JP2002257721A - Method and analyzer for analyzing specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To effcieintly analyze a specimen containing plural kinds of mixed components or a specimen showing a continuous characteristic distribution, when conducting analysis using evanescent field excitation. SOLUTION: This method is provided with a develpoment step S1 for developing the components contained in the specimen into a developing area of one dimesion or more, a transfer step S2 for transferring the components developed in the developing area onto an analytical area of one dismension or more while keeping a developed result, and an analytical step 3 for analyzing the components transferred to the analytical area by a method using the excitation by an evanescent field.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample analysis method and apparatus used for analyzing components contained in a sample, for example, in the fields of biochemical analysis and medical analysis. Evanecent Field)
TECHNICAL FIELD The present invention relates to a method and an apparatus for analyzing a sample using the excitation by the sample.

[0002]

2. Description of the Related Art In the above technical field, proteins and D
As a technique for accurately analyzing various substances including biological substances such as NA even in a minute amount, an analysis technique using near-field light, in particular, an analysis technique using evanescent field excitation is known.

As shown in FIGS. 8A and 8B, when a light wave is diffracted or totally reflected at an interface between two materials A and B having different refractive indexes, an energy field is present near both sides of the interface. (Evanescent field) occurs. This analysis technique uses the excitation light (evanescent wave) generated by this energy field to analyze a substance, and examples thereof include surface plasmon resonance analysis, evanescent field excitation fluorescence, and surface enhanced Raman scattering analysis. No.

[0004] Surface plasmon resonance analysis (Surface Plas
mon Resonance (SPR) is, as shown in FIG. 9A, the analyte D based on the resonance between the surface plasmon wave and the evanescent wave generated at the interface between the insulator B 'and the metal C.
For example, as disclosed in Japanese Patent Laid-Open No. 7-174.
There is a method described in Japanese Patent Application Laid-Open No. 693 and Japanese Patent Application Laid-Open No. 10-311831. This method does not require labeling with a fluorescent substance, an antibody, or the like, and also allows kinetic analysis of an analyte.

[0005] The evanescent field excitation fluorescence method is shown in FIG.
As shown in (b), this is a method of performing an analysis based on fluorescence generated when a fluorescent substance bound to an analyte D 'is excited by an evanescent field.
The outline is described in JP-A-693 and JP-A-10-311831. This technique requires labeling with a fluorescent substance, but allows for highly sensitive analysis,
Kinetic analysis is also possible because there is no need to wash and separate excess label.

[0006] Surface Enhanced Rman scattering (Surface Enhanced R
The aman scattering (SERS) analysis method is a method of performing analysis using a phenomenon in which Raman scattering is amplified when a Raman active substance is adsorbed on the surface of a metal particle, and is disclosed in, for example, Japanese Patent Application Laid-Open No. 9-222395. There is a method described. This technique, in addition to labeling with Raman actives,
Since it is necessary to wash and separate excess label, kinetic analysis is impossible, but very sensitive analysis is possible.

[0007] In any of the above methods, after the analyte is immobilized on the element for analysis, the analyte is directly analyzed, or a certain reagent is added to the analyte to be analyzed. This is a method for detecting a reaction between an analyte and a reagent, and enables quantitative analysis of a trace amount of an analyte. Further, depending on the characteristics of the analyte and the reagent, the analyte can be re-analyzed by removing the reagent from the analyte after the analysis.

[0008]

By the way, in the above-mentioned conventional analysis technique utilizing evanescent field excitation, it is necessary to assemble a chip including an analysis element on which an analyte (analyte) is immobilized prior to analysis. There is, but Figure 10
As shown in (a) and (b), when the analyte is immobilized on the analysis element E1, after assembling the chip E, a liquid sample containing the analyte as a component is placed on the surface of the analysis element E1. (See FIG. 10A), or by assembling the chip E after spotting such a sample on the surface of the analysis element E1 before assembling the chip E (see FIG. 10A). (B)), the analyte is immobilized on the element for analysis. In FIGS. 10A and 10B, E2 and E3 are components of the chip.

However, in the former method of immobilization, only one kind of sample can be used. Therefore, the combination and characteristics (for example, concentration) of analytes that can be immobilized are as follows.
There is a problem that it is limited to a single one. In addition, in the latter immobilization method, although a plurality of types of specimens can be used, their dropping needs to be performed separately and independently.
There is a problem that the analyte is fixed discretely and the combination and characteristics of the analyte in each spot are also limited to a single one.

Therefore, in the conventional analysis technique based on evanescent field excitation using these immobilization techniques, when a plurality of types of mixed components or components having a continuous characteristic distribution are handled, electrophoresis, chromatography, and the like are required. Using a well-known technique, these components are developed and separated in advance, and components having desired component elements and desired characteristics are extracted therefrom, and then analyzed using these as analytes.

However, in the conventional procedure of extracting and developing a desired component as an analyte after developing and separating by a technique such as electrophoresis or chromatography as described above,
When the extraction operation is complicated, and when analyzing each component element for a plurality of types of mixed components, or when analyzing a component showing a continuous characteristic distribution in detail, multiple extraction operations must be performed. There is a problem that it takes time and effort.

In order to accurately extract a desired analyte, the position where the desired analyte exists is determined by visualizing the developed and separated components or referring to another database. There is a problem that the extraction operation must be specified, the extraction operation becomes more complicated, and it is difficult or impossible to specify an accurate position depending on the type of the analyte.

The present invention has been made in view of the above problems, and has been made in consideration of the above problems. An evanescent field capable of efficiently analyzing a sample containing a plurality of types of mixed components and a sample having a continuous characteristic distribution is provided. It is an object of the present invention to provide a sample analysis method and apparatus using excitation.

