The present application claims priority from Japanese application JP 2004-347986 filed on Dec. 1, 2004, the content of which is hereby incorporated by reference into this application.
The present invention relates to an analyzer and a detection system in which a reaction solution containing at least a molecule that interacts with at least a biomolecule or at least a tissue section containing the biomolecule fixed on a substrate is stirred in a reaction space.
In the field of current molecular biology, attention is focused on expression analysis and functional analysis of genes and proteins as an important task. To perform this analysis, DNA chip, DNA microarray, protein array, tissue microarray, and the like that are immobilized with nucleic acids, proteins, or tissue sections on a slide glass substrate have come into widespread use. In order to carry out a reaction such as hybridization or antigen-antibody reaction on the slide glass substrate, it is generally necessary to cover the substrate with cover glass or to keep it in a wet chamber or in a closed container for prevention of evaporation of a reaction solution during a reaction requiring a long time (more than 12 hours) after dropping the reaction solution containing a nucleic acid probe or antibody on the substrate. Since mixing the reaction solution is effective for shortening the reaction time, enhancing sensitivity in signal detection, and enhancing reproducibility of detection signal, a reaction vessel or apparatus provided with mixing function is used.
As a conventional apparatus, for example, as disclosed in Patent Document 1 (U.S. Pat. No. 6,238,910), a hybridization apparatus for DNA microarray in which hybridization reactivity is improved by carrying out reciprocal shaking of a reaction solution in a reaction vessel by means of an installed pump function is described.
In Patent Document 2 (U.S. Patent Application No. 20040115097), a method in which surface acoustic waves stimulated on the surface of a piezoelectric solids by surface distortion of the piezoelectric body arising from application of an electric field to interdigital electrodes deposited on the piezoelectric solids is utilized for mixing of a small quantity of liquid is disclosed.
Further, in Patent Document 3 (JP-A No. 248008/2003), a method in which mixing of a reaction solution in a micro-reaction vessel is carried out by allowing magnetic beads to be present in the reaction solution in the micro-reaction vessel and providing the magnetic beads with magnetic changes externally to fluidize them in the reaction vessel.
Since efficiency of hybridization is improved in a reaction apparatus of conventional technology having a function of mixing of a reaction solution compared with a case in which mixing is not performed, it is considered that mixing of a reaction solution by the conventional technology is an effective technique. However, when reciprocal shaking of a reaction solution is carried out by a pumping function provided to the apparatus, an extra volume of the reaction solution corresponding to the solution retained in the volume of syringe pump and that of a flow path between the pump and the reaction vessel is required in addition to the volume of the solution retained in the reaction vessel on the slide glass substrate, resulting in wasting a sample or probe contained in the reaction solution. Further, it is necessary to arrange a flow path connecting the pump to the reaction vessel, thereby making the mechanism of the apparatus more complicated.
On the other hand, in the method disclosed in Patent Document 2 in which the flow path to connect a pump portion for mixing a reaction solution to the reaction vessel is not required, vibration amplitude of the surface acoustic wave utilized for mixing the solution is only several nanometers and very small, and therefore a region in which mixing can be performed is limited in the depth direction of the reaction vessel. Further, not only is the surface sound wave very weak but also its traveling direction is limited to the direction perpendicular to the interdigital electrodes, and thus arrangement of a plurality of surface acoustic wave-generating portions is required to stir and mix uniformly the whole region on the slide glass substrate, resulting in making the mechanism of the apparatus more complicated.
In the technique disclosed in Patent Document 3 described above, magnetic beads are moved toward the upper side of the micro-reaction vessel (the inside of the cover), and therefore liquid movement caused by the movement of the beads is satisfactory in the upper side of the micro-reaction vessel, whereas liquid movement, that is, mixing efficiency of the liquid in the lower side of the vessel near the surface of the slide glass substrate is decreased. Furthermore, contact of the magnetic beads with the slide glass substrate in the micro-reaction vessel detaches fixed nucleic acid, protein, or tissue section from the substrate, and the possibility that intensities of signals to be detected are influenced cannot be denied.
An object of the present invention is to provide an analyzer and a detection system that do not need a complicated device mechanism, achieve a uniform mixing of a reaction solution over an entire region on a slide glass substrate, and have a way of mixing that gives high reaction efficiency.
An apparatus characterized in that a substrate holder to hold a substrate where at least part of the surface is fixed with at least a substance that binds specifically to at least a target analyte, a cover member that faces the substrate holder and covers the substrate, a liquid inlet to introduce a liquid between the substrate and the cover member, a liquid outlet to discharge the liquid introduced between the substrate and the cover member, and an actuating unit that makes contact with the cover member are provided and the actuating unit deforms at least part of the cover member is provided. Here, the cover member may have a concave portion facing the substrate and make contact with the actuating unit on its surface not facing the substrate. The material for the cover member is desirably a material having elasticity. Specifically, it may be rubber such as synthetic rubber and elastic rubber or a material classified as elastomer. The present apparatus may be used either as a solution mixing type apparatus or an analysis system equipped with a detection system.
