JP2004020386A - Hybridization method and hybridization apparatus for selectively coupling substance, and base for immobilizing the selectively coupling substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an efficient hybridization method without any deviation in a reaction by reducing the reaction time of a selectively coupling substance. <P>SOLUTION: In a process for immobilizing the selectively coupling substance onto the base, bringing a sample solution to be inspected containing a corresponding selectively coupling substance to be selectively coupled with the selectively coupling substance into contact with the immobilized selectively coupling substance, and coupling the selectively coupling substance with the corresponding selectively coupling substance for reaction, an AC voltage is applied between two electrodes for parallel movement that are arranged outside of both ends of the selectively coupling substance immobilization region and oppose each other. A voltage is applied between a lower electrode for vertical movement being arranged immediately below a site where the selectively coupling substance in the base is immobilized, and an upper electrode for vertical movement being arranged at a position that opposes the selectively coupling substance immobilization surface via the sample solution to be inspected, thus performing the coupling reaction. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides a method for binding reaction between a substance that selectively binds to a test substance (herein, a “selective binding substance”) and a test substance (herein, a “corresponding selective binding substance”), The present invention relates to an apparatus and a substrate for that purpose.
[0002]
[Prior art]
Research on genetic information analysis of various organisms has begun, including information on human genes and many other genes, their base sequences, the proteins encoded by the gene sequences, and the sugar chains secondary to these proteins. Is rapidly being revealed. The functions of macromolecules such as genes, proteins, sugar chains and the like whose sequences have been revealed can be examined by various methods. Mainly, for nucleic acids, it is possible to examine the relationship between various genes and their biological functions by utilizing complementation between various nucleic acids / nucleic acids, such as Northern hybridization or Southern hybridization. Regarding proteins, the functions and expression of proteins can be examined using a protein-protein reaction, as typified by Western hybridization.
[0003]
In recent years, a new analysis method or methodology called a DNA microarray method (DNA chip method) has been developed as a technique for analyzing a large number of gene expressions at a time, and has attracted attention. These methods are basically the same as the conventional methods in that they are nucleic acid detection and quantification methods based on a nucleic acid / nucleic acid hybridization reaction. It can also be applied to the detection and quantification of proteins and sugar chains based on the hybridization between chains / proteins. These technologies have a great feature in that a large number of DNA fragments, proteins, and sugar chains are arranged and fixed at high density on a flat substrate piece called a microarray or chip. As a specific method of using the microarray method, for example, a sample in which an expressed gene or the like of a research target cell is labeled with a fluorescent dye or the like is hybridized on a piece of a flat substrate, and nucleic acids (DNA or RNA) complementary to each other are bound to each other. A method of labeling the portion with a fluorescent dye or the like and then reading it at high speed with a high-resolution analyzer, or a method of detecting a response such as a current value based on an electrochemical reaction. Thus, the type of the gene contained in the sample can be quickly estimated.
[0004]
On the other hand, since the nucleic acid solution used for nucleic acid / nucleic acid hybridization is precious, it is desirable to carry out the hybridization reaction with the amount of nucleic acid as small as possible. Therefore, it is necessary to improve the efficiency in hybridization with a low-concentration nucleic acid solution. However, conventionally used microarrays in which a large number of selective binding substances such as DNA fragments, proteins, and sugar chains are aligned and immobilized on a flat substrate piece at a high density, or the selective binding substance is placed inside a porous hollow fiber. The immobilized porous hollow fibers are bound and fixed, and cut in a direction intersecting with the fiber axis of the array to form a thin piece, thereby forming a two-dimensional high-density fiber array in which the selective binding substance is fixed inside the fiber. In a microarray, or a microarray in which the selective binding substance is aligned and fixed at a high density on the fiber surface, and the fibers are arranged as a three-dimensional structure, the hybridization reaction depends on the natural diffusion of the selective binding substance. Uses a solution containing a small amount of a selective binding substance to efficiently initiate a hybridization reaction, effectively It is difficult to use.
[0005]
Therefore, a sample nucleic acid fixing site having a conductor layer is provided on the substrate of the microarray, a positive potential is applied to the sample nucleic acid fixing site to create an electric field, and the sample nucleic acid in the nucleic acid solution is fixed to the sample nucleic acid solution. Attempts have also been made to increase the hybridization efficiency by attracting to the vicinity of the site and locally increasing the nucleic acid concentration in the vicinity of the sample nucleic acid immobilization site (JP-A-8-154656).
[0006]
Microarrays using proteins and sugar chains are expected to have the same effects as microarrays using these nucleic acids.
[0007]
However, in the method using the conductor layer, since the sample nucleic acid and the sample nucleic acid fixed to the sample nucleic acid fixing site are electrically adsorbed to the sample nucleic acid fixing site, the movement of the nucleic acid is limited, and the hybridization is not performed. Saturation due to collisions between nucleic acids during the period when nucleic acids are attracted to the sample nucleic acid fixation site, many unreacted nucleic acids remain even if the hybridization time is extended, and a small amount of sample nucleic acid is effectively used in the hybridization reaction Has limitations. Therefore, even the method of increasing the efficiency of the hybridization reaction of the selective binding substance by the electric suction invented to solve this inefficiency was not sufficiently efficient.
[0008]
[Problems to be solved by the invention]
Under such circumstances, establishing a hybridization method that can effectively use a small amount of a high-molecular sample such as an expensive nucleic acid, protein, sugar chain, antibody, or antigen is considered to be increasingly important in the future. It is strongly required for polymer analysis.
[0009]
The present invention solves the conventional drawbacks described above, and uses a small amount of a selective binding substance effectively to perform a hybridization reaction in a short time. It is intended to provide a substrate.
[0010]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-described problems, and as a result, during the hybridization reaction period, the corresponding selective binding substance was attached to the microarray substrate, or in the vicinity of the selective binding substance immobilized on the fiber. It has been found that the efficiency of the hybridization reaction can be increased by increasing the probability of collision between the selective binding substance and the corresponding selective binding substance by constantly moving the selective binding substance and the homogeneity of the binding reaction. Reached.
[0011]
That is, the present invention
(1) immobilizing a selective binding substance on a substrate, bringing a test sample solution containing a corresponding selective binding substance that selectively binds to the selective binding substance into contact with the immobilized selective binding substance, In the step of causing the selective binding substance to react with the corresponding selective binding substance, in the direction intersecting with an axis perpendicular to the plane of the immobilization site where the selective binding substance is immobilized, and immobilizing the selective binding substance. An AC voltage is applied between two opposing parallel movement electrodes disposed outside both ends of the portion, and a vertical movement lower electrode disposed immediately below the portion of the base material on which the selective binding substance is immobilized. A method for applying a voltage between upper electrodes for vertical movement arranged at a position facing the surface for immobilizing the selective binding substance via the test sample solution to perform the binding reaction, ,
(2) Immobilizing a selective binding substance on a substrate, and bringing a test sample solution containing a corresponding selective binding substance that selectively binds to the selective binding substance into contact with the immobilized selective binding substance. An apparatus for performing a binding reaction between the selective binding substance and the corresponding selective binding substance, in a direction intersecting a vertical axis of a base on which the base material is installed and a surface on which the selective binding substance is immobilized. And two opposing parallel-moving electrodes disposed outside of both ends of the selective-bonding substance-immobilized region, AC voltage applying means for applying an AC voltage between the parallel-moving electrodes, A vertical movement lower electrode disposed immediately below the site where the selective binding substance is immobilized, and a vertical movement upper electrode disposed at a position facing the selective binding substance immobilizing surface via the test sample solution An electrode, the upper electrode for vertical movement and the vertical Hybridization apparatus selective binding substance having a DC-AC application means for applying a DC voltage and / or AC voltage between the movement for the lower electrode,
(3) Immobilizing a selective binding substance on a substrate, and bringing a test sample solution containing a corresponding selective binding substance that selectively binds to the selective binding substance into contact with the immobilized selective binding substance. A substrate for allowing the selective binding substance to react with the corresponding selective binding substance, wherein the selective binding substance is immobilized on the substrate; Two opposing parallel movement electrodes disposed outside both ends of the substance fixing region, a vertical movement lower electrode disposed immediately below the selective binding substance fixing region, and a vertical movement lower electrode. It is a base material for fixing a selective binding substance having a conductive pattern wiring connected thereto.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
The method for hybridization of a selective binding substance of the present invention comprises the steps of: constantly moving a corresponding selective binding substance near a fixed selective binding substance during a hybridization reaction; The hybridization reaction is carried out efficiently by increasing the concentration of the selective binding substance, in a direction intersecting the axis perpendicular to the plane of the immobilization site where the selective binding substance is immobilized, and outside the both ends of the selective binding substance immobilization site. AC voltage is applied between two opposing parallel-moving electrodes arranged at the same position, and selectively coupled via the test sample solution to the vertical-moving lower electrode disposed immediately below the site where the selective-binding substance is immobilized. It is important to apply a voltage between the upper electrodes for vertical movement arranged at a position facing the surface where the immobilized substance is fixed.
