JP2008128774A - Substrate for substance analysis and system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new two-dimensional separation/migration technique for executing separation, detection, recovery, and analysis (measurement) of a solute molecule group in a specimen in a short time, simply, and highly accurately. <P>SOLUTION: This substrate for substance analysis is equipped at least with a specimen introduction part 2 provided on a disk-like substrate 1, a first dimensional separation path 3 communicating with the introduction part 2 and capable of separating solute molecules in the specimen by isoelectric focusing electrophoresis, a second dimensional separation path 4 communicating with the separation path 3 and extended radially to the outside of the substrate, and a recovery part 5 recovering a separated solution through the separation path 4. In this system, the same substrate is handled. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel two-dimensional separation of solute molecules that can be performed on a substrate and a detection technique thereof. More specifically, the present invention relates to a technique for accurately detecting a separated solute molecule by performing sedimentation separation (centrifugation) following isoelectric focusing on a disk-shaped substrate.

  There is a technique for detecting and detecting biochemical processes such as various hybridizations and interactions or reactions between molecules in a region or a channel formed in a predetermined shape on a substrate. Sensor chips called DNA chips (DNA microarrays) and protein chips are typical examples. The form and structure of such a sensor chip substrate vary depending on the purpose. Among them, a substrate in which a reaction field or a flow path is formed on a disk-like substrate has been proposed.

  For example, Patent Document 1 discloses a bioassay substrate having a structure in which a plurality of radial groove structures are formed on a disk-shaped substrate. One application of the bioassay substrate is a DNA chip.

  Patent Document 2 discloses a technique devised to apply an electric field to a reaction field disposed on a disk-shaped substrate. This technique is aimed at improving the accuracy of the hybridization reaction.

Patent Document 3 discloses a technique for performing electrophoresis of a sample by filling a gel-like medium in a capillary formed on a substrate. The capillary extends from the inner diameter side to the outer diameter side of the substrate, forms a gel flow in the capillary by centrifugal force, and performs electrophoresis in a direction opposite to the gel flow.
JP 2004-28992 A. JP 2004-45376. JP 2004-361198 A.

  Next, “two-dimensional electrophoresis” is known as a method for separating (fractionating) a plurality of types of solute molecules contained in a sample solution based on their properties and molecular weight. This two-dimensional electrophoresis method, in a broad sense, refers to a method of separating and analyzing solute molecules by sequentially performing electrophoresis in two directions (dimensions), but in a narrow sense, performing isoelectric focusing. This means a method of performing SDS-polyacrylamide gel electrophoresis (hereinafter abbreviated as SDS-PAGE) in the second dimension, and this method is currently widely used for separation and analysis of many kinds of proteins. In isoelectric focusing, when a solute molecule (amphoteric electrolyte) is electrophoresed in the presence of a pH gradient, it moves to the isoelectric point (the point at which the charge becomes 0) and stops, and each solute molecule is isoelectric. SDS-PAGE is a molecular sieve in which sodium dodecyl sulfate (SDS) is bound to a solute molecule in a molecular sieve polymerized in a mixed solution of acrylamide and N, N'-methylenebisacrylamide. This is the principle of applying a negative charge corresponding to, and moving it to the anode side for fractionation.

In recent years, a technique for performing the above two-dimensional electrophoresis method on a substrate has been proposed. For example, an electric field is formed on the first electrophoretic separation medium using a substrate on which a first electrophoretic separation medium and a second electrophoretic separation medium are formed with a space therebetween, and then the first electrophoretic separation medium is formed. A technique for separating into a second separation medium by applying an electric field in a direction perpendicular to the longitudinal direction of the electrophoretic separation medium has been proposed (see Patent Document 4). There has also been proposed a two-dimensional electrophoresis method in which an electrophoresis electric field 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 (see Patent Document 5).
JP 2006-162405 A. Japanese Patent Application Laid-Open No. 2004-150899.

  In the present invention, the morphological characteristics of the disk-shaped substrate can be effectively utilized to perform separation, detection, recovery, and analysis (measurement) of a solute molecule group in a sample in a short time with high accuracy. The main purpose is to provide a novel two-dimensional separation electrophoresis technique.

  In the present invention, first of all, a sample introduction part provided at a predetermined position of a disk-shaped substrate and a first dimension that communicates with the sample introduction part and can separate solute molecules in the sample by isoelectric focusing. A separation path, a second dimension separation path composed of a capillary communicated with the first dimension separation path and extending radially outward of the substrate, and a recovery unit for recovering the separation solution from the second dimension separation path; , At least a substrate for substance analysis.