[0014]

In order to achieve the above object, a sample analysis method of the present invention (claim 1) is a method for analyzing a sample containing one or more components, and is included in the sample. A development step of developing the components in a development area of one or more dimensions, a transcription step of transferring the components developed in the development area to an analysis area of one or more dimensions while maintaining the previous development result, An analysis step of analyzing a component transferred to the region by a method using excitation by an evanescent field is provided.

In the developing step, a gel may be used as a developing area, and the developing may be performed by gel electrophoresis (claim 2) or may be performed by chromatography. Further, in the developing step, the image may be developed in the two-dimensional developing area by two-dimensional electrophoresis, and in the transferring step, the image may be transferred to the two-dimensional analyzing area while maintaining the two-dimensional developing result ( Claim 3).

Further, in the transfer step, the transfer may be performed by bringing the development area and the analysis area into contact with each other and applying an electric field, and may be an intermediate step in transferring the components from the development area to the analysis area. A membrane may be used as a medium. In the former case, an electric field may be applied while using the analysis area as an electrode. In the developing step, while the gel as the developing area is in contact with the analysis area, the components are subjected to electrophoresis in the vicinity of the analysis area of the gel, and the gel is analyzed in the transfer step. The transfer may be performed by fixing a portion in the vicinity of the analysis area to the analysis area.

In the transfer step, the components transferred to the analysis area may be fixed to the analysis area. In this case, a substance having a functional group capable of binding to a component may be previously bound to the surface of the analysis area, and immobilization may be performed by binding the component to this functional group. A substance capable of adsorbing a component may be bound to the surface of the region in advance, and immobilization may be performed by adsorbing the component to the substance. Immobilization may be performed by crosslinking or covering the surface of the region and the components.

In the transfer step, an element for surface plasmon resonance analysis may be used as an analysis area, and in the analysis step, analysis may be performed by surface plasmon resonance analysis. In the transfer step, an element for evanescent field excitation fluorescence analysis may be used as an analysis area, and in the analysis step, analysis may be performed by evanescent field excitation fluorescence analysis.

In the transfer step, an element for surface enhanced Raman scattering analysis may be used as an analysis area, and in the analysis step, analysis may be performed by surface enhanced Raman scattering analysis. In the analysis step, a reagent is added to the previous analysis area, and a reaction between the reagent and the component in the analysis area is performed.
Detection may be performed by a method using excitation by an evanescent field.

On the other hand, the sample analyzer of the present invention (claim 7)
Is an apparatus for realizing the above-described sample analysis method of the present invention, which is an apparatus for analyzing a sample containing one or more components, wherein the components contained in the sample are developed in a one-dimensional or more development area. And a transfer unit that transfers the components to the one-dimensional or more analysis area while maintaining the expansion result, and an analysis unit that analyzes the components of the analysis area by a method using excitation by an evanescent field. It is characterized by:

[0021]

Embodiments of the present invention will be described below with reference to the drawings. [1] Description of Sample Analyzer as One Embodiment of the Present Invention FIG. 1 is a block diagram showing a functional configuration of a sample analyzer as one embodiment of the present invention. The sample analyzer 1 is a device for analyzing a sample containing one or more components, and includes a developing unit 2, a transfer unit 3, and an analyzing unit 4.

The developing section 2 is used for storing a sample containing various substances such as biological substances such as proteins and DNA as components.
It is developed in a one-dimensional or more development area. Here, as a method of development performed by the development unit 2, the components after the development are analyzed by an element for analysis (the surface area of the element for analysis corresponds to the analysis area of the present invention. Details of this analysis element) Will be described later). Examples thereof include gel electrophoresis such as polyacrylamide gel electrophoresis and agarose gel electrophoresis, and chromatography such as thin layer chromatography and paper chromatography.

The developing region may be a solid or semi-solid (one or more dimension) solid or semi-solid (hereinafter, referred to as one or more) which permits the development of the components in the sample and the transfer to the analysis region while maintaining the developed result. , A developing phase) may be used. Among the above-mentioned development methods, in the case of gel electrophoresis, the internal region of the gel such as polyacrylamide gel or agarose gel is used, and in the case of chromatography, the internal region or surface in the stationary phase such as a thin layer or filter paper is used. The region will have the role of the deployment region in the present invention.

Although the expansion area described in the above description is two-dimensional, this expansion area is not limited to two dimensions, but may be other dimensions, for example, one dimension. I do not care. The dimension of the development area and the dimension in which the component is developed may not be the same. For example, as shown in FIG. 11, when electrophoresis is performed using a gel F having a two-dimensional development area, one direction or two or more specimens are subjected to one-dimensional electrophoresis in one direction of the two-dimensional area of the gel F. (See FIG. 11 (a)), or a single sample may be developed in both directions of the two-dimensional region of the gel F by two-dimensional electrophoresis (FIG. 11 (b)). reference). If transfer is possible while maintaining the development result, the dimension of the development area and the dimension of the development result are both arbitrary.

With the above configuration, the developing unit 2 has a function of developing the components contained in the sample into a one-dimensional or more developing area. The transfer unit 3 transfers the components developed by the developing unit 2 to a one-dimensional or more analysis element (analysis area) while maintaining the developed result. Here, as a method of transferring the developed component to the element for analysis, the developed component can be analyzed later by a method using evanescent field excitation while maintaining the one-dimensional or more developed result by the developing unit 2. A method that can be transferred to the analysis element is used. For example, there are (a) a method using gravity, (b) a method using an electric field, (c) a method using an intermediate medium, and (d) a method of fixing the developed components as they are.

