JP2005331411A - Isoelectric point electrophoretic chip and apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an isoelectric point electrophoretic chip not causing the diffusion of an electrode liquid in performing isoelectric point electrophoresis. <P>SOLUTION: A flow channel 16 for allowing a sample to flow is formed on a glass substrate 11, and reservoirs 14 and 15 for injecting the electrode liquid for producing a pH gradient and holes 12 and 13 for introducing or discharging the sample are formed to a substrate 10 at a position separate from the position of the reservoirs 14 and 15. Ion permeable semipermeable membranes 18 such as polymer semipermeable membranes or the like are fixed to the bottoms of the reservoirs 14 and 15 by an adhesive and the electrode liquid is prevented from penetrating in the flow channel. Electrodes are inserted in the reservoirs 14 and 15 to apply voltage and the flow channel is irradiated with light such as UV rays or the like from a light source and detected by a unidimensional detector such as a photodiode array or the like arranged on the side opposite to the light source to detect the convergence of protein to be measured. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an isoelectric focusing apparatus that separates and analyzes amphoteric electrolytes such as peptides and proteins at their isoelectric point (PI), and isoelectric focusing that is particularly suitable for proteomic analysis using a microchip. This relates to an electrophoresis chip.

  For protein research, SDS-PAGE (SDS-polyacrylamide gel electrophoresis) is used as a separation method based on the difference in molecular size and charge, and isoelectric focusing is used as a separation method using an isoelectric point. Yes.

In recent years, in the field of analytical chemistry, research on μTAS (Micro Total Analysis Systems) has become active, and it is expected to use a microchip to achieve high-speed analysis, sample reduction, and solvent saving.
In particular, since proteome analysis uses expensive and precious samples, high throughput is required, and protein analysis by isoelectric focusing using a microchip is particularly useful.

  Isoelectric focusing is one of the protein analysis methods. By applying a voltage using an amphoteric electrolyte solution that forms a pH gradient, the protein is converged at the isoelectric point of the protein for identification and quantification. (See Non-Patent Document 1).

In conventional isoelectric focusing, it is necessary to add an electrode solution for passing hydrogen ions and hydroxide ions to both ends of the channel filled with the amphoteric electrolyte solution. It was a problem that the electrode liquid entered the flow path due to the difference between the water head and the water head at the time of injecting the electrode liquid. Therefore, the electrode liquid was flowed by adding a highly viscous reagent to the electrode liquid. To prevent intrusion.
IEEJ Technical Committee Materials CHS-03-53

When performing isoelectric focusing, if the pH of the sample is extremely high or low, the electrode solution is decomposed by hydrolysis, and operations such as washing the electrophoresis device due to the high viscosity of the electrode solution. There was a drawback that it was difficult.
An object of the present invention is to provide an isoelectric focusing device having excellent operability and an electrophoresis chip using a microchip.

In the present invention, in an isoelectric focusing chip having a flow channel inside a plate-like member and having liquid inlets and outlets at both ends of the channel, the liquid inlet / outlet is separated from the liquid inlet / outlet at two positions along the channel. An electrode solution introduction hole for forming a pH gradient is formed, and the hole and the flow path are connected via a material through which ions pass.
Here, the “chip” does not necessarily mean that it is small, but means that a flow path is formed inside a plate-like member.

The preferred material is a semipermeable membrane. As the semipermeable membrane, for example, from the group consisting of regenerated cellulose, cellulose acetate, polyacrylonitrile copolymer, polymethyl methacrylate, ethylene vinyl alcohol copolymer, aromatic polysulfone, aromatic polyamide, and carbonate-ethylene oxide copolymer. A selected polymer compound can be used.
The material can be fixed to the plate member with an adhesive.

  The isoelectric focusing device of the present invention includes an isoelectric focusing chip of the present invention, a power supply device for applying a voltage to a flow path between electrode liquid introduction holes of the isoelectric focusing chip, and the flow path. A light source for irradiating light, and a detector for detecting light from the flow path by the light of the light source.