  In this invention, a series of operation steps of sample introduction → first-dimensional separation by isoelectric focusing → second-dimensional separation in the capillary → sample recovery from the capillary (second-dimensional separation path) are performed on the same substrate. Thus, it is possible to carry out while controlling the execution timing of each process.

  In the present invention, the sample separation means and method in the second dimension separation path are not particularly limited. For example, in the second dimension separation path, sedimentation is caused by centrifugal force obtained when the substrate is rotated. It is good also as a structure in which the concentration gradient depending on the molecular weight at the time of equilibrium is formed. That is, the second dimension separation may be performed based on the sedimentation equilibrium method.

  In this “sedimentation equilibrium method”, when a solute molecule settles due to the action of centrifugal force and a concentration gradient occurs, a diffusion force works against the concentration gradient, and finally the balance between centrifugal force and nucleic acid force (precipitation equilibrium). Is a method based on the principle that a concentration gradient depending on the molecular weight is formed based on the molecular weight of the solute molecule, the molecular weight of the solute molecule, the dissociation constant (or equilibrium constant), the stoichiometry, and the molecular weight. The molecular shape can be estimated from the friction coefficient obtained from the sedimentation coefficient.

  The ultracentrifugation analysis includes the sedimentation equilibrium method performed by relatively low-speed centrifugation and the sedimentation velocity method performed at high speed. This “sedimentation rate method” is a method that can be performed in a shorter time (1-2 hours) than the sedimentation equilibrium method, and is more sensitive to molecular homogeneity than the equilibrium method. For this reason, it is generally used for a sample uniformity test, and at the same time, the sedimentation coefficient of each component can be determined. Given the sedimentation coefficient and molecular weight, the rough shape of the molecule can also be estimated. In principle, not only the sedimentation coefficient but also the diffusion coefficient can be measured at the same time by examining the time-dependent change in the shape of the sedimentation interface.As a result, the molecular weight can be obtained from the Svedberg equation. A method of calculating molecular weight relatively accurately ("SVEDBERG", described later) by simulating with a numerical solution of Lamm's equation and simultaneously obtaining the sedimentation coefficient s and the diffusion coefficient D by the method of least squares has been proposed. This method can be used when the sedimentation equilibrium method cannot be applied in a system in which the association gradually progresses while centrifuging for a long time. If the molecular weight is not so large, a relatively good molecular weight estimate can be obtained. Obtainable.

  Next, the present invention provides a system for handling the substance analysis substrate having the above-described configuration. Specifically, a substrate rotation driving means that can rotate while controlling the disk-like substrate at a predetermined speed according to the purpose, a sample supply means for the sample introduction unit, a voltage application means for the first-dimensional separation path, Provided is a substance analysis system comprising at least detection means for detecting solute molecules separated in the second-dimensional separation path based on the centrifugal force obtained by the rotation.

  In this material analysis system, sample supply to the substrate → voltage application during isoelectric separation in the first dimension separation path → sedimentation equilibrium by centrifugal force obtained by rotating the substrate (second dimension separation) → sedimentation equilibrium Thus, a series of operation steps of detecting the separated solute molecules forming a concentration gradient can be automatically performed on the same substrate while controlling the execution timing of each step by a computer.

  Further, in this system, (1) means for promoting the transfer of the sample from the first-dimensional separation path to the second-dimensional separation path, (2) means for promoting the recovery of the sample from the two-dimensional separation path, You may devise so that any one or both of (1) and (2) may be provided. The specific method of any of the promotion means (1) and (2) is not particularly limited. By adopting these means, it is possible to smoothly transfer the sample from the first dimension separation path to the second dimension separation path, or to smoothly collect the sample from the second dimension separation path. .

  The detection means of the present system includes, for example, a means for irradiating a selected position of the two-dimensional separation path with fluorescence excitation light and a fluorescence emitted from a fluorescently labeled molecule existing in the two-dimensional separation path. It is possible to employ a means including at least a means for capturing.

  According to the present invention, introduction (supply) of a sample containing a solute molecule for analysis, two-dimensional separation of a solute molecule group, detection / recovery / analysis (measurement) of a two-dimensionally separated solute molecule, etc. can be performed in a short time. It can be carried out easily and with high accuracy on the substrate.

