JP2008107259A - Capillary isoelectric point electrophoresis passage, substance separation method using the passage, and substrate for substance separation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a capillary isoelectric point electrophoresis technology having high separation accuracy. <P>SOLUTION: The technology provides a capillary isoelectric point electrophoresis passage, at least comprising a solid-phased amphoteric carrier on the surface of the inner wall of an electrophoresis capillary interposed between counter electrodes. Furthermore, an isoelectric point electrophoresis method comprises formation of solution pH gradient in the capillary isoelectric point electrophoresis passage; a two-dimensional separation method that uses isoelectric point electrophoresis method; and in addition, a basis for substance separation for use in the two-dimensional separation method. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a capillary isoelectric focusing technique. More particularly, the present invention relates to a capillary isoelectric focusing path in which an amphoteric carrier is solid-phased on the inner wall surface of a capillary, a substance separation method using the electrophoresis path, and a substance separation substrate.

  “Isoelectric point” refers to the pH at which the net charge (total) becomes zero in so-called ampholytes that dissociate in aqueous solution and exhibit both acid and base properties, such as amino acids and proteins. . In an aqueous solution in which different types of amphoteric electrolyte groups are mixed, the amphoteric electrolyte groups can be separated based on the difference in isoelectric point.

  When electrophoresis is performed in an electrophoresis gel forming a pH gradient, the amphoteric electrolyte can be moved (focused) to an electrophoresis position corresponding to each isoelectric point and aligned in the electrophoresis gel. Generally, it is called “isoelectric focusing method”. Further, a method of performing SDS-polyacrylamide gel electrophoresis (hereinafter abbreviated as SDS-PAGE) in the second dimension following this isoelectric focusing method is generally referred to as “two-dimensional electrophoresis method”. In particular, it is widely used for the separation and analysis of many kinds of proteins.

  Here, as a general method for preparing a pH gradient electrophoresis gel, a method of applying an electric field by adding an amphoteric carrier to the gel, or using an acrylamide derivative having various isoelectric side chains, the pH gradient is simultaneously generated. There is a method of forming (IPG (Immobilized pH gradient) method). In general, research in the field of proteomics often uses the latter IPG method, which is excellent in resolution and reproducibility.

  On the other hand, capillary electrophoresis is a general term for methods of performing electrophoresis in capillaries (capillaries). When electrophoresis is performed in a solution state without using a carrier such as an electrophoresis gel, it is difficult to handle because convection easily occurs due to Joule heat, but if the electrophoresis is performed in a capillary, the convection phenomenon can be prevented, There is an advantage that heat dissipation is also easy.

In recent years, there has been a two-dimensional electrophoresis method in which an electrophoresis electric field filled with a buffer for electrophoresis is formed in a separation channel of a microchip, and a minute amount of protein sample is simultaneously separated by molecular weight and isoelectric point. It has been proposed (see Patent Document 1).
Japanese Patent Application Laid-Open No. 2004-150899.

  In the conventional isoelectric focusing method, the adjustment work of the pH gradient electrophoresis gel is complicated and time-consuming, and there is room for improvement in the resolution by the pH gradient electrophoresis gel. Assuming a configuration in which isoelectric focusing is performed in a solution environment in the capillary, since the pH gradient electrophoresis gel is not used, the gel adjustment work itself can be omitted, but there is a problem to be improved in terms of separation accuracy. there were.

  Accordingly, the main object of the present invention is to provide a capillary isoelectric focusing technique with high separation accuracy.

  In the present invention, firstly, a capillary isoelectric focusing path having at least a configuration in which an amphoteric carrier is solid-phased on the inner wall surface of an electrophoresis capillary interposed between counter electrodes is provided. The amphoteric carrier has a functional group that can ionize hydrogen ions (for example, a carboxyl group) and a functional group that receives hydrogen ions (for example, an amino group).

  Secondly, the present invention provides an isoelectric focusing method which is performed by forming an aqueous solution pH gradient in the capillary isoelectric focusing path having the above-described configuration.

  Third, a first-dimensional separation step of performing isoelectric focusing in a capillary isoelectric focusing path having a structure in which an amphoteric carrier is solid-phased on the inner wall surface of the capillary, and an isoelectric focusing liquid obtained from the step A two-dimensional separation method is provided that performs at least a second-dimensional separation step in which centrifugal force is applied to perform separation based on molecular weight.