Hereinafter, these transfer methods will be described with reference to FIGS.
This will be described with reference to FIG. In addition, in the following drawings, the code | symbol 5 represents the development phase which has the area | region for a development, and the code | symbol 6 represents an element for an analysis. 2 to 5, an element for surface plasmon resonance analysis is used as an example of the element 6 for analysis. This element 6 for surface plasmon resonance analysis
Are the element unit 6-1, the metal thin film 6-2, and the organic transfer layer 6-3.
It is composed of Here, the organic transfer layer 6-3 is for immobilizing the components transferred to the surface, and is unnecessary when a part of the transfer method described below or the immobilization method is used. There is. Of course, the following transfer method is not limited to the element 6 for surface plasmon resonance analysis,
It can be used for other analysis elements.

In the method (a), as shown in FIG. 2, a developing phase 5 such as a gel or a stationary phase having a developing area is placed on an analysis element 6 to diffuse the developed component itself, or In this method, the component is moved to the analysis element 6 using the gravity applied to the developed component. In this method, the result of component development may be slightly distorted due to the time required for movement,
Further, it is difficult to transfer all the components to the analysis element 6, but depending on the type of the developing phase 5 and the type of the component to be transferred, the transfer can be efficiently performed by a simple operation.
It is particularly effective when used in combination with a technique for fixing to an analysis element described later.

In the method (b), as shown in FIGS. 3 (a) and 3 (b), an electric field is generated while a developing phase 5 such as a gel or a stationary phase is in contact with an analysis element 6. By
This is a method of moving components to the analysis element 6. This method can be used only when the component to be transferred is a substance having an electric charge.However, it is possible to transfer quickly without breaking the development result, and it is possible to transfer many of the developed components. It is very effective because it can.

FIG. 3 shows an electric field generation method.
As shown in (a), two electrodes 7-1 and 7- are sandwiched between the developing phase 5 and the analysis element 6 which are brought into contact with each other from both sides.
2, an electric field may be generated in such a direction that the component to be transferred moves from the developed phase 5 to the analysis element 6. Here, as will be described later, when an analysis element coated with a metal thin film is used (for example, an element for surface plasmon resonance analysis), transfer is performed by applying an electric field while using this analysis element as one of the electrodes. May be performed. That is, as shown in FIG. 3B, the analysis element 6 and one electrode 7-
By disposing the developing phase 5 between the first and second elements 2 and generating an electric field using the analyzing element 6 and the electrode 7-2, it is possible to perform transfer with a simpler configuration.

In the method (c), as shown in FIG. 4, a medium having an adsorptivity, for example, an adsorption film 8 such as a nitrocellulose film or a polyfluoroethylene film is used as an intermediate medium to analyze components for analysis. This is a method of moving the element. Specifically, first, the components are transferred from a developing phase 5 such as a gel or a stationary phase to an adsorption film 8 as an intermediate medium and adsorbed, and then the components transferred and adsorbed to the adsorption film 8 are analyzed. Move to element 6. These operations may be performed using gravity as in the method (a), or may be performed using an electric field as in the method (b).

According to this method, although the operation is somewhat complicated, a medium such as an adsorption film widely used for collecting and removing various biological components can be used as an intermediate medium, and a well-known method such as a blotting method can be used. Since the apparatus used in the above method can also be used, efficient transfer can be performed by utilizing existing media and apparatuses. Also, the preservation state of the developed result and the transfer efficiency of the components are good.

The method (d) is a method in which a component developed in the developing phase 5 such as a gel or a stationary phase is directly fixed to the analysis element 6. Specifically, for example, when the developing unit 2 performs gel electrophoresis as a developing method, electrophoresis is performed near the surface or bottom of the gel 5 as a developing phase, and after the gel electrophoresis is completed, the gel 5 Component is analytical element 6
There is a method of fixing the gel 5 to the analysis element 6 in such a direction that the gel 5 comes into contact with the analysis element 6. In this case, the gel 5 in a portion where the analysis element 6 does not exist may be removed, or a thin gel 5 may be prepared from the beginning and fixed to the analysis element as it is.

As shown in FIG. 5, when the developing section 2 performs gel electrophoresis, electrophoresis is performed in a state where the gel 5 is fixed to the analysis area 6 in advance, and the analysis element is The gel 5 in a portion where 6 is not present may be removed, or the thin gel 5 may be used as it is for subsequent analysis. Of course, this method can also be applied when the developing unit 2 uses another method (for example, thin film chromatography) as the developing method. Further, by combining this method with the method (c), the intermediate medium such as the adsorption film 8 to which the components of the developing phase 5 are transferred and adsorbed may be fixed to the analysis element 6 as it is.

According to this method, the transferred component is the gel 5
Or in a state surrounded by a transfer area such as
Is fixed to the analysis element 6 in a state of being adsorbed by an intermediate medium such as the above, so that the subsequent analysis is restricted depending on the type of the developing region 5 or the intermediate medium (adsorption film) 8 or the type of the component to be transferred. In some cases. However, when analyzing a specific component, an appropriate type of developing phase 5 or intermediate medium 8 may be used.
Is used, it is possible to transfer (fix) it to the analysis element 6 while keeping the development result of the component exactly as it is,
Also, almost all components can be transferred (fixed) and provided for later analysis.

When the transfer is performed by the transfer unit 3,
The analysis area to which the component is to be transferred is a one-dimensional or more analysis element that allows the component to be transferred using the evanescent field excitation while maintaining the expansion result of one or more dimensions of the component. Are used. For example, when components are analyzed by the surface plasmon resonance analysis method in the analysis unit 4 later, an element for surface plasmon resonance analysis (such as an analysis element in which a gold thin film is coated on the surface of a diffraction grating) is used in the analysis unit 4. When components are analyzed by the evanescent field excitation fluorescence analysis, the surface areas of the evanescent field excitation fluorescence analysis element each have a role of an analysis area in the present invention.