Using as another construction an apparatus having a substrate holder to hold a substrate where at least part of the surface is fixed with at least a probe or at least a tissue section that binds selectively to at least a target analyte in a sample solution, a cover member that has a concave portion so as to face the substrate holder and form a space to retain a solution on the surface of the probe or the tissue section fixed on the substrate and covers the substrate, a liquid inlet to introduce a liquid into the space formed between the substrate and the cover member, a liquid outlet to discharge the liquid introduced into the space formed between the substrate and the cover member, and an actuating unit that makes contact with the cover member and exerts a force on the cover member externally, mixing of the solution retained in the space may be carried out by deforming at least part of the cover member by the actuating unit.
According to the above construction, it becomes possible to stir the solution retained in the space under various conditions by deforming an arbitrary place of the deformable cover member with an arbitrary force and in an arbitrary number of times and magnitude of movement. This mixing allows uniform mixing of the reaction solution to be accomplished over the entire region on the slide glass substrate, and high reaction efficiency is also obtained. It should be mentioned that even when the volume of the solution retained in the space is small, not only can uniform mixing be accomplished but also uniformity in reaction efficiency can be obtained by the above construction.
Further, an increase in the number of reaction processing of target analyte is achieved by providing a plurality of the concave portions on the cover member to form reaction spaces and mixing each of the reaction spaces via deformation of the cover member.
Furthermore, it becomes possible to detect reaction signals continuously or concurrently with the reaction by providing a window portion on the substrate holder as well as a detection unit to detect the reaction with the target analyte. Owing to a short time between a reaction and its detection, the number of analyzable target analyte can be increased.
According to the present invention, there is an effect of enhancing signal intensities due to an improvement in reaction efficiency by mixing in the reaction space a solution containing at least a molecule interacting with at least a biomolecule fixed on a substrate or the biomolecule localized on at least a tissue section fixed thereon. Even when a plurality of the reaction spaces may be provided and the number or the kind of test samples may differ, there is also an effect that reactions can be run at the same time.
Hereinafter, embodiments of the present invention are explained referring to
The construction of an analyzer according to the present invention is shown in
The height of the space 5 formed between the substrate 1 and the cover member 7 that has a concave portion 6 so as to face the substrate holder 4 and form the space 5 to retain a solution on the probe or the tissue section fixed on the surface of the substrate 1 and covers the substrate 1 as shown in
In order to stir the solution in the space 5, the actuating units 10 and 11 that make contacts with the cover member 7 and exert an external force on the cover member are used as shown in
With the use of the analyzer according to the present invention, an example of the results obtained from performing immunohistochemical staining with a monoclonal antibody is shown in
Next, another example of the actuating unit shown in
The present invention may also take the following constructions:
(1) An analyzer characterized in that a substrate holder to hold a substrate where at least part of the surface is fixed with at least a probe or at least a tissue section that binds selectively to at least a target analyte in a sample solution, a cover member that has a concave portion so as to face the substrate holder and form a space to retain a solution on the surface of the probe or the tissue section fixed on the substrate and covers the substrate, a liquid inlet to introduce a liquid into the space formed between the substrate and the cover member, a liquid outlet to discharge the liquid introduced into the space formed between the substrate and the cover member, and an actuating unit that makes contact with the cover member and exerts a force on the cover member externally are provided, and mixing of the solution retained in the space is carried out by deforming at least part of the cover member by the actuating unit.
(2) The solution mixing type analyzer described in (1) characterized in that the concave portion faces the surface of the substrate on which the probe or tissue section is fixed, and the actuating unit makes contact with an arbitrary location on the surface of the concave portion not facing the substrate and deforms the cover member.
(3) The solution mixing type analyzer described in (1) characterized in that a motor or actuator is provided as the actuating unit.
(4) The solution mixing type analyzer described in (1) characterized in that a member susceptible to magnetic influence is attached to an arbitrary location on the surface of the concave portion not facing the substrate as the actuating unit, and the cover member is deformed by moving the member susceptible to magnetic influence through a change of magnetic field externally.
(5) The solution mixing type analyzer described in (4) characterized in that the change of magnetic field is performed by moving a permanent magnet arranged in the vicinity of the member susceptible to magnetic influence or by magnetizing and de-magnetizing an electromagnet.
(6) The solution mixing type analyzer described in (1) characterized in that the thickness of the space formed between the substrate and the concave portion provided on the cover member is from 20 micrometers to one millimeter.