[0013]
Further, the hybridization device of the present invention is a preferable device for performing the above method, a base on which a substrate is placed, and a direction intersecting a vertical axis of a surface on which the selective binding substance is immobilized, and Two opposing parallel-moving electrodes disposed outside both ends of the selective-bonding substance-immobilized region, AC voltage applying means for applying an AC voltage between the parallel-moving electrodes, and a selective-binding substance on the substrate A lower electrode for vertical movement disposed immediately below the portion on which is fixed, an upper electrode for vertical movement disposed at a position opposite to the surface on which the selective binding substance is immobilized via the test sample solution, and DC / AC applying means for applying a DC voltage and / or an AC voltage between the upper electrode and the lower electrode for vertical movement.
[0014]
In the present invention, platinum, platinum, or the like can be used for the parallel-moving electrode, the vertical-moving upper electrode, and the vertical-moving lower electrode (hereinafter, referred to as the vertical-moving electrode when the latter two are referred to). Simple metals such as gold, silver, chromium, titanium, nickel, aluminum, copper, and palladium, or oxides, nitrides, or alloys of these metals, carbon or carbon compounds, or conductive polymers, and the like. It suffices that at least one selected from the following is included.
[0015]
The properties of the metal alone or the oxides, nitrides or alloys of these metals can be obtained by applying a voltage between the electrodes for parallel movement and / or between the electrodes for vertical movement provided using these materials. Since current flows between the parallel movement electrodes and / or between the vertical movement electrodes via the test sample solution containing the corresponding selective binding substance, it reacts with the test sample solution and A material from which metal ions are hardly eluted is preferable.
[0016]
Typical examples of the carbon compound include graphite and fullerene.
[0017]
Representative examples of the conductive polymer include polyacetylene, polypyrrole, polythiophene, polyaniline, and the like, and a composite conductive plastic or the like having improved conductive properties by mixing these conductive polymers with the metal, a carbon compound, and the like. .
[0018]
The material of the electrode for parallel movement and / or the upper and lower electrodes for vertical movement is made of platinum, titanium, gold, silver, aluminum, copper, palladium alone or an alloy thereof, carbon or carbon compound, or conductive material. It is preferably at least one selected from polymers.
[0019]
The electrode for parallel movement is preferably formed in advance on a substrate for immobilizing a selective binding substance for the reason described below, but has an electrode on the hybridization device side and is used for immobilizing the selective binding substance in a hybridization preparation stage. A mode in which an electrode is mounted on a substrate may be used.
[0020]
Therefore, as the selective binding substance fixing substrate preferably used in the present invention, the selective binding substance fixing site for immobilizing the selective binding substance on the substrate, and from both ends of the selective binding substance fixing region. It has two opposing electrodes for parallel movement arranged on the outside, a lower electrode for vertical movement provided immediately below the site for fixing the selective binding substance, and a conductive pattern wiring connected to the lower electrode for vertical movement. Things.
[0021]
The lower electrode for vertical movement disposed immediately below the selective binding substance fixing portion attracts the corresponding selective binding substance to the selective binding material fixing portion by flowing a current between the lower electrode and the upper electrode for vertical movement. In order to have a function, it is necessary to form on a substrate for fixing a selective binding substance in advance.
[0022]
When a metal is used as an electrode material, a mask having an opening in the shape of a parallel movement electrode and / or a vertical movement lower electrode is arranged on the base material as a means for placing an electrode on the base material using these materials. Then, an electrode is formed by a sputtering method, an evaporation method, or a thick film electrode is formed by a plating method, and further, an electrode is formed by bonding a metal foil or a thin metal plate to a base material with an adhesive. Method. In the case of using a carbon compound, a mask having an opening in the shape of an electrode is provided over a base material, and the electrode can be formed by a sputtering method. In the case of using a conductive polymer, a conductive polymer in the form of a paste is applied using a printing method such as silk screen printing, and the paste is cured using a photocuring method using ultraviolet light to form an electrode.
[0023]
In the case where the parallel movement electrode is disposed on the hybridization device side in advance, a thin plate made of the metal material, the carbon compound, and the conductive polymer is disposed on the base of the hybridization device. An electrode can be formed by placing the substrate for fixing a selective binding substance in a base shape and then mounting the electrode plate on the substrate for fixing a selective binding substance.
[0024]
Next, a hybridization method, a hybridization device, and a substrate for immobilizing a selective binding substance of the present invention will be described with reference to the drawings.
[0025]
FIG. 1A is a cross-sectional view of one embodiment of the hybridization device of the present invention, and FIG. FIG. 2 is a principle view showing the operation of the selective binding substance in the present invention. Note that the present invention is not limited to this example.
[0026]
As shown in FIGS. 1 and 2, the hybridization apparatus of the present invention comprises a substrate 1 for immobilizing a selective binding substance placed on a base 14, a cover substrate 6, and an AC voltage applying means 9. And an upper electrode 7 for vertical movement mounted on the cover substrate 6, and a DC and AC voltage applying means 12. On the lower electrode 4 for vertical movement provided on the substrate 1 for immobilizing a selective binding substance, a selective binding substance immobilizing portion 5 is provided. Are fixed, and the selective binding substance immobilized areas having a plurality of selective binding substances 15 are arranged in an array to form a selective binding substance array area 8. Further, on the substrate 1 for fixing the selective binding substance, parallel movement electrodes 2 and 3 are provided on both sides of the selective binding substance array region 8, and the conductive pattern wiring 10 is connected thereto. A cover substrate 6 is provided on the moving electrodes 2 and 3. A test sample solution 13 containing the corresponding selective binding substance 16 is filled in a space sandwiched between the selective binding substance fixing substrate 1 and the cover substrate 6 via the parallel movement electrodes 2 and 3. Each lower electrode 4 for vertical movement is connected to the DC voltage and AC voltage applying means 12 by contact between the conductive pattern wiring 10 and the connector 11. Similarly, the vertical movement upper electrode 7 is also connected to the DC voltage and AC voltage application means 12, and the parallel movement electrodes 2 and 3 are connected to the AC voltage application means 9.
[0027]
As the substrate 1 for fixing a selective binding substance, a glass substrate or a resin such as PMMA or polycarbonate can be used. In particular, when used in a system for detecting the result of hybridization by fluorescence, non-fluorescent glass and low autofluorescence are used. PMMA or the like is desirable. The cover substrate 6 is not particularly limited as long as it has good chemical resistance to a solution used for hybridization, but glass, polypropylene, polycarbonate, or the like can be used.
[0028]
In the present invention, there is a selective binding substance array region 8 in which a plurality of selective binding immobilization sites are arranged as described above, and the parallel-moving electrodes 2 and 3 are provided at both ends of the selective binding substance array region 8. It is preferable to be located more outside.
[0029]
The form in which the selective binding substances 15 are arranged in the selective binding substance array region 8 is preferably arranged on grid points on a two-dimensional plane. Alternatively, the position of the selective binding substance fixing portion 5 may be different from the surface of the selective binding substance fixing substrate 1 in height, so that a three-dimensional arrangement may be employed.
[0030]
Next, a method for hybridizing a selective binding substance of the present invention using the above-described hybridization device will be described. After the test sample solution 13 is filled, a voltage is applied between the lower electrode 4 for vertical movement and the upper electrode 7 for vertical movement using the DC voltage and AC voltage applying means 12. The voltage applied here is that the lower electrode 4 for vertical movement has a positive potential with respect to the upper electrode 7 for vertical movement, or the lower electrode 4 for vertical movement has a positive potential with respect to the upper electrode 7 for vertical movement. It is preferable to have a voltage application pattern which is a potential and in which a DC component and an AC component are mixed. As a result, an electric field is generated between the vertical movement upper electrode 7 and the vertical movement lower electrode 4, and the corresponding selective binding substance 16 naturally diffused in the test sample solution 13 has a negative charge. It is drawn in the direction of the vertical movement lower electrode 4 having a potential.