As the analysis area, a continuous area of one dimension or more, which can transfer components while maintaining the developed result, is used. The dimension of the analysis area is arbitrary and may not be the same as the dimension of the development area or the dimension of the development result, as long as the components can be transferred without reducing the dimension of the development result. For example, after a component is one-dimensionally developed by one-dimensional electrophoresis using a gel having a two-dimensional development region, the component is converted from the gel to a two-dimensional analysis region ( It is also conceivable to perform the analysis by transferring the data to the surface area of the analysis element).

By the way, depending on the transferred components and the type of the analysis element, the analyte (component) transferred from the development area onto the analysis element may be bound simply by being transferred onto the analysis element. May not be able to withstand subsequent analysis. Therefore, in order to be able to carry out the subsequent analysis more reliably, the transfer unit 3 has the above-described (a).
When transfer is performed using each of the methods (1) to (d), the transferred component is immobilized on the analysis element according to the type of the transferred component and the analysis element.

Here, as a technique for immobilizing the transferred component to the element for analysis, the transferred component is later subjected to evanescent field excitation while maintaining the expansion result of one or more dimensions by the expansion unit 2. A method that can be immobilized on an analysis element so that analysis can be performed by a method is used. For example, (i) a method using a functional group, (ii) a method using adsorption, (iii)
A method using cross-linking, and (iv) a method using inclusion. These methods are described below with reference to FIG.
This will be described with reference to (b) and (c).

In the method (i), a substance having a functional group capable of binding to the component is previously bound to the surface of the analysis element 6, and the component is bound to the functional group, whereby the transferred component is removed. This is a method of fixing. Specifically, FIG.
As shown in (a), a substance serving as a scaffold for immobilization, for example, a substance 6-3a such as thioctic acid having a thiol group capable of binding to gold and a carboxyl group that can be used for binding to a protein, It is fixed on the surface of the analysis element 6 in advance. Subsequently, depending on the type of the substance 6-3a serving as a scaffold, the scaffold substance 6-3a is activated so that the transcribed component can be bound to the scaffold substance 6-3a only by contact. For example, the substance 6-3a serving as this scaffold
Carbodiimide so that the transcribed component can bind to the scaffold 6-3a only by contact. In this state, the transferred component is immobilized on the analysis element 6 by performing the transfer from the development area onto the analysis element 6.

In the method (ii), a substance capable of adsorbing the transferred component on the surface of the analysis element 6 is previously bound,
In this method, the transferred component is immobilized by adsorbing the component on the substance. Specifically, as shown in FIG. 6B, a substance capable of adsorbing the transferred component, for example, an adsorbing substance 6-3b such as nitrocellulose or polyfluorinated ethylene is previously placed on the surface of the element 6 for analysis. To be fixed. In this state, when the transfer is performed from the development area onto the analysis element 6, the transferred component is adsorbed by the adsorbing substance 6-3b on the analysis element 6, and is fixed on the analysis element.

The method (iii) is a method in which the transferred component is brought into contact with the surface of the analysis element, and the transferred component is immobilized by cross-linking the surface of the analysis area with the component. . Specifically, as shown in FIG. 6 (c), first, as a preparation for immobilization, a substance 6-3c serving as a pedestal for cross-linking having a functional group capable of cross-linking with the transferred component is preliminarily placed on the analytical element. 6 is immobilized on the surface. Subsequently, the transferred component is bonded to the surface of the analysis element 6 by a relatively weak force such as an intermolecular force or an electrostatic force. Further, the transferred component is cross-linked with the functional group of the crosslinked pedestal material 6-3c in a state where the component is immobilized on the surface of the analytical element with a weak force, so that the transferred component is analyzed by the analytical element. 6 is firmly fixed to the surface.

In the method (iv), the transferred component is brought into contact with the surface of the element for analysis 6 and the surface of the element for analysis 6 and the component are covered to immobilize the transferred component. It is. Specifically, first, the transferred component is bonded to the surface of the analysis element 6 by a relatively weak force such as an intermolecular force or an electrostatic force. Subsequently, in a state where the components are immobilized on the surface of the analysis element 6 with a weak force, the transferred components and the surface of the analysis element 6 are covered with a substance such as agarose gel to thereby transfer the transferred components. Is securely immobilized on the surface of the analysis element 6. That is, this is a fixing method common to the above-described transfer method (d).

With the above configuration, the transfer unit 3 has a function of transferring the components developed in the one-dimensional or more development area by the development unit 2 to the one-dimensional or more analysis area while maintaining the developed result. Will be. The analysis unit 4 analyzes the component transferred to the one-dimensional or more analysis element (analysis area) by the transfer unit 3 by a method using excitation by an evanescent field.

Here, as a method of analyzing the transferred component, one-dimensional or more continuous analysis of the component transferred to the one-dimensional or more analysis element while maintaining the one-dimensional or more developed result is performed. An analysis method using evanescent field excitation that can be performed is used. Examples include surface plasmon resonance analysis, evanescent field excitation fluorescence analysis, and surface enhanced Raman scattering analysis.

The analysis targets include the characteristics of the components that can be analyzed by the transcribed components alone, such as the presence, position, and amount of the components in the development result, and the presence or absence of the reaction between the components and the specific reagents, and the reaction speed. And the characteristics of components that can be analyzed based on the result of the reaction between the reagent and the component added to the analysis region. In particular, when the latter analysis is performed, a reagent is added to the analysis area to which the component has been transferred, the reaction between the reagent and the component is detected, and the analysis is performed based on the detection result. As a detection reagent to be added, a reagent corresponding to each of the above-described methods is used. For example, when analysis is performed using evanescent field excitation fluorescence analysis, a reagent to which a fluorescent dye that emits light by evanescent field excitation is bound, and when analysis is performed using surface enhanced Raman scattering analysis, gold colloid is used. A reagent to which a Raman active substance such as described above is bound is used. In the case of performing analysis using surface plasmon resonance analysis, there is no particular limitation on the reagent.