  Embodiments of a substance analysis substrate according to the present invention will be described below with reference to the accompanying drawings. First, FIG. 1 is a diagram (upper view, plan view) showing a simplified configuration of a substrate for substance analysis according to the present invention.

  A substrate for substance analysis (hereinafter simply referred to as “substrate”) denoted by reference numeral 1 in FIG. 1 has a disk-like form as viewed from above. The aperture size and layer structure of the substrate 1 can be appropriately selected according to the purpose, and the configuration on the substrate can also be designed or modified within the scope of the object of the present invention.

  The substrate 1 is made of a material that can transmit excitation light (for example, fluorescence excitation light) having a predetermined wavelength. For example, the substrate 1 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 1 is electrically connected to positive and negative electrodes arranged at predetermined positions in the substrate 1 for the purpose of performing isoelectric focusing described later in the first-dimensional separation path, and from an external power source (not shown). Is provided with a conductor layer (not shown). This conductor layer can be formed of a light-transmitting and conductive material such as ITO (indium-tin-oxide) or aluminum. For example, ITO or aluminum is formed into a predetermined thickness (for example, about 150 nm) by sputtering or electron beam evaporation. Since this conductor layer has optical transparency, an optical pickup from the back side of the substrate can be employed.

In accordance with the object of the present invention, a region and a channel having a predetermined shape are formed on the substrate 1. For example, when the upper layer of the substrate is formed of a polyimide resin, it is more than that of an inorganic oxide film such as SiO _2. It is advantageous in terms of raw material costs, and since it is easier to form a thick film than the inorganic oxide film or the like, there is an advantage that the film formation cost is low.

  In particular, when the upper layer of the substrate is formed of a photosensitive polyimide resin, a region having a predetermined shape and size, a capillary, a channel, and the like can be easily formed by performing a surface treatment using a photoresist. For example, a photosensitive polyimide resin is applied to a substrate to a thickness of about 5 μm by spin coating or the like, and pattern exposure and development are performed using a laser beam exposure apparatus or the like, whereby a predetermined shape and size region, capillary, flow A road or the like can be easily formed.

  Subsequently, the configuration on the substrate will be specifically described. First, a central hole H into which a rotation shaft (not shown) capable of holding and rotating the substrate 1 can be introduced is provided at the center of the substrate 1. The rotating shaft may be a voltage supply means, and the conductor layer exposed on the inner peripheral wall surface of the center hole H may be energized.

  In the vicinity of the center hole H, a sample introduction unit 2 capable of introducing a predetermined volume of sample is provided, and this sample introduction unit 2 communicates with the first-dimensional separation path 3 through a flow path 21. ing. The first-dimensional separation path 3 is formed, for example, so as to form a capillary, and a solute molecule group (for example, a protein group that is an ampholyte) in the sample is separated in the capillary by isoelectric focusing. It has a possible configuration.

  Here, the procedure of isoelectric focusing will be described with reference to FIGS. FIG. 2 is a workflow diagram showing the work procedure of isoelectric focusing, and FIG. 3 is a diagram showing the configuration concept of the first-dimensional separation path 5 and the principle of isoelectric focusing. Note that the “electrophoresis path” in FIG. 2 means a first-dimensional separation path.

  First, in the first step (S1 in FIG. 2), a sample including a solute molecule group to be separated by isoelectric focusing is introduced from the sample introduction unit 2 into the first-dimensional separation path 3. A solute molecule group in a sample can be a separation target if it is an amphoteric electrolyte, and usually a protein is often a substance to be separated.

  Next, in the second step (S2 in FIG. 2), the acidic solution 311 is injected into the positive electrode 31 side, and the basic solution 321 is injected into the negative electrode 32 side, which is arranged in the first-dimensional separation path 3 so that a voltage can be applied. To do. 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, a voltage is applied from the external power source V to the positive electrode 31 and the negative electrode 32 in the third stub (see S3 in FIG. 2).

  Through the above steps S1 to S3, a pH gradient is formed in the first-dimensional separation path 3 with the positive electrode 31 side being acidic and the negative electrode 32 side being basic, and the solute molecule group P has isoelectricity of each solute molecule. Focusing is performed on a migration position corresponding to a point.