  Here, the second dimensional separation step of performing separation by molecular weight using centrifugal force may include a step of forming a concentration gradient depending on the molecular weight by the sedimentation equilibrium method in addition to the usual centrifugal separation method. And

  Fourth, a substance separation substrate is provided in which at least the capillary isoelectric focusing path and a second-dimensional separation path connected to the capillary isoelectric focusing path are provided. For example, the substrate may be a disc-shaped substrate, and the second-dimensional separation path may be a centrifugal separation path extending outward in the substrate radial direction. In the centrifugal separation path, the centrifugal separation method and the sedimentation equilibrium method can be performed based on the centrifugal force obtained by the rotation of the substrate.

  According to the present invention, capillary isoelectric focusing or two-dimensional separation can be performed with high separation accuracy.

  Embodiments of the method according to the present invention will be described below with reference to the accompanying drawings.

  First, the procedure of isoelectric focusing using the capillary isoelectric focusing path according to the present invention will be described with reference to FIGS. FIG. 1 is a workflow diagram showing a work procedure, and FIG. 2 is a schematic diagram showing a capillary isoelectric focusing path.

  First, in the first procedure, a substance to be separated 2 to be separated by isoelectric focusing is injected into the capillary isoelectric focusing path 1 (see S1 in FIG. 1). The substance to be separated 2 can be an object of separation if it is an amphoteric electrolyte, and usually a protein is often used as a substance to be separated. In the second procedure, an acidic solution 311 is injected on the positive electrode 31 side of the capillary isoelectric focusing path 1 and a basic solution 321 is injected on the negative electrode 32 side (see S2 in FIG. 1). As the acidic solution 311, a phosphoric acid solution and a sodium hydroxide solution are generally used as the basic solution 321, but any acidic and basic solutions can be used. Subsequently, in the third procedure, a voltage is applied from the external power source V to the positive electrode 31 and the negative electrode 32 of the capillary isoelectric focusing path 1 (see S3 in FIG. 1).

  Through the above steps S1 to S3, a pH gradient is formed in the capillary isoelectric focusing electrophoresis path 1 with the positive electrode 31 side being acidic and the negative electrode 32 side being basic, and the substances to be separated 2 have their respective isoelectric points. Focusing is performed to the migration position corresponding to.

  In general, isoelectric focusing is performed under constant current conditions, but after the start of isoelectric focusing, the formation of an ion gradient proceeds and moves between the positive electrode 31 and the negative electrode 32 of the capillary isoelectric focusing path 1. As the ionic substance to be reduced decreases, the electrical resistance of the capillary isoelectric focusing path 1 gradually increases.

  Therefore, in order to maintain the constant current, it is necessary to apply a large voltage between the positive electrode 31 and the negative electrode 32. At this time, a large amount of Joule heat is generated in the capillary isoelectric focusing path 1. This Joule heat causes convection in the capillary isoelectric focusing path 1 to destabilize the ion gradient, and changes the chemical properties of the substance to be separated, particularly when the substance to be separated is a protein. There is also a risk of affecting.

  Here, as shown in FIG. 2, a temperature management unit 4 is provided on the outer wall of the capillary isoelectric focusing path 1 to dissipate Joule heat, thereby destabilizing the ion gradient and thermal denaturation of the substance to be separated. We devised to prevent.

  The temperature management unit 4 can be composed of a heat conducting part 41 and a heat exchanging part 42, a heat conducting material such as a copper plate or a heat pipe is used for the heat conducting part 41, and a Peltier element or the like is used for the heat exchanging part 4. Heat exchange means such as a heat pump, a heat radiating plate and a fan can be employed without any particular limitation.

  Furthermore, it is preferable to give the temperature management unit 4 not only a function of radiating Joule heat, but also a function of keeping the entire capillary isoelectric focusing path 1 at a constant temperature. As a result, variations in temperature conditions for each isoelectric focusing can be eliminated, and the reproducibility of isoelectric focusing can be improved.

  Next, the configuration of the inner wall surface of the capillary isoelectric focusing path 1 according to the present invention will be described. As described above, the amphoteric carrier is solid-phased on the inner surface of the capillary isoelectric focusing path 1. FIG. 3 is a cross-sectional view of the capillary isoelectric focusing path 1, and shows the inner wall of the capillary isoelectric focusing path 1, the amphoteric carrier solid-phased on the inner wall, and the carboxyl group and amino group of the amphoteric carrier. The group is shown schematically.

  In FIG. 3, an amphoteric carrier 111 is uniformly solid-phased on the inner wall of the capillary isoelectric focusing path 1 indicated by reference numeral 11. The amphoteric carrier 111 is, for example, a polymer compound in which glycine, glycylglycine, amine, epichlorohydrin, or the like is polymerized, and has a carboxyl group and an amino group in its molecular structure. Here, the amphoteric carrier 111 is shown as a schematic diagram having one carboxyl group and one amino group, but the amphoteric carrier 111 may have a plurality of carboxyl groups and amino groups.