[0031]
Here, the relationship between the voltage application to each electrode and the hybridization effect will be described with reference to FIG. FIG. 5 shows a voltage application pattern to each electrode in the present invention. When the voltage applied to both the vertical movement electrodes in the DC and AC voltage ON section 28 of the DC voltage and AC voltage application means 12 is a DC potential, the corresponding selective binding substance 16 is oriented in the direction of the vertical movement lower electrode 4. And the concentration of the corresponding selective binding substance 16 near the selective binding substance fixing site 5 disposed on the lower electrode 4 for vertical movement locally increases. Since the collision probability between the immobilized selective binding substance 15 and the corresponding selective binding substance 16 is increased, the efficiency of hybridization is improved. However, in this case, since the electrostatic attraction always acts in the direction of the lower electrode 4 for vertical movement, if the DC voltage application time becomes longer, most of the corresponding selective binding substance 16 becomes the selective binding substance fixing portion. 5, the degree of freedom of operation may be reduced, and the efficiency of hybridization gradually decreases. As a measure to alleviate this, in the present invention, as shown in the DC and AC voltage application pattern 26, the vertical movement lower electrode 4 side has a positive potential with respect to the vertical movement upper electrode 7, and the DC component and the AC By applying a voltage in a pattern in which components are mixed, the corresponding selective binding substance 16 is attracted to the vicinity of the selective binding substance fixing site 5 by a strong attractive force in a high DC voltage section, While increasing the concentration of the corresponding selective binding substance 16, it is possible to prevent the degree of freedom of operation of the selective binding substance 15 and the corresponding selective binding substance 16 from being impaired. Therefore, the efficiency of hybridization does not decrease even if the voltage application time is long.
[0032]
As described above, in addition to locally increasing the concentration of the corresponding selective binding substance 16 in the vicinity of the selective binding substance fixing portion 5 using the DC voltage and AC voltage applying means 12, the present invention In the ON section 29 of the AC voltage applying means, the AC voltage applying means 9 is used to interpose between the electrodes 2 and 3 for translation in a direction intersecting the vertical axis of the surface on which the selective binding substance is immobilized. A voltage is applied (AC voltage application pattern 27). As a result, an electric field is generated between the parallel movement electrodes 2 and 3, and the corresponding selective binding substance 16 crosses the selective binding substance array region 8 according to the direction of the electric field generated between the parallel movement electrodes 2 and 3. Repeat the movement in the direction. Specifically, when the parallel-moving electrode 2 has a positive potential and the parallel-moving electrode 3 has a negative potential, the corresponding selective binding substance 16 is attracted to the parallel-moving electrode 2, and the parallel-moving electrode 2 has a negative potential. When the parallel movement electrode 3 has a positive potential, the corresponding selective binding substance 16 is attracted to the parallel movement electrode 3. By moving the corresponding selective binding substance 16 across the selective binding substance array region 8 in this manner, the selective binding substance 15 and the corresponding selective binding substance 16 Are contacted in the region with little deviation depending on the location, and hybridization occurs uniformly.
[0033]
As described above, the gate signal 25 is input to the AC voltage applying means 9 and the DC voltage and AC voltage applying means 12, and the DC voltage and AC voltage applying means ON section 28 and the AC voltage applying means ON section 29 are alternately generated. Accordingly, even when the test sample solution 13 having a low concentration is used, highly efficient and uniform hybridization can be realized, which is preferable.
[0034]
Note that, as the voltage applied to the electrode is higher, the electric attractive force or electric repulsive force that the selective binding substance 15 and the corresponding selective binding substance 16 having a negative charge receive from the electrode becomes stronger, and the corresponding selective binding substance 16 Needless to say, the effect of contact with the selective binding substance 15 due to the movement increases, but if a high voltage is applied for a long time, the selective binding substance 15 and the corresponding selective binding substance 16 may be damaged. In the present invention, the voltage is preferably set between 5 V and 50 V per 1 cm electrode spacing, and more preferably between 10 V and 25 V per 1 cm electrode spacing in order to obtain more stable hybridization results.
[0035]
As described above, in the method of the present invention for improving the efficiency of hybridization by using the AC electric field generated in the horizontal direction and the AC and DC electric fields generated in the vertical direction, the selective binding substance 15 and the corresponding selective binding substance 16 are used. Are constantly relatively moved during the period of hybridization, and the collision and contact are repeated, so that the efficiency of hybridization is increased.
[0036]
Here, as the selective binding substance fixing site, a position on a plane provided on the base material, irregularities provided on the base material are preferably used, and a hole penetrating the base material 1 for fixing the selective binding substance is preferably used. A similar effect can be expected even if a rod-shaped resin, glass, metal, fiber, or the like is inserted and used as the resin, glass, metal, or fiber tip as a selective binding substance fixing portion, and particularly provided on the base material. It is preferably a fiber or a fiber bundle inserted into the hole.
[0037]
Further, in order to reduce the amount of the expensive test sample solution 13, the electrodes 2 and 3 for parallel movement are preferably made as thin as possible, and more preferably 5 μm to 200 μm. In order to form such an electrode that is very thin and has little unevenness in thickness, the electrode is preferably formed in advance on a substrate for fixing a selective binding substance, but has an electrode on the hybridization device side, An electrode may be mounted on the selective binding substance-immobilizing substrate 1 at the stage of preparing for hybridization.
[0038]
Next, a conventional hybridization method and apparatus will be described with reference to FIGS. FIG. 3 is a cross-sectional view of a conventional hybridization device, and FIG. 4 is a principle diagram showing an operation of a selective binding substance in the conventional hybridization device.
[0039]
As described above, in the conventional method, since the hybridization reaction depends on the spontaneous diffusion of the selective binding substance, the probability of contact between the selective binding substance and the corresponding selective binding substance is low. Efficiency was low. Therefore, what has been proposed to solve this inefficiency is a hybridization method of a selective binding substance by electric suction as shown in FIGS.
[0040]
3 and 4, the selective binding substance 15 ′ is fixed on the selective binding substance fixing portion 20 provided on the selective binding substance fixing substrate 17. On the base material 17 for fixing the selective binding substance, a support material 18 is further provided outside the region where the selective binding substance 15 'is arranged, and a cover base material 21 is laid on the support material 18. You. A test sample solution 23 containing the corresponding selective binding substance 16 ′ is filled in a space sandwiched between the selective binding substance fixing substrate 17 and the cover substrate 21 via the support material 18. After the test sample solution 23 is filled, a negative potential is applied to the vertical movement upper electrode 22 by using the voltage application means 24, and a positive potential is applied to the vertical movement lower electrode 19 disposed immediately below the selective binding substance fixing portion 20. By applying a potential, an electric field is generated between the two electrodes, and the corresponding selective binding substance 16 ′ spontaneously diffused into the test sample solution 23 has a negative charge. It is sucked in the direction of 20. As a result, the concentration of the corresponding selective binding substance 16 ′ in the vicinity of the selective binding substance fixing site 20 increases, or the selective binding substance 15 ′ is absorbed in the process of adsorption of the corresponding selective binding substance 16 ′ to the selective binding substance fixing site 20. 'And the corresponding selective binding substance 16' come into contact, and hybridization occurs.
[0041]
However, in the method shown in FIG. 3, the corresponding selective binding substance 16 'is adsorbed to the selective binding substance fixing site 20 by electric force, and further, the selective binding substance having the same negative charge as the corresponding selective binding substance 16' is added. The selective substance 15 ′ is also adsorbed on the surface of the selective binding substance fixing portion 20, and there is no relative movement between the selective binding substance 15 ′ and the corresponding selective binding substance 16 ′. The dynamic contact probability between the selective binding substance and the corresponding selective binding substance is low, and sufficient efficiency of the hybridization reaction cannot be obtained.
[0042]
In the present invention, “selective binding substance” means a substance capable of selectively binding directly or indirectly to a test substance, and typically includes nucleic acids, proteins, saccharides, and other antigens. Compounds. The nucleic acid may be DNA or RNA. A single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a single-stranded nucleic acid having a base sequence complementary to the base sequence or a part thereof. Substance ". As proteins, antibodies, Fab fragments and F (ab ') ₂ Antigen binding fragments of antibodies, such as fragments, as well as various antigens can be mentioned. An antibody or an antigen-binding fragment thereof selectively binds to a corresponding antigen, and an antigen selectively binds to a corresponding antibody. The saccharide is preferably a polysaccharide, and includes various antigens. In addition, substances having antigenicity other than proteins and saccharides (antigenic compounds) can also be immobilized. The “selective binding substance” is preferably at least one selected from nucleic acids, proteins, saccharides, antibodies and antigenic compounds, and particularly preferred are nucleic acids, antibodies and antigens. The selective binding substance used in the present invention may be a commercially available substance, or may be a substance obtained from living cells or the like.