A sensor device is used to detect the characteristics (existence, amount, etc.) of the components in the analysis area or the reaction between the components and the reagent. Such sensor devices have been developed according to the types of analysis methods such as evanescent field excitation fluorescence analysis, surface enhanced Raman scattering analysis, and surface plasmon resonance analysis. As a detection method, detection at one point or a plurality of points is the mainstream, but by performing detection while scanning the analysis area using such a point detection type sensor device, continuous detection of one or more dimensions is performed. Detection can be performed for an area. Further, a one-dimensional and two-dimensional detection type sensor device capable of simultaneously detecting a region having a one-dimensional or two-dimensional spread has been developed (for example, Japanese Patent Application Laid-Open No. H11-344637 and Benno Rothenha
usler, et al. "Surface-plasmon microscopy": NATURE
VOL. 332 14 April 1988, p.615-617, describes a related technique). By using such a one-dimensional sensor device or a two-dimensional sensor device, a continuous area of one dimension or more can be obtained. Can be detected at once.

With the above configuration, the analyzing section 4 has a function of analyzing the components transferred to the analysis area by the transferring section 3 by a method using excitation by an evanescent field. [2] Description of Sample Analysis Method as One Embodiment of the Present Invention Next, the flowchart of FIG. 7 shows a sample analysis procedure (sample analysis method of the present embodiment) executed by the sample analyzer 1 of the present embodiment. It will be described with reference to FIG.

The sample to be analyzed by the sample analyzer 1 of the present embodiment is expanded into a one-dimensional or more expansion area by the expansion unit 2, transferred to a one-dimensional or more analysis area by the transfer unit 3, and It is a substance that can be analyzed by a method using evanescent field excitation by the analysis unit 4. Specifically, various substances, mainly biological substances, such as proteins, lipids, carbohydrates, nucleic acids, low molecular organic compounds, complexes, viruses, microorganisms and cells, and composites thereof are the analytes.

Actually, a sample containing such an analyte as a component is subjected to analysis by the sample analyzer 1 of the present embodiment. As the sample, the components contained therein are developed by the developing unit 2 into a one-dimensional or more developing area, transferred by the transfer unit 3 to the one-dimensional or more analyzing area, and analyzed by the analyzing unit 4. What can analyze by the method using evanescent field excitation is used. Specifically, it is a sample in which the above-mentioned analyte is dissolved or suspended in a liquid phase medium.

As shown in FIG. 2, when a sample to be analyzed is input to the sample analyzer 1 of this embodiment, first, the developing unit 2 converts components contained in the sample into one or more dimensions. (Step S1).
Specifically, the components contained in the sample are converted into the development area (gel electrophoresis, chromatography, etc.) according to the type of the component (analyte) to be analyzed (analyte) contained in the sample and the content of the analysis. Gel or stationary phase). As described above, by selecting a developing method according to the sample to be analyzed and performing the developing, it is possible to efficiently perform the developing and separating according to the characteristics of the component to be developed.

Further, the components are developed in a one-dimensional or more development area in one or more dimensions according to the development method (for example, in the case of two-dimensional electrophoresis, the components are two-dimensionally formed on a two-dimensional gel). The expansion result of one or more dimensions is directly processed later (transfer by the transfer unit 3, analysis unit 4).
Analysis). As described above, by transferring the components while maintaining the development results of one or more dimensions, it is possible to analyze the components while utilizing the detailed development results in the one or more dimensions area.

Next, the components developed in the development area in step S1 are transferred to the one-dimensional or more analysis area by the transfer unit 3 while maintaining the one-dimensional or more development result (step S2). ). Specifically, the components developed in the development area (gel, stationary phase, etc.) are transferred according to the type of the component and the content of the analysis (such as a method using gravity,
Is transferred to the analysis area by a method using an electric field. As described above, by performing the transfer by selecting the transfer method according to the sample to be analyzed, it becomes possible to perform the transfer more efficiently in accordance with the characteristics of the component to be transferred.

Further, depending on the type of the component to be transferred and the type of the analysis area, an immobilization method (a method using a functional group, a method using adsorption, etc.) is used.
The transferred component is fixed to the analysis area. Thus, by performing immobilization according to the sample to be analyzed,
The transferred component is securely fixed to the analysis area, and the subsequent analysis can be performed reliably.

Finally, the component transferred to the analysis area in step S2 is analyzed by the analysis unit 4 by a method using excitation by an evanescent field (step S2).
3). Specifically, the components transferred to the analysis element while maintaining the development results are analyzed according to the components to be analyzed and the content of the analysis by using an evanescent field excitation analysis method (surface plasmon resonance analysis, Evanescent field excitation fluorescence analysis, surface enhanced Raman scattering analysis).

As described above, the components contained in the sample are developed in a one-dimensional or more development area, and the components are transferred to a one-dimensional or more analysis area while maintaining the developed result, and are excited by an evanescent field. By performing analysis using the method, the analysis results corresponding to the development results of the components contained in the sample can be obtained at once using the development results of the components contained in the sample. Therefore, for a sample containing a plurality of types of mixed components or a sample showing a continuous characteristic distribution, there is no need to perform a complicated analyte extraction operation or a plurality of repetitive analyzes, thereby enabling efficient analysis. Become.

Further, by analyzing the components transferred to the analysis element while maintaining the development result by an analysis method using evanescent field excitation, a sample containing a plurality of types of mixed components and a continuous characteristic distribution can be analyzed. The sample shown can be efficiently analyzed while enjoying the advantages associated with the analysis using evanescent field excitation. Specifically, by using an element for surface plasmon resonance analysis as an analysis area and performing analysis by surface plasmon resonance analysis, a sample containing a mixture of a plurality of types of components and a sample showing a continuous characteristic distribution can be obtained. Efficiently, components can be analyzed extremely quickly without the need for labeling and washing, and kinetic analysis of components can be performed.