  Here, in general, isoelectric focusing is performed under a constant current condition, but after the start of isoelectric focusing, the formation of an ion gradient proceeds, and the first-dimensional separation path (isoelectric focusing path) 3 When the ionic substance moving between the positive electrode 31 and the negative electrode 32 of the first electrode 31 decreases, the electrical resistance of the first-dimensional separation path 3 gradually increases. Therefore, in order to maintain the constant current, the positive electrode 31 A large voltage needs to be applied between the negative electrode 32 and the negative electrode 32, and a large amount of Joule heat is generated in the first-dimensional separation path 3.

  This Joule heat generates convection in the first-dimensional separation path 1 and destabilizes the ion gradient. In particular, when the solute molecule group P to be separated is a protein, its chemical properties change. There is a risk.

As shown in FIG. 3, the first-dimensional separation path 3 is provided with a temperature management unit U to dissipate the Joule heat so as to prevent destabilization of the ion gradient and thermal denaturation of the substance to be separated. To do. The temperature management unit U includes a heat conducting unit U ₁ and a heat exchanging unit U _{2. The} heat conducting unit U ₁ includes a heat conductive material such as a copper plate or a heat pipe, and the heat exchanging unit U ₂ includes a Peltier element. It is possible to employ heat exchange means such as a heat pump, a heat radiating plate, and a fan without any particular limitation.

  Furthermore, it is preferable to give the temperature management unit U not only a function of radiating Joule heat but also a function of keeping the entire first-dimensional separation path 3 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, a preferred configuration of the inner wall surface of the first-dimensional separation path (isoelectric focusing electrophoresis path) 3 will be described with reference to FIG. A so-called amphoteric carrier 34 is solid-phased on the inner wall surface 33 of the first-dimensional separation path 3. In FIG. 4, the amphoteric carrier immobilized on the inner wall surface 33 of the first-dimensional separation path 3 and the carboxyl group and amino group of the amphoteric carrier are schematically shown.

  An amphoteric carrier 34 is uniformly solid-phased on the inner wall surface 33 of the first-dimensional separation path 3. The amphoteric carrier 34 is a polymer compound in which, for example, glycine, glycylglycine, amine, epichlorohydrin and the like are polymerized, and has a carboxyl group and an amino group in its molecular structure. FIG. 4 schematically shows a configuration in which the amphoteric carrier 34 has one carboxyl group and one amino group, but the amphoteric carrier 34 may have a plurality of carboxyl groups and amino groups.

  The carboxyl group present in the molecular structure of the amphoteric carrier 34 ionizes and releases hydrogen ions depending on the pH of the solvent in the first-dimensional separation path 3. On the other hand, depending on the pH of the solvent, the amino group takes in hydrogen ions in the solvent by coordination bonds, and forms a state where there are many hydroxide ions in the solvent.

  As described above, when a voltage is applied to the positive electrode 31 and the negative electrode 32 of the first dimension separation path 3 (see S3 in FIG. 2), the positive electrode 31 side is acidic and the negative electrode 32 side is defined in the first dimension separation path 3. A basic pH gradient is formed. At this time, the carboxyl group and amino group of the amphoteric carrier 34 release hydrogen ions into the solvent solution or take them in reverse, as described above, when the pH of the surrounding solvent changes with the formation of the pH gradient. Thus, the function of promoting the formation of a pH gradient is exhibited. And even when 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 it in reverse. .

  Thus, in the configuration in which the amphoteric carrier 34 is solid-phased on the inner wall surface 33 of the first-dimensional separation path 3, a pH gradient can be quickly formed in the solvent of the first-dimensional separation path 3, and as a result, stable Thus, it is possible to perform isoelectric focusing with high accuracy.

  Here, referring again to FIG. 1, the substrate 1 is provided with a second-dimensional separation path 4 provided so as to communicate with the first-dimensional separation path 3. The second-dimensional separation path 4 extends in the radial direction of the substrate 1 (see arrow X in FIG. 1), and a plurality of second-dimensional separation paths 4 are radially viewed from above. (See FIG. 1).

  The second-dimensional separation path 4 is a capillary configured to perform a second-dimensional separation process using centrifugal force. That is, the substrate 1 has a center hole H at the center thereof (as described above), and after inserting a rotation shaft (not shown) into the center hole 6 at a predetermined speed in the direction of arrow R in FIG. When rotated, a centrifugal force can be applied to the second-dimensional separation path 4 formed in the radial direction of the substrate 1. In FIG. 1, only a total of eight second-dimension separation paths 4 are shown, but the number of second-dimension separation paths 4 can be arbitrarily designed.