[0043]
DNA or RNA is prepared from living cells by a known method. For example, DNA is extracted by the method of Blin et al. (Blin et al., Nucleic Acids Res. 3: 2303 (1976)). The extraction can be performed by the method of Fabaloro et al. (Favalolo et al., Methods Enzymol. 65: 718 (1980)). Examples of the nucleic acid to be immobilized include a linear or circular plasmid DNA or chromosomal DNA, a DNA fragment obtained by cutting them with a restriction enzyme or chemically, a DNA synthesized by an enzyme or the like in a test tube, or a chemically synthesized oligo. Nucleotides and the like can also be used.
[0044]
In the present invention, it is preferable that the selective binding substance and the corresponding selective binding substance are single-stranded nucleic acids, and the binding reaction is hybridization between nucleic acids.
[0045]
Usually, one type of selective binding substance is fixed to each of the selective binding substance fixing sites. For example, when it is desired to bind multiple types of genes having mutations to the same selective binding substance fixing site. In addition, a plurality of types of selective binding substances can be immobilized on one selective binding substance immobilizing site.
[0046]
Further, the selective binding substances immobilized on the plurality of selective binding substance fixing sites may be different types of selective binding substances or the same selective binding substances. In addition, one type of selective binding substance is immobilized on some of the plurality of selective binding substance-immobilized sites, and one of the plurality of selective binding substance-immobilized sites is immobilized on the other plurality of selective binding substances. Another type of selective binding substance can be immobilized. The type and order of the selective binding substance are not limited by the position in the selective binding substance sequence region. It is also effective to fix the same selective binding substance on a plurality of selective binding substance fixing sites to increase the measurement sensitivity.
[0047]
The selective binding substance can be fixed to the selective binding substance fixing site by a known method. In the case where an unmodified selective binding substance is immobilized on the selective binding substance immobilizing site, the immobilization can be performed by baking or ultraviolet irradiation after the selective binding substance and the selective binding substance immobilizing site act on each other. In Examples described later, DNA is fixed to a slide glass substrate by this method. When the selective binding substance modified with an amino group is fixed to the selective binding substance fixing site, a crosslinking agent such as glutaraldehyde or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is used. Can be used to bind to the functional group of the selective binding substance fixing site. The temperature at which the sample containing the selective binding substance is allowed to act on the selective binding substance-immobilized site is preferably 5C to 95C, more preferably 15C to 65C.
[0048]
In the present invention, the selective binding substance may be directly immobilized on the selective binding substance-immobilized site, or a derivative obtained by chemically modifying the selective binding substance or a nucleic acid that is denatured as necessary may be immobilized. You may. Amination, biotinylation, digoxigenation and the like are known as chemical modifications of nucleic acids [Current Protocols In Molecular Biology, Ed. Frederick M .; Ausubel et al. (1990), De-isotope experiment protocol (1) DIG hybridization (Shujunsha)], and in the present invention, these modification methods can be adopted.
[0049]
As an example, introduction of an amino group into a nucleic acid will be described. The bonding position between the aliphatic hydrocarbon chain having an amino group and the single-stranded nucleic acid is not particularly limited, and is not limited to the 5 ′ end or 3 ′ end of the nucleic acid, but also in the nucleic acid chain (for example, a phosphodiester bond). Site or base site). This single-stranded nucleic acid derivative can be prepared according to the method described in Japanese Patent Publication No. 3-74239, U.S. Pat. No. 4,667,025, U.S. Pat. No. 4,789,737, and the like. In addition to this method, for example, using a commercially available reagent for introducing an amino group [for example, Aminolink II (trade name); PE Biosystems Japan, Amino Modifiers (trade name); Clontech], or using DNA It can be prepared according to a well-known method of introducing an aliphatic hydrocarbon chain having an amino group into the 5'-terminal phosphoric acid (Nucleic Acids Res., 11 (18), 6513- (1983)).
[0050]
The substrate for fixing a selective binding substance obtained by the above-mentioned method can be subjected to an appropriate treatment after the selective binding substance is fixed to the selective binding substance fixing region. For example, by performing a heat treatment, an alkali treatment, a surfactant treatment, or the like, the fixed selective binding substance can be modified. Alternatively, when a selective binding substance obtained from a biological material such as a cell or a microbial cell is used, unnecessary cell components may be removed. Then, the substrate for fixing a selective binding substance after the treatment can be used as a detection material for the selective binding substance. Note that these processes may be performed separately or simultaneously. Further, it may be appropriately performed before the sample containing the selective binding substance is fixed to the selective binding substance fixing region.
[0051]
The substrate for immobilizing a selective binding substance of the present invention, in which the selective binding substance is arranged in an array, interacts with the test substance by using the immobilized selective binding substance as a probe, whereby a specific substance in the sample is obtained. An analyte can be detected. The two types of test samples can be labeled (as distinguished) as described below, and the differences can be compared.
[0052]
For the detection of the corresponding selective binding substance in the test sample, which selectively binds to the selective binding substance, a known means capable of specifically recognizing the binding can be used. For example, a label such as a fluorescent substance, a luminescent substance, or a radioisotope is bound to the corresponding selective binding substance in the sample, and after the selective binding reaction and washing, the label can be detected. Regarding the type of these labels and the method of introducing the labels, fluorescent substances and luminescent substances used for immunoassay or measurement of nucleic acid hybridization are well known in this field, and various types are commercially available. Therefore, these commercially available fluorescent substances and luminescent substances can be used.
[0053]
After the binding reaction between the selective binding substance immobilized on the selective binding substance fixing site and the corresponding selective binding substance in the test sample, or simultaneously with the binding reaction, the selective binding substance is selectively bound to the corresponding selective binding substance. The labeled free measurement substance is further reacted, and after washing, the label of the measurement substance bound to the selective binding substance-immobilized site via the corresponding selective binding substance and the selective binding substance is measured. Can also be detected. For example, when a nucleic acid having a specific base sequence as a selective binding substance is immobilized on the selective binding substance fixing site, and the corresponding selective binding substance is a nucleic acid containing a region complementary to the nucleic acid, the corresponding selective binding substance In the nucleic acid as a substance, a nucleic acid complementary to a region other than the region complementary to the selective binding substance can be labeled and used as a measurement substance. In addition, an antigen is immobilized as a selective binding substance on the selective binding substance-immobilized site, and when the corresponding selective binding substance is an antibody that reacts with the antigen by an antigen-antibody, a second antibody that reacts with the antibody by an antigen-antibody is labeled. This can be used as a measurement substance.
[0054]
Further, when a material having electrical conductivity is used for the selective binding substance fixing portion, a method of detecting a response such as a current value based on an electrochemical reaction can be used. In this case, the selective binding substance and the corresponding selective binding substance immobilized on the selective binding substance immobilization site serving as an electrode are reacted in a reaction solution that promotes or suppresses the reaction between the two, and all or one of the materials is reacted. The part is contained in the bound selective binding substance and the corresponding selective binding substance, and by measuring the value of the current flowing through the reacted electrode, that is, the selective binding substance fixing site, the It can detect the presence or absence of the binding of the selective binding substance and the degree of the binding.
[0055]
In the measurement method using the substrate for immobilizing a selective binding substance applied to the hybridization device of the present invention, the test substance to be provided as the selective binding substance and the corresponding selective binding substance, a nucleic acid to be measured, for example, Examples include, but are not limited to, genes such as pathogenic bacteria and viruses, genes causing genetic diseases and parts thereof, various biological components having antigenicity, antibodies against pathogenic bacteria and viruses, and the like. Examples of samples containing these test substances include body fluids such as blood, serum, plasma, urine, stool, cerebrospinal fluid, saliva, various tissue fluids, various foods and drinks, and dilutions thereof. It is not limited to these. The nucleic acid to be the test substance may be a nucleic acid extracted from blood or cells by a conventional method, or may be a nucleic acid amplified by a nucleic acid amplification method such as PCR using the nucleic acid as a template. In the latter case, the measurement sensitivity can be greatly improved. When a nucleic acid amplification product is used as a test substance, the amplified nucleic acid can be labeled by performing amplification in the presence of a nucleoside triphosphate labeled with a fluorescent substance or the like. When the test substance is an antigen or an antibody, the test substance antigen or antibody may be directly labeled by an ordinary method, or the test substance antigen or antibody may be bound to the selective binding substance. After that, the selective binding substance-immobilized site on which the selective binding substance is immobilized is washed, and the antigen or antibody is reacted with a labeled antibody or antigen that undergoes antigen-antibody reaction, and hybridizes with the antigen or antibody as the test substance This makes it possible to measure the label bound to the selective binding substance-immobilized site.