(7) The solution mixing type analyzer described in (1) characterized in that the magnitude of deformation of the cover member by the actuating unit is in the range from at least 20% to less than 100% of the thickness of the space.
(8) The solution mixing type analyzer described in (1) characterized in that a plurality of the concave portions are provided on the cover member in order to form a plurality of spaces between the substrate and the concave portion provided on the cover member.
(9) The solution mixing type analyzer described in (8) characterized in that, for the cover member having a plurality of the concave portions, the cover member on each concave portion is deformed by the actuating units that make contacts independently with the surfaces of each concave portion not facing the substrate.
(10) The solution mixing type analyzer described in (1) characterized in that the cover member is made of a flexible and deformable resin material which can be quickly deformed in response to the shape of the actuating unit, the magnitude of its movement, the speed of its movement, and the like, and is preferably a silicone resin, an elastomer containing silicone, or a silicone rubber, and mixing of the solution retained in the space is carried out by deformation by the actuating unit.
(11) The solution mixing type analyzer described in (1) characterized in that the target analyte is a single strand or double strand nucleic acid, antibody, antigen, receptor, ligand, or enzyme when the probe fixed on the substrate is a nucleic acid probe, antigen, antibody, ligand, receptor, or substrate, or the target analyte is a single strand nucleic acid or antibody when the tissue section is fixed on the substrate.
(12) A detection system characterized in that a substrate holder to hold a substrate where at least part of the surface is fixed with at least a probe or at least a tissue section that binds selectively to at least a target analyte in a sample solution, a cover member that has a concave portion so as to face the substrate holder and form a space to retain a solution on the surface of the probe or the tissue section fixed on the substrate and covers the substrate, a liquid inlet to introduce a liquid into the space formed between the substrate and the cover member, a liquid outlet to discharge the liquid introduced into the space formed between the substrate and the cover member, a detection unit to detect a reaction between the fixed probe or tissue section and the target analyte in the sample solution, and an actuating unit that makes contact with the cover member and exerts a force on the cover member externally are provided, and mixing of the solution retained in the space is carried out by deforming at least part of the cover member by the actuating unit.
(13) The detection system described in (12) characterized in that the substrate holder has a window portion, and the detection unit detects the reaction through the window portion.
(14) The detection system described in (12) characterized in that the concave portion faces the surface of the substrate on which the probe or tissue section is fixed, and the actuating unit makes contact with an arbitrary location on the surface of the concave portion not facing the substrate and deforms the cover member.
(15) The detection system described in (12) characterized in that a motor or actuator is provided as the actuating unit.
(16) The detection system described in (12) characterized in that a member susceptible to magnetic influence is attached to an arbitrary location on the surface of the concave portion not facing the substrate as the actuating unit, and the cover member is deformed by moving the member susceptible to magnetic influence through a change of magnetic field externally.
(17) The detection system described in (16) characterized in that the change of the magnetic field is performed by moving a permanent magnet arranged in the vicinity of the member susceptible to magnetic influence or by magnetizing and de-magnetizing an electromagnet.
(18) The detection system described in (12) characterized in that the thickness of the space formed between the substrate and the concave portion provided on the cover member is from 20 micrometers to one millimeter.
(19) The detection system described in (12) characterized in that the magnitude of deformation of the cover member by the actuating unit is in the range from at least 20% to less than 100% of the thickness of the space.
(20) The detection system described in (12) characterized in that a plurality of the concave portions are provided on the cover member in order to form a plurality of spaces between the substrate and the concave portion provided on the cover member.
(21) The detection system described in (20) characterized in that, for the cover member having a plurality of the concave portions, the cover member on each concave portion is deformed by the actuating units that make contacts independently with the surfaces of each concave portion not facing the substrate.
(22) The detection system described in (20) characterized in that, for the plurality of spaces formed, the window portion is arranged corresponding to each of the spaces to detect the reaction.
(23) The detection system described in (20) characterized in that the cover member is made of a flexible and deformable resin material which can be quickly deformed in response to the shape of the actuating unit, the magnitude of its movement, the speed of its movement, and the like, and is preferably a silicone resin, an elastomer containing silicone, or a silicone rubber, and mixing of the solution retained in the space is carried out by deformation by the actuating unit.
(24) The detection system described in (12) characterized in that the target analyte is a single strand or double strand nucleic acid, antibody, antigen, receptor, ligand, or enzyme when the probe fixed on the substrate is a nucleic acid probe, antigen, antibody, ligand, receptor, or substrate, or the target analyte is a single strand nucleic acid or antibody when the tissue section is fixed on the substrate.
Number | Date | Country | Kind |
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2004-347986 | Dec 2004 | JP | national |