Further, by using an element for evanescent field excitation fluorescence analysis as an analysis area and performing analysis by evanescent field excitation fluorescence analysis, a sample containing a plurality of types of mixed components or a sample showing a continuous characteristic distribution is obtained. Can efficiently and quickly analyze components with high sensitivity without the need for washing, and can perform kinetic analysis of components.

Further, by using a surface-enhanced Raman scattering analysis element as an analysis area and performing analysis by surface-enhanced Raman scattering analysis, a sample containing a plurality of types of mixed components or a sample showing a continuous characteristic distribution is obtained. Can efficiently analyze the components with extremely high sensitivity.
Depending on the characteristics of the component to be analyzed, a reagent is added to the analysis area after the component is transferred, and the reaction between the reagent and the component is detected by a method using evanescent field excitation. Analyzes based on the reaction between components and reagents can be performed efficiently even for samples containing multiple types of mixed components or samples that show a continuous characteristic distribution. Subsequent desorption of the reagents from the components also allows for reanalysis of the components. In addition, when the components are known, it is possible to select a reagent (compound) that reacts with a specific component, which is also useful for drug screening.

[3] Others Although the sample analysis method and the sample analyzer according to one embodiment of the present invention have been described above, the present invention is not limited to this, and does not depart from the gist of the present invention. ,
It can be implemented in various forms.

[0060]

EXAMPLES Hereinafter, specific embodiments of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and may be carried out in various forms unless departing from the gist thereof. It is possible to implement. [1] Example 1 As a first example, consider the case where a sample containing a plurality of proteins is kinetically analyzed for the interaction with a reagent.

First, in the developing unit 2, a plurality of proteins contained in the sample are developed on a two-dimensional gel by two-dimensional electrophoresis, and each protein is separated. Subsequently, in the transfer unit 3, the protein spread on the two-dimensional gel is
The image is transferred onto a two-dimensional surface plasmon resonance analysis element while maintaining the two-dimensional development result. Finally, in the analysis unit 4, a reagent whose interaction is to be analyzed is added to the two-dimensional surface plasmon resonance analysis element onto which the protein has been transferred, and the interaction between this reagent and the protein is analyzed by surface plasmon resonance analysis. Perform analysis by method.

[2] Embodiment 2 As a second embodiment, a case will be considered where the distribution of all proteins is analyzed for a specimen containing a plurality of types of proteins.
First, in the developing unit 2, a plurality of types of proteins contained in a sample are developed on a two-dimensional gel by two-dimensional electrophoresis, and each protein is separated.

Subsequently, the transfer unit 3 transfers the protein developed on the two-dimensional gel to the two-dimensional surface plasmon resonance analysis element while keeping the two-dimensional development result. Finally, in the analysis unit 4, a reagent that reacts with all proteins is added to the two-dimensional surface plasmon resonance analysis element onto which the protein has been transferred, and the reaction between the reagent and the protein is determined by surface plasmon resonance analysis. To analyze the distribution and quantity of total protein.

[3] Example 3 As a third example, consider a case in which the distribution of a specific protein is analyzed for a sample containing a plurality of types of proteins. First, in the developing unit 2, a plurality of types of proteins contained in a sample are developed on a two-dimensional gel by two-dimensional electrophoresis, and each protein is separated.

Subsequently, in the transfer section 3, the protein developed on the two-dimensional gel is transferred to the two-dimensional surface plasmon resonance analysis element while keeping the two-dimensional development result. Finally, in the analysis unit 4, a reagent that reacts only with a specific protein is added to the two-dimensional surface plasmon resonance analysis element onto which the protein has been transferred, and the reaction between the reagent and the specific protein is determined by surface plasmon resonance. Analyze by resonance analysis to analyze the distribution and amount of specific proteins.

[4] Embodiment 4 As a fourth embodiment, a case will be considered where the distribution of DNA molecules having a specific sequence is analyzed for a sample containing a plurality of DNA molecules as components. First, in the developing unit 2, a plurality of DNA molecules contained in a sample are developed on a two-dimensional gel by one-dimensional electrophoresis, and each DNA molecule is separated.

Subsequently, in the transfer section 3, the protein developed on the two-dimensional gel is transferred to the two-dimensional evanescent field excitation fluorescence analyzing element while maintaining the one-dimensional development result. Finally, in the analysis unit 4, a fluorescently labeled DNA molecule having a sequence complementary to a specific sequence is added to the two-dimensional evanescent field-excited fluorescence analysis element to which the DNA molecule has been transcribed. Is analyzed by an evanescent-field-excited fluorescence analysis element to determine the binding of D having a specific sequence to the specific arrangement on the analysis element.
The distribution of NA is analyzed.

[5] Example 5 Finally, as a more specific example, a sample containing biotinylated porcine serum albumin as a component was subjected to SDS-PAGE.
E (sodium dodecyl sulfate polyacrylamide-gel elec
trophoresis (SDS polyacrylamide gel electrophoresis), and then analyze the antigen-antibody reaction with anti-porcine serum albumin antibody by surface plasmon resonance analysis after one-dimensional development.

[Preparation of Analytical Element] A gold thin film was formed by sputtering on a resin having a diffraction grating with a pitch of about 800 nm. A buffer A (10
mM HEPES, 0.15 M NaCl, 3 mM ED
PIERSE DTSSP (3,3'-dithiobis (s) dissolved in TA • 2Na, pH 7.4) to 5 mM.
ulfosuccinimidyl-propionate)) was dropped 400 μL,
It was left at room temperature for 30 minutes. After washing with pure water and drying, NeutrAvidin manufactured by PIERCE, also adjusted to 100 μg / mL in buffer A, was added to 400 A.
μL was added dropwise and left at room temperature for 1 hour. After washing and drying with pure water, 400 μL of Kishida Chemical's ethanolamine adjusted to 1M was added dropwise to pure water, left at room temperature for 30 minutes, and further washed and dried with pure water. An analytical element for surface plasmon resonance analysis in which NeutrAvidin was bonded to a metal surface was prepared.