[0056]
The step of allowing the test substance to interact with the immobilized selective binding substance can be performed in exactly the same manner as in the prior art. The reaction temperature and time are appropriately selected depending on the chain length of the nucleic acid to be hybridized, the type of antigen and / or antibody involved in the immune reaction, and the like. In the case of nucleic acid hybridization, usually 50 ° C to 70 ° C. C. for about one minute to several hours, and in the case of an immune reaction, it is usually about room temperature to about 40 ° C. for about one minute to several hours.
[0057]
By the above method, a test substance such as a nucleic acid, an antibody, or an antigen that selectively binds to the immobilized selective binding substance can be measured. That is, when a nucleic acid is immobilized as a selective binding substance, a nucleic acid having a sequence complementary to this nucleic acid or a part thereof can be measured. When an antibody or antigen is immobilized as a selective binding substance, an antigen or antibody immunoreacting with the antibody or antigen can be measured. The “measurement” referred to in the present specification includes both detection and quantification.
[0058]
By using the present invention, the expression of genes, proteins, and sugar chains in various organisms can be efficiently, rapidly, and simply examined. For example, after labeling nucleic acids extracted from normal human liver and hepatitis virus-infected liver, each of the selective binding substance-immobilized sites in which various known human genes are immobilized on the selective binding substance-immobilizing substrate of the present invention. Perform hybridization. By comparing the degree of binding of the normal liver nucleic acid and the hepatitis liver nucleic acid to the various known human genes, it is possible to examine changes in gene expression in the hepatitis liver.
[0059]
Similarly, by binding a labeled normal brain extract protein and an Alzheimer's brain extract protein to a fiber array to which various monoclonal antibodies as proteins are bound, and comparing the bound proteins to normal, abnormal expression of the protein in the Alzheimer's brain You can find out.
[0060]
【Example】
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.
[0061]
Example 1
In order to confirm that the immobilization and hybridization of nucleic acids can be reliably performed on the glass substrate using the hybridization apparatus shown in FIG. 1 used in Examples of the present invention, no cross-reaction was performed on the biological sample, An experiment was performed using digoxigenin as a label, which is thermally stable and does not decompose due to the heat applied during hybridization.
[0062]
An actin gene nucleic acid solution (manufactured by Takara Shuzo Co., Ltd.) (the nucleic acid concentration of 10 μg / ml) was placed on an amino-introduced slide glass substrate (76 mm × 26 mm × 1 mm) (manufactured by Matsunami Glass Industry Co., Ltd.) to form individual immobilized sites Spotting was performed so that the diameter was about 200 μm, dried in the air, and then subjected to ultraviolet treatment (using a UV crosslinker manufactured by Stratagene) to obtain a substrate on which the nucleic acid was immobilized. An oligonucleotide complementary to a part of the used nucleic acid sequence was synthesized and labeled with digoxigenin (DIG: Digoxigenin, Roche Diagnostics Co., Ltd.).
[0063]
The terminally aminated oligonucleotide was dissolved in a 100 mM borate buffer (pH 8.5) to a final concentration of 2 mM. An equal amount of digoxigenin-3-O-methylcarbonyl-α-aminocaproic acid-N-hydroxy-succinimide ester (26 mg / ml dimethylformamide solution) was added, and the mixture was allowed to stand at room temperature overnight. Ethanol precipitation was performed using glycogen (Roche Diagnostics Co., Ltd.) as a carrier, and the precipitate was air-dried and then dissolved in 100 μmol of 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. The DIG-labeled oligonucleotide thus obtained was used as a model of the sample nucleic acid.
[0064]
The prepared nucleic acid-immobilized substrate is placed on the base of the hybridization device shown in FIG. 1 so that the nucleic acid-immobilized portion faces upward, and a solution obtained by dissolving the DIG-labeled oligonucleotide in EDTA is placed on the nucleic acid-immobilized substrate. Dropping, sealing with a cover base material, hybridization without applying voltage to the electrode for vertical movement and the electrode for parallel movement, according to a standard method (performed according to Roche Diagnostics Co., Ltd., product manual) Was done.
[0065]
After the hybridization was completed, the nucleic acid-immobilized substrate was washed, and an anti-DIG enzyme-labeled antibody solution was added to allow an antigen-antibody reaction. After the reaction, the nucleic acid-immobilized substrate was washed to remove unbound antibodies. DIG detection reagent was added and equilibrated. When water was removed and a light signal was detected, a signal was detected in accordance with the immobilization of the nucleic acid.
[0066]
As a result, it was confirmed that the hybridization apparatus of the present invention had no structural or functional problems and could be reliably used as a hybridization apparatus by a conventional method in which no AC electric field was applied.
[0067]
Example 2
Pretreatment of selective binding substance fixed substrate
A slide glass (76 mm × 26 mm × 1 mm) (manufactured by Matsunami Glass Industry Co., Ltd.) was cleaned with a mixed solution of pure water, ethanol, and NaOH, and then washed with pure water. Further, in order to form a selective binding substance immobilization site on the slide glass, circular holes having a hole diameter of 200 μm are arranged in a grid pattern at intervals of 500 μm in an area of 5 mm × 5 mm, and each opening is an opening pattern having a width of 10 μm. And a stainless steel mask with the opening pattern converged on an opening for forming an electrode portion connected to an electrode provided in the hybridization apparatus is placed close to the slide glass, and is sputtered. A gold electrode corresponding to the shape of the opening of the mask was provided on a slide glass. The surface on which the electrode pattern is disposed is immersed in a mixed solution of pure water and poly-L-lysine (composition: 10% poly-L-lysine), and an amino group is added to the surface of the slide glass including the site for fixing the selective binding substance. Was introduced.
[0068]
Two types of nucleic acid solutions (“Takara Control & Primer Set-A” manufactured by Takara Shuzo Co., Ltd .; product number TX803 (λ DNA fragment of about 1000 bp), and “Human TFR (1 kb) Template & Primer Set” manufactured by Takara Shuzo Co., Ltd.) Each nucleic acid was amplified by PCR based on product number TX806 (human transferrin receptor DNA fragment of about 1000 bp). As the primers used in the PCR method, those included in each product were used. This was purified to obtain a purified nucleic acid solution. The two types of purified nucleic acid solutions were spotted on the site for immobilizing the selective binding substance on the slide glass, dried in the air, and subjected to UV crosslink (120 mJ) to immobilize the two types of nucleic acids on the site for immobilizing the nucleic acid. A nucleic acid-immobilized substrate was obtained. Next, in order to block excess amino groups on the surface of the slide glass that has not reacted with the nucleic acid, a solution of a mixture of boric acid, pure water, NaOH for pH adjustment, succinic anhydride, and 1-methyl-2-pyrrolidone (anhydrous 3 g of succinic acid was dissolved in 187 ml of 1-methyl-2-pyrrolidone, and the surface on which the nucleic acid was fixed was immersed in 17 ml of 1 M Na-borate (pH 8.0) solution immediately before use, and shaken. Then, it was washed.
[0069]
RNA processing
An RNA solution (“λpolyA + RNA-A” manufactured by Takara Shuzo Co., Ltd .; product number TX802) was prepared. It has a base sequence complementary to one of the above nucleic acids (TX803). This was treated with reverse transcriptase "Superscript II" (manufactured by GIBCO BRL; product number 18064-071), 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 1.0 mM dTTP, Cy5-dUTP (Amersham Pharmacia; Product No. PA55022), and incubated at 42 ° C. for 1 hour to perform reverse transcription to obtain a cDNA solution incorporating Cy5 dye.
[0070]
Similarly, an RNA solution ("Human TFR RNA (1 kb)" manufactured by Takara Shuzo Co., Ltd .; product number TX805) was prepared, and Cy5-dUTP was changed to Cy3-dUTP (manufactured by Amersham Pharmacia; product number PA53022). Reverse transcription was performed under the same conditions as above to obtain a cDNA solution in which the Cy3 dye was incorporated. The cDNA into which the Cy3 dye has been incorporated has a base sequence complementary to one of the above nucleic acids (TX806).