  Here, the two-dimensional separation method using the substrate 1 will be described more specifically with reference to FIGS.

  First, in the central region of the substrate 1, a first-dimensional separation path (electric focusing electrophoresis path) 3 provided in a circular shape (ring) as viewed from above is an insulating portion 33 (see FIG. 1), and a positive electrode 31 and a negative electrode 32 are disposed with the insulating portion 33 therebetween. In accordance with the operation steps (described above) shown in FIG. 2, the sample, the acidic solution 311 and the basic solution 312 are injected into the first-dimensional separation path (electric focusing electrophoresis path) 3, and the voltage is applied to the positive electrode 31 and the negative electrode 32. Is applied, a pH gradient is formed between the positive electrode 31 and the negative electrode 32, and the solute molecule group P (see FIG. 3) in the sample is focused on the isoelectric point of each solute molecule, and the first-dimensional separation path (electrical current) Point electrophoresis path 3).

  Following such first-dimensional separation, the solute molecule group P arranged in the circumferential direction in the first-dimensional separation path (electric focusing electrophoresis path) 3 is introduced into the second-dimensional separation path 4. In the second-dimensional separation 4, the second-dimensional separation using the centrifugal force obtained by rotating the substrate 1 is performed.

  At this time, an isoelectric point separation liquid (first-dimensional separation liquid) containing the solute molecule group P is introduced from the first-dimensional separation path 3 to the second-dimensional separation path 4. This isoelectric point separation liquid introduction method includes a method of moving using a capillary phenomenon, a method of moving using a centrifugal force, a negative pressure suction, a positive pressure extrusion, or a control method using a philicity principle. It is not limited and can be adopted.

  Among these methods, the control method using the amphiphilic principle is that a solute is present in the connecting portion M (see FIGS. 1 and 5) between the first-dimensional separation path (electric focusing electrophoresis path) 3 and the second-dimensional separation path 4. This is a method of controlling the movement of the isoelectric focusing liquid from the first dimension separation path 3 to the second dimension separation path 4 by providing an area where the passage of molecules can be controlled by the amphiphilic principle.

  Specifically, the connecting portion M is set as a hydrophobic region, the passage of the hydrophilic solute molecule is blocked, and the hydrophobic region is converted to hydrophilic at an appropriate timing to thereby convert the hydrophilic solute molecule. Pass through. 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 connection part M is formed of titanium oxide, the movement of the isoelectric point separation liquid to the second dimension separation path 4 at the stage where the first dimension separation process is performed can be blocked, and the first dimension separation is performed. After the process is completed, the isoelectric focusing liquid can be reliably and smoothly introduced into the second-dimension separation path 4 by irradiating the connection part M with ultraviolet rays and converting the hydrophobicity to the hydrophilicity. It becomes.

  In this way, the isoelectric separation liquid introduced from the first dimension separation 3 into the second dimension separation path 4 is centrifuged in the second dimension separation path 5 using the centrifugal force obtained by the rotation of the substrate 1. Or the second-dimensional separation step by sedimentation equilibrium method.

  In the “centrifugation method”, separation is performed based on the molecular weight of the substance to be separated. An example is sucrose concentration gradient centrifugation. According to this sucrose concentration gradient centrifugation method, a sucrose solution is filled in the second-dimension separation path 4 and centrifuged to form a sucrose concentration gradient. Separation can be based on molecular weight. In this way, the solute molecule group that has been subjected to the second-dimensional separation is separated from the second-dimensional separation path 4 and can be subjected to subsequent analysis such as identification by a mass spectrometer, for example.

  In addition, the “sedimentation equilibrium method” is a method in which a substance (solute) present in a solvent is precipitated by the action of centrifugal force, and this sedimentation force and the diffusive force of the solute against this equilibrium (balance sedimentation). In this method, the concentration distribution of the solute is measured by an interference optical system, a photoelectric scanning device, or the like to obtain the molecular weight of the solute.

  The molecular weight determined by this 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 dimensional separation step after isoelectric focusing, and can be said to be a method suitable as the second dimensional separation step performed in the second dimensional separation path 4. Solute molecules whose molecular weight has been measured by the sedimentation equilibrium method can be subjected to subsequent analysis such as substance identification analysis based on molecular weight. In the present invention, a sedimentation rate method may be used.