[Preparation of Sample] M of 25 mM, pH 6.0
SIGMA pig serum albumin (10 mg) was dissolved in es buffer (0.9 mL), and PIERCE NHS-biotin (Ez-Link Sulfo-NHS-LC-) was adjusted to 10 mg / mL in the same Mes buffer. Biotin)
After adding 0.1 mL and stirring, it was left still at 4 ° C. for 2 hours. Thereafter, dialysis with a PBS buffer was performed three times for one hour each to obtain a 10 mg / mL biotinylated porcine serum albumin solution (sample) adjusted under non-reducing conditions and non-boiling conditions.

[Development of Specimen to Development Area] 5 μL of the above biotinylated porcine serum albumin solution was added to 12% SDS-
Electrophoresis was performed using a PAGE gel, and developed in a one-dimensional region (developing region) in the gel.

[Transfer of Sample from Development Area to Analysis Area] After the development of the sample, the metal surface of the analytical element is brought into contact with the surface of the gel, and in this state, the gel and the analytical element are attached to the Bio-Rad. The blotting buffer was set in a blotting device (Western blotting: 2.9 g, Tris 5.8 g, SDS 0.37 g,
By adding water to 200 mL of methanol and adding a buffer to 1 L) and performing electrophoresis at a constant current of 100 mA for 2 hours, the sample present in the developing area in the gel is removed from the primary surface on the metal surface of the analytical element. The image was transferred to the original area (area for analysis).

Before and after the transfer of the specimen, the resonance angle of surface plasmon resonance at points # 1 to # 10 on the analysis area was measured using an angle transition type surface plasmon resonance measuring apparatus. Here, points # 1 to # 10 are in this order:
Equally spaced (1 mm) on the analysis area, which is a one-dimensional area
It is a point that exists side by side. Table 1 below shows the amount of change in the resonance angle at points # 1 to # 10 before and after the transfer of the sample.

[Analysis of Sample Existing in Analytical Area] After transfer of the sample, 400 μL of SIGMA β-casein adjusted to be 0.1% in buffer A was dropped on the metal surface of the analytical element. Then, after leaving still at room temperature for 30 minutes, it was washed and dried with pure water, and a treatment for suppressing non-specific binding to NeutrAvidin to which biotinylated porcine serum albumin was not bound was performed.

Subsequently, a 20 μg / mL anti-porcine serum albumin antibody (manufactured by Yagai Co., Ltd.) was brought into contact with the metal surface of the analytical element, and biotinylated porcine serum albumin, which is a component in the sample existing on the analysis area, was removed. The antigen-antibody reaction was performed, and before and after the reaction, the resonance angles of surface plasmon resonance at points # 1 to # 10 in the analysis region of the analysis element were measured online using an angle transition type surface plasmon resonance measurement device. . Table 1 below shows the amount of change in the resonance angle at points # 1 to # 10 before and after the antigen-antibody reaction.

[0076]

[Table 1]

According to the results in Table 1, points # 4 to # 8
The positions are close to each other, and the amount of change in the resonance angle before and after transcription is large, so that it corresponds to the position where the biotinylated porcine serum albumin in the sample was developed, and that the transcribed biotinylated porcine serum albumin is present. Conceivable.
Actually, at these points, the amount of change in the resonance angle before and after the antigen-antibody reaction was large, and it was confirmed that the antigen-antibody reaction occurred.

On the other hand, at points # 1 to # 3, # 9 and # 10, since the amount of change in the resonance angle before and after the transcription was small, no biotinylated porcine serum albumin was present, and NeutrAv was not present.
It is thought that non-specific binding to idin is suppressed by β-casein. Actually, at these points, the amount of change in the resonance angle before and after the antigen-antibody reaction was small, and it was confirmed that the antigen-antibody reaction did not occur.

From these results, in this example, biotinylated porcine serum albumin, which is a component in the sample, was analyzed by SDS.
-It can be seen that the image is transferred to the analysis area on the analysis element while maintaining the result of development on the development area in the gel by PAGE electrophoresis. It can also be seen that, for this biotinylated porcine serum albumin, the development result by SDS-PAGE electrophoresis and the analysis result of the antigen-antibody reaction by surface plasmon resonance analysis were obtained at the same time.

[0080]

As described above in detail, according to the sample analysis method and the sample analyzer of the present invention, components contained in a sample are developed in a one-dimensional or more development area, and the primary result is maintained while maintaining the development result. By transferring the components to the original or larger analysis area and analyzing them by a method using excitation by an evanescent field, the corresponding analysis results can be obtained at once using the development results of the components contained in the sample. . Therefore, for a sample containing a plurality of types of mixed components or a sample showing a continuous characteristic distribution, there is no need to perform a complicated analyte extraction operation or a plurality of repetitive analyzes, thereby enabling efficient analysis. (Claims 1 and 7).

Here, by selecting a developing method such as gel electrophoresis (claim 2) or chromatography according to the sample to be analyzed and performing the developing, more efficient developing can be performed in accordance with the characteristics of the component to be developed. -Separation can be performed. In addition, by developing components using two-dimensional electrophoresis and transferring components while maintaining the two-dimensional development results, it is possible to analyze components while taking advantage of the detailed development results in the two-dimensional area. Is possible (claim 3).

Further, by bringing the development area and the analysis area into contact with each other and applying an electric field to perform the transfer, it is possible to perform the transfer by a simple method while reliably maintaining the development results of the components. At this time, by using the analysis area as an electrode, transfer can be performed with a simpler configuration. In addition, by performing transfer using the film as an intermediate medium, transfer can be performed easily and inexpensively using a medium used for another method.