In the isoelectric focusing chip of the present invention, in order to prevent the diffusion of the electrode liquid and the water head difference at the time of injecting the electrode liquid, the electrode liquid for forming a pH gradient and the sample to be electrophoresed are separately disposed, By disposing an ion-permeable membrane at the interface where the liquid inlet and the flow path inside the plate-like member are in contact, the electrode liquid does not diffuse or enter the flow path, so that the analysis operation can be performed easily. .
In addition, since it is not necessary to use a highly viscous electrode solution that has been used to prevent the electrode solution from entering the flow path, it is possible to increase the speed of analysis even in electrophoresis devices, sample washing, etc. Become.

  As described above, by using the present invention, isoelectric focusing analysis can be performed simply, at high speed and with high accuracy.

FIG. 1 illustrates an embodiment of an isoelectric focusing chip of the present invention. (A) is a plan view, and (B) is a cross-sectional view at the XX line position. The substrates 10 and 11 are glass substrates, for example, quartz glass substrates. Both glass substrates 10 and 11 are joined by hydrofluoric acid or the like to form a chip. On the surface of the glass substrate 11 on the bonding surface side, a minute flow channel 16 having a width and depth of several hundred μm or less and used as a liquid sample analysis flow channel is formed.
In the glass substrate 10, through holes 12 and 13 for introducing or discharging a liquid sample are formed at positions corresponding to both ends of the flow path 16.

  Further, reservoirs 14 and 15 serving as electrode solution introduction holes are formed at two positions along the flow path 16 between the holes 12 and 13 in the glass substrate 10, and the bottoms of the reservoirs 14 and 15 are made of, for example, cellulose acetate or the like. An ion-permeable polymer semipermeable membrane 18 is fixed with an adhesive and is connected to the flow path 16 through the membrane. The reservoirs 14 and 15 store an electrode solution that causes a pH gradient.

  The isoelectric focusing chip of the present invention is not limited to the structure shown in FIG. 1, but has a structure in which the flow path 16, the holes 12, 13 and the reservoirs 14, 15 are formed on the same substrate, or a combination of three substrates. As long as the sample introduction port and the reservoir are separately arranged, such as a structure in which a flow path is formed inside one glass substrate. Further, the shape of the flow path is not limited to that of the embodiment.

  Examples of ion permeable membranes include polymer compounds such as regenerated cellulose, cellulose acetate, polyacrylonitrile copolymer, polymethyl methacrylate, ethylene vinyl alcohol copolymer, aromatic polysulfone, aromatic polyamide, and carbonate / ethylene oxide copolymer. The dialysis efficiency can be improved by reducing the film thickness and decreasing the salt film resistance on the dialysate side.

  In the production method of an ion permeable membrane, a spinning solution in which a polymer is dissolved in a solvent is guided to a coagulation bath from an annular hollow nozzle directly or via air, and gelled, and the polymer and the solvent are phase-separated to form a hollow. Wet spinning method for yarn, gelling by cooling during running in the air, then dry-wet separation method in which solvent-free phase separation is performed in a coagulation bath, and spinning solution obtained by adding a plasticizer or the like to a polymer and thermally melting it from an annular hollow spinning port There are three types of melt spinning methods in which the plasticizer is removed in an aqueous solution after extrusion into air and cooling phase separation. In addition, as the hollow fiber forming agent used at this time, an inert gas or a solidifying or non-solidifying liquid is used. The membrane structure of the hollow fiber thus obtained is mainly determined by the size and aggregation of polymer particles formed in the phase separation process of the polymer and the solvent during film formation. It is determined by the method and conditions during film formation.

