, and a step of forming a hydrophobic region and a step of binding and capturing the protein that has been denatured and inactivated by the method. [Effects of the Invention]

[0009] According to the present invention, whether a protein has been denatured and inactivated can be determined by separating normal proteins from denatured and inactivated proteins simply by passing the proteins through a flow path device without enzymatic reaction. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a schematic diagram showing the entire flow channel device for separating proteins in one embodiment. [Figure 2] FIG. 1 is a schematic diagram showing a reservoir-shaped portion for separating proteins in a channel device for separating proteins according to one embodiment. [Figure 3] FIG. 1 is a schematic diagram showing the behavior of normal proteins and denatured and inactivated proteins when an aqueous solution containing proteins is injected into a flow channel device for separating proteins according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0011] A flow channel device for separating proteins according to an embodiment of the present invention will be described below with reference to the drawings. The drawings are schematic and differ in size, proportions, and the like from the actual device. Furthermore, the embodiments shown below exemplify configurations for embodying the technical concept of the present invention, and the technical concept of the present invention is not limited to the following in terms of the constituent materials, shape, structure, and the like. The technical concept of the present invention can be modified in various ways within the technical scope defined by the claims.

[0012] FIG. 1 is a schematic diagram showing the entire flow channel device for separating proteins of the present invention.

[0013] 2 is a schematic diagram showing an enlarged view of the reservoir-shaped portion of the flow channel device. An aqueous solution containing proteins is injected from an inlet 10 and discharged from an outlet 11 through a reservoir-shaped portion 14. Normal proteins flow through the flow channel as they are, but denatured and inactivated proteins are absorbed by the hydrophobicity of the reservoir-shaped portion 14. region The protein is captured by the nano-processed discharge flow channel 13, which is much thinner than the fibrous protein, and is then trapped in the liquid reservoir 14. Furthermore, small protein clumps, which are formed when the hydrophobic portions of the protein bind together and the surface becomes hydrophilic, are detected by the spectroscopic detection system 17 in the discharge flow channel 13.

[0014] In Figure 3, if the inlet channel 12 and outlet channel 13 are hydrophilically coated, hydrophobic proteins will not stick to them under flowing water conditions, and hydrophilic proteins will flow along with the aqueous solution. Therefore, by coating the area around the inlet 10 with a hydrophilic coating, normal proteins 20 will flow along with the aqueous solution, and denatured and inactivated proteins with exposed hydrophobic amino acid portions will flow along with the aqueous solution. Protein 21 also flows through the channel.

[0015] The hydrophobic coating is applied to the hydrophilic coating portion 16. region 15. Hydrophobic region The hydrophobic coating portion 15 is a liquid pool-shaped portion 14 having a larger volume than the hydrophilic coating portion 16. region Protein 21, which has exposed hydrophobic amino acids and passes near the wall of 15, binds to the wall by the action of hydrophobic aggregation. The curved shape of the liquid pool makes it easy for the aqueous solution containing the protein to form a vortex. This creates a structure that makes it easy for the protein to accumulate. The accumulated protein is hydrophobic. region If there are two or more exposed hydrophobic amino acids, they will be connected to form a fiber. If the hydrophobic amino acid is exposed at the tip of this fibrous protein, the hydrophobic amino acid will be exposed while the liquid pool is stagnating. region 15 and is captured.

[0016] The curved shape of the liquid pool is preferably sharply curved rather than gently curved, as this makes it easier to create a vortex. Also, by providing pillar shapes on the curved wall of the liquid pool, it becomes easier to create a vortex.

[0017] However, if the protein is a fibrous protein without exposed hydrophobic amino acids at the tip, region Therefore, for proteins that are large in length or volume, the discharge side flow channel 13 can be made thin using nano-level processing, thereby making it possible to create a structure that prevents proteins from flowing to the discharge side.

[0018] Examples of materials used for the flow channel of the present invention include glass, dimethylpolysiloxane, etc. Dimethylpolysiloxane is preferred because it is a hydrophobic material.

[0019] The material used for the hydrophilic coating portion can be obtained by grafting the surface of dimethylpolysiloxane with a hydrophilic polymer layer or by modifying it with plasma, or it can be another hydrophilic material.

[0020] Hydrophobic region Examples of materials used for the electrode include glass and dimethylpolysiloxane.

[0021] The following describes embodiments of the present invention, but the present invention is not limited to the following. [Example]

[0022] Hereinafter, specific embodiments of the present invention will be described.