When the electrophoresis is performed by gel electrophoresis, the components are electrophoresed while the gel is in contact with the analysis area, and after the electrophoresis, the gel portion where the components are present is analyzed. By performing immobilization on the region and performing transcription, it becomes possible to perform transcription simply and inexpensively using reagents and tools used for ordinary gel electrophoresis.

Further, the component is bonded to a functional group present on the surface of the analysis region, the component is adsorbed on the surface of the analysis region, or the component is crosslinked or included with the surface of the analysis region. By immobilizing the components in the analysis area,
Transfer can be performed reliably with a simple configuration. In addition, by using a surface plasmon resonance analysis element as an analysis region and performing analysis by surface plasmon resonance analysis, it is possible to efficiently efficiently analyze a sample containing a plurality of types of mixed components and a sample showing a continuous characteristic distribution. In addition, components can be analyzed very quickly without the need for labeling and washing, and kinetic analysis of components can be performed (claim 4).

Further, by using an element for evanescent field excitation fluorescence analysis as an analysis area and performing analysis by evanescent field excitation fluorescence analysis, a sample containing a plurality of types of mixed components or a sample showing a continuous characteristic distribution is obtained. In addition, the component can be analyzed efficiently and rapidly with high sensitivity without the need for washing, and the kinetic analysis of the component can be performed (claim 5).

Further, by using a surface-enhanced Raman scattering analysis element as an analysis region and performing analysis by surface-enhanced Raman scattering analysis, a sample containing a plurality of types of mixed components or a sample showing a continuous characteristic distribution is obtained. It is also possible to efficiently analyze components with extremely high sensitivity (claim 6). After the transfer, a reagent is added to the analysis area, and the reaction between the reagent and the component is detected by a method using evanescent field excitation. Analyzes based on the reaction between a component and a reagent can be performed efficiently even on a sample showing distribution, and depending on the characteristics of the component and the reagent, the reagent can be desorbed from the component after the analysis, so that the Reanalysis is also possible.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a functional configuration of a sample analyzer according to one embodiment of the present invention.

FIG. 2 is a diagram for explaining a method using gravity, which is one of the transfer methods of the present embodiment.

FIGS. 3A and 3B are diagrams for explaining a method using an electric field, which is one of the transfer methods of the present embodiment.

FIG. 4 is a diagram for explaining a method using an intermediate medium, which is one of the transfer methods of the present embodiment.

FIG. 5 is a diagram for explaining a method of fixing a developed component, which is one of the transfer methods of the present embodiment.

FIGS. 6A to 6C are diagrams for explaining a technique for immobilizing a component transferred in the present embodiment on an analysis element.

FIG. 7 is a flowchart for explaining a sample analysis procedure (a sample analysis method as one embodiment of the present invention) of the present embodiment.

FIGS. 8A and 8B are diagrams for explaining excitation by an evanescent field.

FIG. 9 (a) shows the principle of surface plasmon resonance analysis,
(B) is a figure for each explaining the principle of the evanescent field excitation fluorescence method.

FIGS. 10A and 10B are diagrams for explaining a technique for immobilizing an analyte on an analysis element in a conventional analysis technique using excitation by an evanescent field.

FIGS. 11A and 11B are diagrams for explaining the development of components by gel electrophoresis.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Sample analyzer 2 Development part 3 Transfer part 4 Analysis part 5 Development phase (gel, stationary phase) 6 Element for analysis (element for surface plasmon resonance analysis) 6-1 Element part 6-2 Metal thin film 6-3 Organic transfer layer 6-3a Scaffold material 6-3b Adsorbed material 6-3c Base material 7-1, 7-2 Electrode 8 Intermediate medium (adsorption film) A, B Materials with different refractive indices B 'Insulator C Metal D, D' Analytical Product E Chip E1 Element for analysis E2, E3 Components of chip F Gel

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/543 595 G01N 33/561 33/561 27/26 315H 315E 325A 315J

Claims (7)

[The claims] 1. A method for analyzing a sample containing one or more components, comprising: a developing step of developing the component contained in the sample into a one-dimensional or more developing region; Transferring the above-mentioned components to a one-dimensional or more analysis area while maintaining the developed result; andanalyzing the components transferred to the analysis area by a method using excitation by an evanescent field. A sample analysis method, comprising: 2. The sample analysis method according to claim 1, wherein in the developing step, a gel is used as the developing area, and the developing is performed by gel electrophoresis. 3. In the developing step, developing is performed on a two-dimensional developing area by two-dimensional electrophoresis, and in the transferring step, transferring is performed on a two-dimensional analyzing area while maintaining the two-dimensional developing result. The method according to claim 1, wherein the method is performed. 4. The method according to claim 1, wherein in the transferring step, an element for surface plasmon resonance analysis is used as the analysis area, and in the analysis step, analysis is performed by surface plasmon resonance analysis. 2. The sample analysis method according to 1. 5. In the transferring step, an element for evanescent field excitation fluorescence analysis is used as the analysis area, and in the analysis step, analysis is performed by evanescent field excitation fluorescence analysis. The sample analysis method according to claim 1. 6. In the transferring step, an element for surface enhanced Raman scattering analysis is used as the analysis area, and in the analysis step, analysis is performed by surface enhanced Raman scattering analysis. The sample analysis method according to claim 1. 7. An apparatus for analyzing a sample containing one or more components, comprising: a developing unit for developing the component contained in the sample into a one-dimensional or more developing region; A transfer unit that transfers the component developed in the region to a one-dimensional or more analysis region while maintaining the development result, and excites the component transferred to the analysis region by the transfer unit by an evanescent field. A sample analyzer, comprising: an analyzer for performing analysis by a used method.