  The carboxyl group present in the molecular structure of the amphoteric carrier 111 ionizes and releases hydrogen ions depending on the pH of the solution in the capillary isoelectric focusing path 1. On the other hand, depending on the pH of the solution, the amino group takes in hydrogen ions in the solution by coordination bonds, and forms a state where there are many hydroxide ions in the solution.

  As described above, when a voltage is applied to the positive electrode 31 and the negative electrode 32 of the capillary isoelectric focusing path 1 (see S3 in FIG. 1), the positive electrode 31 side becomes acidic and the negative electrode 32 in the capillary isoelectric focusing path 1. A pH gradient with a basic side is formed. At this time, the carboxyl group and amino group of the amphoteric carrier 111 release hydrogen ions into the solution as described above when the pH of the surrounding solution changes with the formation of the pH gradient, or reversely incorporate it. , Exerts the function of promoting the formation of a pH gradient.

  And even if the pH gradient once formed changes for some reason, the function of stably maintaining the pH gradient is exhibited by releasing hydrogen ions into the solution or taking them in reverse. Therefore, in the capillary isoelectric focusing path 1 in which the amphoteric carrier 111 is solid-phased on the inner wall 11, a pH gradient can be quickly formed and stable isoelectric focusing is performed with high accuracy. It becomes possible.

  FIG. 4 is a schematic view showing an embodiment of a substance separation substrate according to the present invention.

  In FIG. 4, the substrate for substance analysis (hereinafter simply referred to as “substrate”) indicated by the symbol A in FIG. 4 has a disk-like form. The aperture size and the layer structure of the substrate A can be appropriately selected according to the purpose, and the configuration on the substrate can be designed or modified within the scope of the object of the present invention.

  The substrate A is made of a material that can transmit excitation light (for example, fluorescence excitation light) having a predetermined wavelength. For example, the substrate A is made of glass such as quartz, or a synthetic resin material such as polycarbonate or polystyrene. In addition, when the synthetic resin material which can be injection-molded is employ | adopted, reduction of a running cost can be achieved.

  The substrate A includes a capillary isoelectric focusing path 1 having a circular shape when viewed from above at the center of the disk. The isoelectric focusing path 1 has the same configuration as the capillary isoelectric focusing path 1 described with reference to FIGS. 1 to 3, and an amphoteric carrier is solidified on the inner surface of the capillary.

  In the substrate A, the first-dimensional separation step by the isoelectric focusing is performed in the capillary isoelectric focusing path 1, and subsequently, the two-dimensionally connected to the capillary isoelectric focusing path 1. The eye separation path 5 is configured so that a second-dimensional separation process using centrifugal force can be performed.

  That is, the substrate A has a center hole 6 at the center thereof, and a rotation shaft (not shown) is inserted into the center hole 6 and then rotated at a predetermined speed in the direction of arrow R in FIG. In the direction, a centrifugal force can be applied to the two-dimensional separation path 5 formed radially upward. In this figure, only eight second-dimensional separation paths 5 are shown, but the number of second-dimensional separation paths 5 can be arbitrarily designed.

  The two-dimensional separation method using the substrate A will be described more specifically below with reference to FIG.

  First, in the capillary isoelectric focusing path 1 which is a first-dimensional separation path provided in a circular shape in the center of the substrate, an insulating part 12 is provided at one point on the circumference. A positive electrode 31 and a negative electrode 32 are disposed at a distance. When a substance to be separated, an acidic solution, and a basic solution are injected into the capillary isoelectric focusing path 1 according to the work procedure shown in FIG. 1 and a voltage is applied between the positive electrode 31 and the negative electrode 32, the positive electrode 31 and the negative electrode 32. A pH gradient is formed between them, and the substances to be separated are focused at the respective isoelectric points and arranged in the capillary isoelectric focusing path 1.

  Here, in the vicinity of the center hole 6, a substance to be separated introduction section 7 that can introduce a substance to be separated in a predetermined volume is provided, and the substance to be separated is passed through the flow path 17 and the capillary isoelectric focusing path. 1 was introduced.

  Subsequently, the substances to be separated arranged circumferentially in the capillary isoelectric focusing path 1 are introduced into the second dimension separation path 5 and the second dimension separation step is performed using centrifugal force. At this time, as a method of introducing the isoelectric focusing liquid containing the substance to be separated from the capillary isoelectric focusing path 1 to the second dimension separating path 5, a method of moving using a capillary phenomenon, a moving by centrifugal force The method of making it, a negative pressure suction, a positive pressure extrusion, or the method of providing a passage control area | region etc. can be employ | adopted without being specifically limited.