[0071]
The two types of cDNA solutions incorporating the above dye were mixed and purified, and further dissolved in a buffer (3.4 × SSC, 0.1% SDS) to obtain a hybridization solution.
[0072]
Hybridization
An electrode for parallel movement in which a nucleic acid-immobilized substrate having two kinds of nucleic acids fixed on its surface is fixed on a base of a hybridization device, and both sides of a region where the nucleic acid is fixed are connected to AC voltage applying means of the hybridization device. Was arranged. In this embodiment, a gold thin plate having a thickness of 200 μm was used as an electrode. The distance between the parallel movement electrodes was 1 cm. 20 μl of the hybridization solution was dropped on the site where the two types of nucleic acids were immobilized, and a cover base material equipped with a vertical movement upper electrode was placed on the parallel movement electrode on both sides of the nucleic acid immobilization site. Sealed to prevent evaporation.
[0073]
First, the voltage pattern shown in the DC and AC voltage application pattern 26 in the DC voltage and AC voltage application means ON section 28 in FIG. 5 was applied for 1 minute between the electrodes for vertical movement to increase the cDNA concentration near the nucleic acid fixing site. . The applied voltage was 1 V at the highest value, 0 V at the lowest value, and 1 Hz in frequency.
[0074]
Thereafter, a DC and AC mixed voltage was applied between the two electrodes for vertical movement and an AC voltage was applied between the electrodes for parallel movement according to the voltage application pattern shown in FIG. 5 in accordance with the gate signal (duty 50%) of 0.1 Hz. The voltage applied here is a DC / AC mixed pattern having a maximum value of 1 V, a minimum value of 0 V, and a frequency of 1 Hz between the vertical movement electrodes, so that the vertical movement lower electrode side has a positive potential. An AC voltage of 10 V and 10 Hz was applied between the parallel movement electrodes, and the system was allowed to stand at 65 ° C. for 10 minutes. Thereafter, the cover substrate and the parallel movement electrode were removed and washed.
[0075]
In order to confirm the effect of the voltage pattern applied between the electrodes for vertical movement, a DC pattern having a maximum value of 1 V and a minimum value of 0 V between the electrodes for vertical movement and a voltage of 10 V between the electrodes for parallel movement were used. After applying an alternating voltage of 10 Hz and allowing the mixture to stand at 65 ° C. for 10 minutes, the cover substrate and the electrode for parallel movement were removed, and a washed sample was prepared.
[0076]
Further, for comparison with the conventional method, a sample was left standing at 65 ° C. for 16 hours without applying a voltage between the electrodes for parallel movement and between the electrodes for vertical movement. Thereafter, the cover substrate and the electrode of the sample were removed and washed.
[0077]
Fluorescence detection
In order to measure the fluorescence from Cy5, the optical system was as follows. First, a laser (wavelength: 635 nm) was used as fluorescence excitation light. First, a bandpass filter (manufactured by Omega Optical; product number X1069) was arranged perpendicular to the optical axis to remove unnecessary light other than the excitation light. Further, a dichroic mirror (manufactured by Omega Optical; product number XF2035) is arranged at an angle of 45 degrees with respect to the optical axis of the laser beam, and the end face of the slide glass opposite to the end face immersed in the condensed beam in the DNA solution. Irradiation. Further, the fluorescence returned from the end surface immersed in the DNA solution is collected on the end surface side on the side to which the excitation light is irradiated, passed through the dichroic mirror (manufactured by OMEGA Optical; product number XF2035), and further passed through the band. Excess excitation light was cut through a pass filter (manufactured by Omega Optical; product number XF3076).
[0078]
Fluorescence from Cy3 is detected by the same method as above except that the dichroic mirror and the band pass filter are for Cy3 (manufactured by Omega Optical; product numbers XF1074, XF2017, and XF3083), and the wavelength of the laser to be irradiated is 532 nm. did.
[0079]
In this way, the fluorescence from the two kinds of nucleic acid-immobilized sites after hybridization was measured for each of Cy5 and Cy3. From the nucleic acid fixing site where the nucleic acid solution of TX803 was spotted, only the fluorescence of Cy5 was observed, and the fluorescence of Cy3 was not detected. From the nucleic acid fixing site where the nucleic acid solution of TX806 was spotted, fluorescence of only Cy3 was observed, and fluorescence from Cy5 was not detected.
[0080]
At this time, the fluorescence intensity obtained from the nucleic acid-immobilized substrate to which the voltage was applied was equivalent to that of the conventional method in which no voltage was applied. Further, when the fluorescence intensity distribution in each nucleic acid fixing site was observed in detail, the method of the present invention showed a more uniform intensity distribution than the conventional method. Thus, the conductive pattern is disposed on the slide glass, and the electric field for increasing the nucleic acid concentration near the nucleic acid fixing site and the voltage for relatively moving the probe nucleic acid and the target nucleic acid and increasing the collision probability are set. It was found that by applying the voltage, a sufficient binding reaction was performed even with a hybridization time of only 10 minutes, and a uniform binding reaction state with little deviation depending on the location was obtained.
[0081]
Example 3
Pretreatment of selective binding substance fixed substrate
A substrate prepared in Example 2 was provided on a slide glass by sputtering, on which a site for selectively binding a substance to be fixed, a conductive pattern for connecting them, and an electrode portion were provided with gold. Two openings and a lower electrode for vertical movement such that 10 mm sides of a 10 mm × 5 mm opening provided for forming a parallel movement electrode on the substrate face in parallel with a space of 10 mm therebetween. Then, a stainless steel mask having an opening pattern for forming conductive pattern wiring was mounted close to the slide glass, and a gold electrode corresponding to the opening shape of the mask was provided on the slide glass by sputtering.
[0082]
The slide glass (76 mm × 26 mm × 1 mm) (manufactured by Matsunami Glass Industry Co., Ltd.) on which the gold electrodes were arranged as described above was cleaned with a mixed solution of pure water, ethanol, and NaOH, and then washed with pure water. Further, the cleaned surface was immersed in a mixed solution of pure water and poly-L-lysine (composition: 10% poly-L-lysine) to introduce amino groups on the surface of the slide glass.
[0083]
Two types of nucleic acid solutions (“Takara Control & Primer Set-A” manufactured by Takara Shuzo Co., Ltd .; product number TX803 (λ DNA fragment of about 1000 bp), and “Human TFR (1 kb) Template & Primer Set” manufactured by Takara Shuzo Co., Ltd.) Each nucleic acid was amplified by PCR based on product number TX806 (human transferrin receptor DNA fragment of about 1000 bp). As the primers used in the PCR method, those included in each product were used. This was purified to obtain a purified nucleic acid solution. The two types of purified nucleic acid solutions were spotted on the surface of the slide glass between the gold electrodes into which the amino groups had been introduced, dried in air, and then subjected to UV cross-linking (120 mJ). To obtain a nucleic acid-immobilized substrate fixed on the substrate. Next, in order to block excess amino groups on the surface of the slide glass that has not reacted with the nucleic acid, a solution of a mixture of boric acid, pure water, NaOH for pH adjustment, succinic anhydride, and 1-methyl-2-pyrrolidone (anhydrous 3 g of succinic acid was dissolved in 187 ml of 1-methyl-2-pyrrolidone, and the surface on which the nucleic acid was fixed was immersed in 17 ml of 1 M Na-borate (pH 8.0) solution immediately before use, and shaken. Then, it was washed.
[0084]
RNA processing
An RNA solution ("PolyA + RNA-A" manufactured by Takara Shuzo Co., Ltd .; product number TX802) was prepared and subjected to the same treatment as the RNA treatment of Example 2 to obtain a cDNA solution containing Cy5 dye and a cDNA solution containing Cy3 dye. And a hybridization solution.
[0085]
Hybridization
A nucleic acid-immobilized substrate having two types of nucleic acids immobilized on the surface was immobilized on a base of a hybridization device, and gold electrodes arranged on both sides of the nucleic acid immobilization site were connected to an AC voltage applying means of the hybridization device. Further, DC voltage and AC voltage applying means were connected to the conductive pattern wiring connected to the lower electrode for vertical movement. 2 μl of the hybridization solution was dropped onto the site where the two types of nucleic acids were fixed, and a cover substrate was provided on the electrodes on both sides of the site where the nucleic acid was fixed, and the hybridization solution was sealed so as not to evaporate. Thereafter, a voltage was applied between the electrodes to carry out hybridization in the same manner as in Example 2, and then the cover substrate and the electrodes were removed and washed.