[0023] [Method of manufacturing a flow channel device] A method for manufacturing the flow channel device for separating proteins shown in Figure 1 will be described. First, a first dimethylpolysiloxane substrate was prepared. Lithography was performed using an electron beam exposure device, and the shape of the flow channel was obtained by dry etching. The area of ​​the flow channel corresponding to the hydrophilic coating region 16 was modified to be hydrophilic using a PDMS surface modification kit (Merck). A second dimethylpolysiloxane substrate was then laminated onto the first dimethylpolysiloxane substrate with the flow channel formed thereon, and the two surfaces were bonded together to produce a flow channel device for separating proteins. Note that, like the first dimethylpolysiloxane substrate, the area of ​​the second dimethylpolysiloxane substrate corresponding to the hydrophilic coating region 16 was modified to be hydrophilic, as shown in Figure 2.

[0024] This flow path device is a wall of the injection side flow path 12 and a second dimethylpolysiloxane substrate side. The upper surface and the lower surface, which is the first dimethylpolysiloxane substrate side, are hydrophilic. The walls, upper surface and lower surface of the shaped portion 14 are hydrophobic region 15, it can bind and capture denatured and inactivated protein 21 with exposed hydrophobic amino acid moieties.

[0025] Furthermore, the upper surface of the reservoir-shaped portion 14, which faces the second dimethylpolysiloxane substrate, may be made of hydrophilic polyethylene terephthalate, or a portion of the second dimethylpolysiloxane substrate corresponding to the hydrophobic coating portion 15 may be modified to be hydrophilic. Because it is hydrophilic, hydrophilic proteins are not captured, making it suitable for observing the interior of the reservoir-shaped portion.

[0026] Next, a separation method using a flow channel device for separating proteins will be described.

[0027] [Embodiment 1 of the separation method] (preparation) First, the flow channel device 1 was placed on a syringe pump 3 via an inlet pipe 4 connected to an inlet 10. A syringe 2 containing an aqueous solution containing protein was fixed to the syringe pump 3. Kimwipe S-200 (manufactured by Nippon Paper Crecia Co., Ltd.) was placed under the outlet part 11 of the flow channel, and the syringe pump 3 was adjusted to allow the aqueous solution to flow at a desired flow rate. A spectroscopic measurement system 17 was placed in the outlet side flow channel 13. An outline of this is shown in Figure 1.

[0028] (Separation method) Next, a syringe pump was used to push the aqueous solution out of the syringe. The aqueous solution containing the protein pushed out of the syringe was sucked into the channel by capillary action, and the sucked aqueous solution was retained inside the reservoir-shaped part due to vortices generated by the curved shape of the reservoir-shaped part. While retained, the denatured and inactivated protein was bound to and captured by the hydrophobic coating site 15. This allowed the normal protein to be separated from the denatured and inactivated protein. A schematic diagram is shown in Figure 3.

[0029] (Measurement with spectroscopic measurement system) The amino acids that make up proteins, tryptophan, tyrosine, and phenylalanine, absorb ultraviolet light around 270 nm, so when the target protein passes through the spectroscopic measurement system, the type of protein that is passing through can be determined from the absorption wavelength and absorbance.

[0030] By using the protein separation flow channel device of the present invention, it became possible to separate normal proteins from denatured and inactivated proteins. This allowed us to understand the state of inactivation without causing an enzymatic reaction, and furthermore, it was possible to selectively bind and capture denatured and inactivated proteins on the wall surface. Furthermore, it was possible to pass only aqueous solutions containing a large amount of normal protein, thereby making it possible to easily increase the proportion of normal protein. [Explanation of symbols]

[0031] 1. Flow path device 2 syringes 3 Syringe Pump 4. Inlet piping 10 Inlet 11 Outlet 12 Injection side flow path 13 Discharge side flow path 14 Liquid pool shape part 15 hydrophobic region 16 Hydrophilic coating area 17 Spectroscopic detection system 20 Normal Proteins 21 Denatured and inactivated proteins

Claims

1. A flow path device based on dimethylpolysiloxane that separates normal proteins from denatured and inactivated proteins, a hydrophilic coating portion obtained by grafting the substrate with a hydrophilic polymer layer to induce an aqueous solution containing a protein; The curved, pool-shaped portion has a hydrophobic region for binding and capturing denatured and inactivated proteins. A flow channel device for separating proteins, comprising:

2. In the flow path device according to claim 1, The pool-shaped portion has a plurality of pillars protruding from an inner surface thereof. A flow path device characterized by:

3. A separation method using the flow path device according to claim 1 or 2, introducing an aqueous solution containing a protein into the hydrophilic coating portion; generating a vortex in the aqueous solution by the pool-shaped portion; a step of binding and capturing the denatured and inactivated protein by the hydrophobic region; A separation method using a flow path device for separating proteins, comprising:

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