  FIG. 5 shows how the solute molecules form a concentration gradient in the second-dimensional separation channel 4 due to sedimentation equilibrium, and the concept of detecting solute molecules present at a predetermined concentration position in the two-dimensional separation channel 4. FIG.

  By detecting at which concentration position in the second dimension separation path 4 the solute molecule exists, the molecular weight of the solute molecule can be known. As an example of the detection means, a fluorescent substance is allowed to exist in the connection portion M between the first-dimensional separation path 3 and the second-dimensional separation path 4. This operation can be performed, for example, after setting the sucrose gradient in the second-dimensional separation path 4.

  Then, the solution is forcibly fed from the first-dimensional separation path 3 to the second-dimensional separation path 4, and the fluorescent substance is bound in the process in which solute molecules (for example, proteins) are precipitated by centrifugal force. Then, with respect to the solute molecule labeled with the fluorescent substance or the solute molecule modified so as to generate fluorescence, the fluorescence excitation light Q having a predetermined wavelength is emitted from the light source 5 and the predetermined concentration position 4a of the two-dimensional separation path 4 is set. The detector 6 can detect the fluorescence excitation light F that is aimed and irradiated from the second-dimensional separation path 4. By this fluorescence detection, it is possible to know whether or not a solute molecule having a predetermined molecular weight exists in the sample, or the abundance of the solute molecule can be determined by fluorescence intensity analysis.

  Although it is expected that the accuracy of sedimentation equilibrium will be adversely affected by fluorescent labeling, the density of solute molecules and fluorescent substances is expected to be about the average density of solute molecules and fluorescent substances. Correction can be performed based on this. Note that it is also possible to detect solute molecules present at a predetermined concentration position in the two-dimensional separation path 4 based on principles such as light absorption of a solute substance and Rayleigh scattering.

  The separation liquid that has undergone the second-dimensional separation as described above is collected from the second-dimensional separation path 4 and stored in the collection unit 5 that communicates with the second-dimensional separation path 4. In addition, the transfer method of the two-dimensional separation liquid from the second-dimensional separation path 4 to the recovery unit 5 is a method of moving using a capillary phenomenon, a method of moving by centrifugal force, negative pressure suction, positive pressure extrusion, or parent A control method using the medicinal principle is not particularly limited and can be employed.

  Among these, the control method using the philicity principle is a region in which the passage of the solute molecules can be controlled by the philicity principle at the connection portion N (see FIG. 1) of the second-dimensional separation path 4 and the recovery portion 5. It is a method of providing and controlling the movement of the two-dimensional separation liquid from the second-dimensional separation path 4 to the recovery unit 5.

  Specifically, as in the case of the connecting portion N, the connecting portion N is made a hydrophobic region to block the passage of hydrophilic solute molecules, and the hydrophobic region is made hydrophilic at an appropriate timing. Convert to allow hydrophilic solute molecules to pass through. 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 connection part M is formed of titanium oxide, it is possible to block the movement of the separation liquid to the recovery part 5 in the stage where the second-dimensional separation process is being performed. By irradiating ultraviolet rays aiming at N and converting hydrophobicity to hydrophilicity, it becomes possible to reliably and smoothly introduce the two-dimensional separation liquid into the recovery unit 5.

  In addition, when the ultraviolet rays are irradiated aiming at only the connection parts M and N of the selected second-dimensional separation path 4, only the two-dimensional separation liquid of the second-dimensional separation path 4 can be collected in one collection unit 5. It becomes.

  Next, FIG. 6 is a figure which shows another embodiment example (code | symbol 10) of the board | substrate for substance analysis based on this invention. The material configuration, layer structure, connection portion configuration, and the like of the substrate are the same as those of the substrate 1 shown in FIG.

  The substrate 10 shown in FIG. 6 is provided with two sample introduction parts 2 and 2 around the center hole H, and with collection parts 5 and 5 and introduction control parts 6 and 6. . The respective locations are selected so as to surround the center hole H and have the same circumferential direction and the same distance from the center hole H. This is to ensure the morphological uniformity of the disk-shaped substrate.

  Here, in addition to the sample containing the solute molecule group for analysis from the sample introduction units 2 and 2, the acidic solution 311 and the basic solution 312 used for isoelectric point separation in the first-dimensional separation path 3, or It is also used for introducing a sucrose solution into the second dimension separation path 4.