[0086]
Further, for comparison with the conventional method, a sample was prepared which was left at 65 ° C. for 16 hours without applying a voltage between the electrodes for parallel movement and between the electrodes for vertical movement. Thereafter, the cover substrate and the electrode of the sample were removed and washed.
[0087]
Fluorescence detection
In order to measure the fluorescence from Cy5 and Cy3, the optical system was the same as the optical system of Example 2, and the fluorescence from Cy5 and Cy3 was detected.
[0088]
In this way, the fluorescence from the two kinds of nucleic acid-immobilized sites after hybridization was measured for each of Cy5 and Cy3. From the nucleic acid fixing site where the nucleic acid solution of TX803 was spotted, only the fluorescence of Cy5 was observed, and the fluorescence of Cy3 was not detected. From the nucleic acid fixing site where the nucleic acid solution of TX806 was spotted, fluorescence of only Cy3 was observed, and fluorescence from Cy5 was not detected.
[0089]
At this time, the fluorescence intensity obtained from the nucleic acid-immobilized substrate to which the voltage was applied was equivalent to that of the conventional method in which no voltage was applied. Further, when the fluorescence intensity distribution in each nucleic acid fixing site was observed in detail, the method of the present invention showed a more uniform intensity distribution than the conventional method. Thus, the conductive pattern is disposed on the slide glass, and the electric field for increasing the nucleic acid concentration near the nucleic acid fixing site and the voltage for relatively moving the probe nucleic acid and the target nucleic acid and increasing the collision probability are set. It was found that by applying the voltage, a sufficient binding reaction was performed even with a hybridization time of only 10 minutes, and a uniform binding reaction state with little deviation depending on the location was obtained.
[0090]
Example 4
Pretreatment of selective binding material fixed substrate
A convex array substrate was prepared in which cylindrical convexes having a diameter of 200 μm and a height of 200 μm were arranged in a grid of 5 mm × 5 mm at intervals of 500 μm on a glass substrate of 76 mm × 26 mm × 1 mm by glass molding. The openings corresponding to the arrangement of the protrusions are connected to the openings by an opening pattern having a width of 10 μm, and the openings are formed in the openings for forming the electrode portions connected to the electrodes provided in the hybridization apparatus. The pattern-focused stainless steel mask is mounted on the convex array substrate in such a manner that the convex and the opening corresponding to the convex are fitted to each other, and the lower electrode for vertical movement is provided on the end of the convex by sputtering, and A gold electrode corresponding to the opening shape was provided. Furthermore, the two openings, the lower electrode for vertical movement, and the conductive pattern are arranged such that the sides of 10 mm of the opening of 10 mm × 5 mm provided for forming the electrodes for parallel movement face in parallel at a distance of 10 mm. A stainless steel mask on which an opening pattern is arranged to form wiring is mounted on the slide glass in close proximity, and a gold electrode corresponding to the opening shape of the mask is formed by sputtering on the convex portion on the convex array substrate. Were provided on both sides of the arrayed area.
[0091]
The slide glass (76 mm × 26 mm × 1 mm) (manufactured by Matsunami Glass Industry Co., Ltd.) on which the gold electrodes were arranged as described above was cleaned with a mixed solution of pure water, ethanol, and NaOH, and then washed with pure water. Further, the cleaned surface was immersed in a mixed solution of pure water and poly-L-lysine (composition: 10% poly-L-lysine) to introduce amino groups on the surface of the slide glass.
[0092]
Two types of nucleic acid solutions (“Takara Control & Primer Set-A” manufactured by Takara Shuzo Co., Ltd .; product number TX803 (λ DNA fragment of about 1000 bp), and “Human TFR (1 kb) Template & Primer Set” manufactured by Takara Shuzo Co., Ltd.) Each nucleic acid was amplified by PCR based on product number TX806 (human transferrin receptor DNA fragment of about 1000 bp). As the primers used in the PCR method, those included in each product were used. This was purified to obtain a purified nucleic acid solution. The two types of purified nucleic acid solutions were spotted on the surface of the slide glass between the gold electrodes into which the amino groups had been introduced, dried in air, and then subjected to UV cross-linking (120 mJ). To obtain a nucleic acid-immobilized substrate fixed on the substrate. Next, in order to block excess amino groups on the surface of the slide glass that has not reacted with the nucleic acid, a solution of a mixture of boric acid, pure water, NaOH for pH adjustment, succinic anhydride, and 1-methyl-2-pyrrolidone (anhydrous 3 g of succinic acid was dissolved in 187 ml of 1-methyl-2-pyrrolidone, and the surface on which the nucleic acid was fixed was immersed in 17 ml of 1 M Na-borate (pH 8.0) solution immediately before use, and shaken. Then, it was washed.
[0093]
RNA processing
An RNA solution ("PolyA + RNA-A" manufactured by Takara Shuzo Co., Ltd .; product number TX802) was prepared and subjected to the same treatment as the RNA treatment of Example 2 to obtain a cDNA solution containing Cy5 dye and a cDNA solution containing Cy3 dye. And a hybridization solution.
[0094]
Hybridization
A convex array substrate having two types of nucleic acids fixed on its surface was fixed on a base of a hybridization device, and gold electrodes disposed on both sides of the nucleic acid fixing site were connected to an AC voltage applying means of the hybridization device. Further, DC voltage and AC voltage applying means were connected to the conductive pattern wiring connected to the lower electrode for vertical movement. 2 μl of the hybridization solution was dropped onto the site where the two types of nucleic acids were fixed, and a cover substrate was provided on the electrodes on both sides of the site where the nucleic acid was fixed, and the hybridization solution was sealed so as not to evaporate. Thereafter, a voltage was applied between the electrodes to carry out hybridization in the same manner as in Example 2, and then the cover substrate and the electrodes were removed and washed.
[0095]
Further, for comparison with the conventional method, a sample was prepared which was left at 65 ° C. for 16 hours without applying a voltage between the electrodes for parallel movement and between the electrodes for vertical movement. After leaving this at 65 ° C. for 16 hours, the cover substrate was removed and washed.
[0096]
Fluorescence detection
In order to measure the fluorescence from Cy5 and Cy3, the optical system was the same as the optical system of Example 2, and the fluorescence from Cy5 and Cy3 was detected.
[0097]
In this way, the fluorescence from the two kinds of nucleic acid-immobilized sites after hybridization was measured for each of Cy5 and Cy3. From the nucleic acid fixing site where the nucleic acid solution of TX803 was spotted, only the fluorescence of Cy5 was observed, and the fluorescence of Cy3 was not detected. From the nucleic acid fixing site where the nucleic acid solution of TX806 was spotted, fluorescence of only Cy3 was observed, and fluorescence from Cy5 was not detected.
[0098]
At this time, the fluorescence intensity obtained from the nucleic acid-immobilized substrate to which the voltage was applied was equivalent to that of the conventional method in which no voltage was applied. Further, when the fluorescence intensity distribution in each nucleic acid fixing site was observed in detail, the method of the present invention showed a more uniform intensity distribution than the conventional method. Thus, the conductive pattern is disposed on the slide glass, and the electric field for increasing the nucleic acid concentration near the nucleic acid fixing site and the voltage for relatively moving the probe nucleic acid and the target nucleic acid and increasing the collision probability are set. It was found that by applying the voltage, a sufficient binding reaction was performed even with a hybridization time of only 10 minutes, and a uniform binding reaction state with little deviation depending on the location was obtained.
[0099]
Example 5
A glass substrate was prepared by arranging through holes having a diameter of about 200 μm in a 5 mm × 5 mm region at intervals of 500 μm in a glass substrate of 76 mm × 26 mm × 1 mm by glass processing. An optical fiber having a diameter of 200 μm was passed through the through hole of the glass substrate, the optical fiber was fixed at a position where the optical fiber protruded from the surface of the glass substrate to a height of 200 μm, and cut at an end surface opposite to the protruding surface. As a result, an optical fiber penetrating substrate having an appearance similar to that of the convex array substrate used in Example 4 was obtained. Using the stainless steel mask used in Example 4, the lower electrode for vertical movement on the end face of the optical fiber, the electrodes for parallel movement on both sides of the region where the optical fibers are arranged, and the optical fiber arrangement were formed by sputtering. A conductive pattern to be connected was provided.
[0100]
The optical fiber penetrating type substrate on which the gold electrodes were arranged as described above was cleaned with a mixed solution of pure water, ethanol and NaOH, and then washed with pure water. Further, the cleaned surface was immersed in a mixed solution of pure water and poly-L-lysine (composition: 10% poly-L-lysine) to introduce amino groups on the surface of the optical fiber penetrating substrate.