  Among these methods, the method of providing a passage control region is to provide a passage control region capable of controlling the passage of a substance at the connection part 15 between the capillary isoelectric focusing path 1 and the second-dimensional separation path 5 so that the capillary isoelectric point can be controlled. This is a method for controlling the movement of the isoelectric point separation liquid from the point electrophoresis path 1 to the second dimension separation path 5.

  Specifically, a hydrophobic passage control region is provided to block the passage of the hydrophilic substance, and at an appropriate timing, the hydrophobic region is converted to hydrophilic to allow the hydrophilic substance to pass therethrough. Such a method can be considered.

  A suitable example is a method of forming a hydrophobic region with titanium oxide. Titanium oxide is a substance that is usually hydrophobic but changes to hydrophilicity when irradiated with ultraviolet rays. Therefore, if the passage control region is formed of titanium oxide, the movement of the isoelectric point separation liquid to the second dimensional separation path 5 can be blocked in the first dimensional separation step, and the ultraviolet light after the first dimensional separation step is completed. Can be introduced into the second-dimension separation path 5 by converting the passage control region into hydrophilicity.

  The isoelectric focusing liquid introduced from the capillary isoelectric focusing path 1 to the second dimensional separation path 5 in this way utilizes the centrifugal force applied by the rotation of the substrate A in the second dimensional separation path 5. To the second-dimensional separation step by the centrifugal separation method and sedimentation equilibrium method.

  In the centrifugal separation method, separation is performed based on the molecular weight of the substance to be separated. One example is sucrose concentration gradient centrifugation. According to this method, the sucrose solution is filled in the second-dimension separation path 5 and centrifuged to form a sucrose concentration gradient. In this concentration gradient, the substances to be separated are separated based on their molecular weights. can do. In this way, the substance to be separated that has been separated in the second dimension separation is separated from the second dimension separation path 5 and is subjected to subsequent analysis such as identification by a mass spectrometer, for example.

  In the sedimentation equilibrium method, the substance (solute) present in the solvent is sedimented by the action of centrifugal force, and the concentration of the solute when this sedimentation force and the diffusive force of the solute against it equilibrate (sedimentation equilibrium). This is a method for determining the molecular weight of a solute by measuring the distribution with an interference optical system or a photoelectric scanning device. The molecular weight determined by the sedimentation equilibrium method is characterized by not depending on the molecular structure of the solute, and even with a highly charged solute, the molecular weight can be accurately measured.

  Therefore, the sedimentation equilibrium method can be suitably employed in the second dimension separation step after isoelectric focusing, and can be said to be a method suitable as the second dimension separation step in the second dimension separation path 5. The substance to be separated whose molecular weight is measured by the sedimentation equilibrium method is subjected to subsequent analysis such as substance identification analysis based on the molecular weight.

It is the workflow figure which showed the work procedure of the isoelectric focusing using the capillary isoelectric focusing path which concerns on this invention. 1 is a schematic diagram of a capillary isoelectric focusing path 1. FIG. 2 is a schematic diagram showing an amphoteric carrier solidified on the inner wall of a capillary isoelectric focusing path 1. FIG. It is a top view of one embodiment of a substrate for separation of chambers concerning the present invention.

Explanation of symbols

A Substance separation substrate V External power source 1 Capillary isoelectric focusing path 11 Inner wall 12 Insulating part 15 Insulating part 15 Amphoteric carrier 2 Substance to be separated 31 Positive electrode 32 Negative electrode 311 Acidic solution 321 Basic solution 4 Temperature management unit 41 Thermal conduction part 42 Heat exchange section 5 Second dimension separation path 6 Center hole

Claims (6)

  A capillary isoelectric focusing path comprising at least a configuration in which an amphoteric carrier is solid-phased on the inner wall surface of an electrophoresis capillary interposed between opposing electrodes.   2. The capillary isoelectric focusing path according to claim 1, wherein the amphoteric carrier has a carboxyl group and an amino group.   An isoelectric focusing method, which is performed by forming an aqueous solution pH gradient in the capillary isoelectric focusing path according to claim 1. A first dimensional separation step of performing isoelectric focusing in a capillary having a configuration in which an amphoteric carrier is solid-phased on an inner wall surface;
Applying a centrifugal force to the isoelectric point separation liquid obtained from the step, and performing a separation by molecular weight;
At least two-dimensional separation method. A capillary isoelectric focusing path comprising at least a configuration in which the inner wall surface of the capillary for electrophoresis interposed between the counter electrodes is solid-phased by an amphoteric carrier;
A second dimensional separation path connected to the capillary isoelectric focusing path;
A substrate for substance separation, characterized in that at least is provided. A disk-shaped substrate,
6. The substance separation substrate according to claim 5, wherein the second dimension separation path is a centrifugal separation path extending outward in the radial direction of the substrate.