  Further, in the present embodiment, all the second-dimensional separation path 4 groups communicate with the recovery channel 51 formed on the outermost peripheral side of the substrate 10, and the recovery channel 51 is connected to the substrate 10. It connects with the collection | recovery parts 5 and 5 located in the center of. Therefore, the two-dimensional separation liquid from the second-dimensional separation path 4 passes through one common collection flow path 51 and is collected in the collection units 5 and 5. In the present embodiment, a portion corresponding to the connection portion N of the substrate 1 (FIG. 1) can be formed at, for example, a connection portion between the recovery flow path 51 and the second-dimensional separation path 4.

  The introduction control units 6 and 6 use a sample solution or other solution (for example, the acidic solution 311 or the basic solution 312 to the first-dimensional separation path 3 or the second-dimensional separation path by means of pressurization or suction). It is used when a sucrose solution, a washing solution, etc.) are introduced into a predetermined location according to the purpose.

  Next, the substance analysis system according to the present invention will be described with reference to FIG. This system is a system that handles the substrate 1 (FIG. 1) and the substrate 10 (FIG. 6) described above. The substrate rotation drive unit A that can rotate the substrate 1 (or 10) while controlling the substrate 1 (or 10) at a predetermined speed; Based on the sample supply unit B for the sample introduction unit 2, the voltage application unit C (see FIG. 3) for the first-dimensional separation path 3 (of the electrodes 31, 32), and the centrifugal force obtained by the rotation, A detection unit D (corresponding to reference numerals 5 and 6 in FIG. 5) for detecting solute molecules separated in the second-dimensional separation path 4, and further, raw data (for example, fluorescence intensity) obtained by the detection means D (Analysis signal E) is provided.

  The substrate rotation drive unit A includes a rotation axis A1, a drive unit A2 that applies a predetermined rotation drive to the rotation axis A1, a control unit (not shown) that controls the rotation speed, and the like. Further, the sample supply unit B has a predetermined solution from a storage unit such as the sample storage unit B1, the isoelectric point pH gradient forming solution storage unit B2, the sucrose solution storage unit B3, and the cleaning solution storage unit (not shown). Is selected and supplied (introduced) to the sample introduction part 2 of the substrate 1 (or 10) at a predetermined timing.

  The voltage application means C plays a role of applying a voltage to the first-dimensional separation path (isoelectric point separation path) 3 (electrodes 31 and 32) on the substrate, and its configuration is as shown in FIG. 3, for example. is there.

  The detection unit D is an optical system member that is arranged in the light path of the fluorescence F acquired from the light source 5 and the detector 6 that emit the fluorescence excitation light described above, and further from the fluorescence excitation light Q and the second-dimensional separation path. (A scraping member for the purpose of wavelength selection, route change, etc.). The analysis unit E analyzes the raw data (for example, fluorescence intensity signal) sent from the detection unit D, analyzes the type, molecular weight, amount, etc. of the detected solute molecules, and lists information on the solute molecules. Create databases and databases. FIG. 8 is a diagram showing a main flow until analysis is performed using the substrate and system according to the present invention.

  FIG. 9 is a diagram showing a concept of a method for identifying a solute molecule obtained from the second-dimension separation path 4 using the substrate or system according to the present invention. The solute molecules separated in the second dimension separation path 4 are collected, and the structure of the solute molecules is identified (substance identification) using, for example, a mass spectrometer (100 in FIG. 9).

  Next, the identified solute molecule (substance) and the observation position (detection position) of the second-dimensional separation path 4 are associated with each other, and a database is created as a conversion table (101 in FIG. 9). An example of this conversion table is shown in FIG. Further, at this time, the detected fluorescence intensity and the quantity relationship between the substances may be acquired and functionalized as shown in FIG. 11, for example. In an actual measurement stage, an expression profile image may be formed from measurement raw data, and an information list regarding all types of substances present in the sample and their abundance may be created using the conversion table. . FIG. 12 shows a format example of the information list.

  Next, FIG. 13 shows an example of the distribution of detection positions (existing positions) of solute molecules in each of the n-dimensional separation path groups 4-1 to 4-n arranged n on the substrates 1 and 10. Show. In the example shown in FIG. 13, there are detection spots at one place in the second-dimension separation path 4-2 and at two places in the second-dimension separation path 4-3. By using a function (see FIG. 11) between substance identification, fluorescence intensity detection, and substance amount separately performed by a mass spectrometer, the probability of dispersion in each second-dimensional separation path can be obtained.