A method for manufacturing a chip according to one embodiment will be described. (A) First, after the glass substrate 11 is washed, a film of Si, Cr or the like is formed, and a photoresist is coated. (B) Next, the photoresist is exposed to UV (ultraviolet) light using a photomask. (C) Thereafter, the photoresist is developed and patterned. (D) Si and Cr are etched and patterned using the patterned photoresist as a mask, and the glass substrate is etched with, for example, a 46% hydrofluoric acid aqueous solution using the pattern of Si and Cr as a mask. Form. (E) The photoresist is removed, and Si, Cr, etc. are removed. (F) The sample introduction hole 12, the discharge hole 13, and the reservoir holes 14 and 15 are formed in the glass substrate 10 by a sandblast method or the like. (G) The glass substrates 10 and 11 are aligned so that the flow channel is inside, and are joined by hydrofluoric acid or the like.
The manufacturing method is not limited to the etching method, and can be performed by mechanical processing, laser processing, molding, or the like.

  FIG. 2 illustrates an embodiment of an isoelectric focusing device using the chip of the embodiment. The channel 16 in the isoelectric focusing chip is filled from the sample inlet 12 with a mixture of the amphoteric electrolyte solution and the protein to be measured. The reservoir 14 is filled with an acidic solution, and the reservoir 15 is filled with an alkaline solution. Electrodes are inserted into the reservoirs 14 and 15, and a voltage is applied to the flow path 16 between the reservoirs 14 and 15 by the power source 24. 20 is a voltmeter that measures the voltage applied to the flow path, and 22 is an ammeter that measures the current flowing through the flow path.

  Using the light source 26, for example, UV light 30 is irradiated along the flow path 16 of the isoelectric focusing chip 1, and one-dimensional such as a photodiode array disposed along the flow path 16 on the opposite side of the light source 26. Using the detector 32, the UV light 30 transmitted through the flow path 16 is detected. The light 30 is not limited to UV light, and may be visible light. After the voltage application, the detector 32 detects that the protein to be measured has converged.

  As the isoelectric focusing device, fluorescence generated from the sample protein in the flow path 16 may be detected. Further, a light source including a plurality of or continuous wavelengths may be used, the detector 32 may be a two-dimensional detector, and a spectroscope may be disposed to detect an absorption spectrum or a fluorescence spectrum due to the sample protein.

  The present invention can be used for isoelectric focusing analysis in which amphoteric electrolytes such as peptides and proteins are separated and analyzed at their isoelectric points.

It is a figure which shows one Example of an isoelectric focusing chip | tip, (A) is a top view, (B) is sectional drawing in the XX position. It is a schematic perspective view which shows one Example of an isoelectric focusing apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10,11 Glass substrate 12 Sample inlet 13 Sample outlet 14,15 Electrode solution reservoir 16 Channel 18 Semipermeable membrane 24 Power source 26 Light source 30 UV light 32 Detector

Claims (5)

In an isoelectric focusing chip having a flow path inside the plate-shaped member and having liquid inlets and outlets at both ends of the flow path,
Electrode solution introduction holes for forming a pH gradient different from the solution inlet / outlet are formed at two positions along the flow path,
An isoelectric focusing chip in which the hole and the channel are connected via a material through which ions pass.   The isoelectric focusing chip according to claim 1, wherein the material is a semipermeable membrane.   The semipermeable membrane is selected from the group consisting of regenerated cellulose, cellulose acetate, polyacrylonitrile copolymer, polymethyl methacrylate, ethylene vinyl alcohol copolymer, aromatic polysulfone, aromatic polyamide, and carbonate-ethylene oxide copolymer. The isoelectric focusing chip according to claim 2, which is a high molecular compound.   The isoelectric focusing chip according to any one of claims 1 to 3, wherein the material is fixed to a plate-like member with an adhesive. 5. The isoelectric focusing chip according to claim 1, a power supply device that applies a voltage to a flow path between the electrode liquid introduction holes, a light source that irradiates light to the flow path, and light from the light source And an isoelectric focusing device comprising a detector for detecting light from the flow path.

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