  Next, FIG. 14 shows an example of a method for estimating the substance amount from each fluorescence intensity data. With respect to a substance having a detection spot at the same position (for example, 201 and 301 in FIG. 13) or a close position in a plurality of second-dimensional separation paths 4, each existence probability (see 201-A and 301-A in FIG. 14). The abundance of each substance can be estimated by applying a parallel row based on 9 (see 400 in Fig. 14) Note that Fig. 15 estimates the abundance of a substance from each fluorescence intensity data. It is the figure which showed the content which generalized the method concept to do.

  In the present invention, introduction (supply) of a sample containing a solute molecule for analysis, two-dimensional separation of a solute molecule group, detection / recovery / analysis (measurement) of a two-dimensionally separated solute molecule, etc. can be performed in a short time. And it can utilize as a technique implemented on a board | substrate with high precision. The two-dimensionally separated solute molecules can be identified by a mass analyzer or the like. For example, it can be used for comprehensive analysis (proteomic analysis) of protein chambers expressed in vivo.

It is a figure (upward view, top view) which shows simply the composition of the substrate for material analysis concerning the present invention. It is a workflow figure showing the work procedure of isoelectric focusing. It is a figure which shows the principle of the structural concept of the first-dimensional separation path 5, and the isoelectric focusing. It is a figure which shows typically the amphoteric carrier solid-phased on the inner wall surface (33) of a 1st-dimensional separation path (3), and the carboxyl group and amino group which this amphoteric carrier has. The figure which shows a concept that a solute molecule forms a concentration gradient by sedimentation equilibrium in the second-dimension separation path (4) and a solute molecule present at a predetermined concentration position in the second-dimension separation path 4 is shown. is there. It is a figure which shows another embodiment example (code | symbol 10) of the board | substrate for substance analysis which concerns on this invention. It is the figure which explained simply the composition concept of the system for substance analysis concerning the present invention. It is a figure which shows the main flows until it analyzes using the board | substrate and system which concern on this invention. It is a figure which shows the concept of the method of identifying the solute molecule acquired from the inside of the 2nd-dimensional separation path 4 using the board | substrate and system which concern on this invention. 6 is a diagram illustrating an example of a conversion table that associates between an identified solute molecule (substance) and an observation position (detection position) of a second-dimensional separation path 4. FIG. It is the image figure which functionalized the quantitative relationship of the detected fluorescence intensity and a substance. It is a figure which shows the format example of the information list regarding the kind of all the substances which exist in a sample, and its abundance. It is a figure which shows the example of distribution of the detection position (existing position) of the solute molecule in each of the 2nd dimension separation path group 4-1 to 4-n arrange | positioned by n board | substrates (1 or 10). It is a figure which shows an example of the method of estimating the amount of substances from each fluorescence intensity data. It is the figure which showed the content which generalized the method concept which estimates the abundance of a substance from each fluorescence intensity data.

Claims (5)

A sample introduction part provided on a disk-shaped substrate;
A first-dimension separation path that communicates with the sample introduction section and is capable of separating solute molecules in the sample by isoelectric focusing;
A second dimension separation path comprising a capillary communicated with the first dimension separation path and extending radially outward of the substrate;
A recovery unit for recovering the separation solution from the second-dimensional separation path;
A substrate for substance analysis comprising at least.   2. The substance according to claim 1, wherein a concentration gradient depending on a molecular weight at the time of sedimentation equilibrium is formed in the second-dimensional separation path by a centrifugal force obtained when the substrate is rotated. Analysis board. A system for handling the substance analysis substrate according to claim 1,
Substrate rotation driving means capable of rotating the substrate while controlling the substrate at a predetermined speed;
A sample supply means for the sample introduction section;
Voltage application means for the first-dimensional separation path;
Detection means for detecting solute molecules separated in the second-dimensional separation path based on the centrifugal force obtained by the rotation;
A material analysis system comprising at least (1) Means capable of controlling the transfer of the sample from the first dimension separation path to the second dimension separation path,
(2) Means capable of controlling the recovery of the sample from the second dimension separation path,
4. The substance analysis system according to claim 3, comprising one or both of the above (1) and (2). The detection means includes
Means for irradiating the selected position of the second dimension separation path with fluorescence excitation light;
Means for capturing fluorescence emitted from fluorescently labeled molecules present in the second-dimension separation path;
The substance analysis system according to claim 3, wherein the substance analysis system comprises at least means.

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