[0101]
Two types of nucleic acid solutions ("Control Template & Primer Set-A" manufactured by Takara Shuzo Co., Ltd .; product number TX803 (λ DNA fragment of about 1000 bp) and "Human TFR (1 kb) Template & Primer Set" manufactured by Takara Shuzo Co., Ltd. Each nucleic acid was amplified by PCR based on product number TX806 (human transferrin receptor DNA fragment of about 1000 bp). As the primers used in the PCR method, those included in each product were used. This was purified to obtain a purified nucleic acid solution. Two types of purified nucleic acid solutions were spotted on the protruding optical fiber end surfaces of the optical fiber penetrating type substrate, dried in air, and then subjected to UV crosslink (120 mJ) to perform nucleic acid fixation of the two types of nucleic acids at the nucleic acid fixing site. A fixed substrate was obtained. Next, in order to block the extra amino group on the end face of the optical fiber that has not reacted with the nucleic acid, a solution (anhydrous succinic anhydride) in which boric acid, pure water, NaOH for pH adjustment, succinic anhydride, and 1-methyl-2-pyrrolidone are mixed. 3 g of the acid was dissolved in 187 ml of 1-methyl-2-pyrrolidone, and the surface on which the nucleic acid was immobilized was immersed in 17 ml of 1 M Na-borate (pH 8.0) solution immediately before use, and shaken. Then, it was washed.
[0102]
RNA processing
An RNA solution ("PolyA + RNA-A" manufactured by Takara Shuzo Co., Ltd .; product number TX802) was prepared and subjected to the same treatment as the RNA treatment of Example 2 to obtain a cDNA solution containing Cy5 dye and a cDNA solution containing Cy3 dye. And a hybridization solution.
[0103]
Hybridization
An optical fiber penetrating substrate having two types of nucleic acids fixed on the surface was fixed on a base of a hybridization device, and gold electrodes arranged on both sides of the nucleic acid fixing site were connected to an AC voltage applying means of the hybridization device. Further, DC voltage and AC voltage applying means were connected to the conductive pattern wiring connected to the lower electrode for vertical movement. 2 μl of the hybridization solution was dropped onto the site where the two types of nucleic acids were fixed, and a cover substrate was provided on the electrodes on both sides of the site where the nucleic acid was fixed, and the hybridization solution was sealed so as not to evaporate. Thereafter, a voltage was applied between the electrodes to carry out hybridization in the same manner as in Example 2, and then the cover substrate and the electrodes were removed and washed.
[0104]
Further, for comparison with the conventional method, a sample was prepared which was left at 65 ° C. for 16 hours without applying a voltage between the electrodes for parallel movement and between the electrodes for vertical movement. Thereafter, the cover substrate and the electrode of the sample were removed and washed.
[0105]
Fluorescence detection
In order to measure the fluorescence from Cy5 and Cy3, the optical system was the same as the optical system of Example 2, and the fluorescence from Cy5 and Cy3 was detected.
[0106]
In this way, the fluorescence from the two kinds of nucleic acid-immobilized sites after hybridization was measured for each of Cy5 and Cy3. From the nucleic acid fixing site where the nucleic acid solution of TX803 was spotted, only the fluorescence of Cy5 was observed, and the fluorescence of Cy3 was not detected. From the nucleic acid fixing site where the nucleic acid solution of TX806 was spotted, fluorescence of only Cy3 was observed, and fluorescence from Cy5 was not detected.
[0107]
At this time, the fluorescence intensity obtained from the nucleic acid-immobilized substrate to which the voltage was applied was equivalent to that of the conventional method in which no voltage was applied. Further, when the fluorescence intensity distribution in each nucleic acid fixing site was observed in detail, the method of the present invention showed a more uniform intensity distribution than the conventional method. Thus, the conductive pattern is disposed on the slide glass, and the electric field for increasing the nucleic acid concentration near the nucleic acid fixing site and the voltage for relatively moving the probe nucleic acid and the target nucleic acid and increasing the collision probability are set. It was found that by applying the voltage, a sufficient binding reaction was performed even with a hybridization time of only 10 minutes, and a uniform binding reaction state with little deviation depending on the location was obtained.
[0108]
【The invention's effect】
By using the selective binding substance hybridization method, hybridization apparatus and selective binding substance-immobilizing substrate of the present invention, hybridization efficiency is improved, and the bias of the reaction at the binding reaction site is reduced, and To complete the hybridization step, and obtain a uniform hybridization result.
[Brief description of the drawings]
FIG. 1 is a cross-sectional view (a) and a plan view (b) of a hybridization device of the present invention.
FIG. 2 is a principle diagram showing the operation of a selective binding substance in the present invention.
FIG. 3 is a cross-sectional view of a conventional hybridization device.
FIG. 4 is a principle view showing the operation of a selective binding substance in a conventional hybridization device.
FIG. 5 shows a voltage application pattern to each electrode in the present invention.
[Explanation of symbols]
1 Substrate for fixing selective binding substances
2 Parallel movement electrode
3 Parallel movement electrode
4 Lower electrode for vertical movement
5 Selective binding substance fixing site
6 Cover base material
7 Upper electrode for vertical movement
8 Selective binding substance sequence region
9 AC voltage application means
10 Conductive pattern
11 Connector
12 DC voltage and AC voltage applying means
13 Test sample solution
14 base
15, 15 'selective binding substance
16, 16 'corresponding selective binding substance
17 Substrate for fixing selective binding substance
18 Supporting materials
19 Lower electrode for vertical movement
20 Selective binding substance fixing site
21 Cover base material
22 Upper electrode for vertical movement
23 Test sample solution
24 Voltage application means
25 Gate signal
26 DC and AC voltage application patterns
27 AC voltage application pattern
28 DC voltage and AC voltage application means ON section
29 AC voltage application means ON section

Claims (16)

Immobilizing a selective binding substance on a substrate, bringing a test sample solution containing a corresponding selective binding substance that selectively binds to the selective binding substance into contact with the immobilized selective binding substance, In the step of causing a substance to react with the corresponding selective binding substance, in a direction intersecting an axis perpendicular to the plane of the immobilization site on which the selective binding substance is immobilized, and at both ends of the selective binding substance immobilization section An AC voltage is applied between two opposing parallel displacement electrodes disposed on the outer side, and a vertical displacement lower electrode disposed immediately below a portion of the substrate on which the selective binding substance is immobilized; A method for hybridization of a selective binding substance, wherein a voltage is applied between a vertical movement upper electrode disposed at a position facing the selective binding substance-immobilized surface via a test sample solution to cause the binding reaction. 2. The method according to claim 1, wherein there is a selective binding substance array region in which a plurality of the selective binding substance immobilization sites are arranged, and the electrode for translation is arranged outside both ends of the selective binding substance array region. 3. A method for hybridization of a selective binding substance. 2. The selective binding substance according to claim 1, wherein a period in which an AC voltage is applied between the parallel movement electrodes and a period in which a voltage is applied between the vertical movement lower electrode and the vertical movement upper electrode alternately exist. 3. Hybridization method. 4. The selective binding substance according to claim 3, wherein a DC voltage applied between the lower electrode for vertical movement and the upper electrode for vertical movement has a positive potential on the side of the lower electrode for vertical movement with respect to the upper electrode for vertical movement. Hybridization method. A voltage applied between the lower electrode for vertical movement and the upper electrode for vertical movement has a positive potential on the lower electrode side for vertical movement with respect to the upper electrode for vertical movement, and a voltage application in which a DC component and an AC component are mixed. The method for hybridizing a selective binding substance according to claim 3, which has a pattern. The method for hybridizing a selective binding substance according to any one of claims 1 to 5, wherein the selective binding substance is at least one selected from nucleic acids, proteins, saccharides, antibodies, and antigenic compounds. The method for hybridizing a selective binding substance according to claim 6, wherein the selective binding substance and the corresponding selective binding substance are single-stranded nucleic acids, and the binding reaction is hybridization between nucleic acids. Immobilizing a selective binding substance on a substrate, bringing a test sample solution containing a corresponding selective binding substance that selectively binds to the selective binding substance into contact with the immobilized selective binding substance, An apparatus for causing a binding reaction between the binding substance and the corresponding selective binding substance, wherein the base on which the base material is installed and a direction intersecting a vertical axis of a surface on which the selective binding substance is immobilized, and Two opposing parallel-moving electrodes disposed outside both ends of the selective-bonding substance-immobilized region, AC voltage applying means for applying an AC voltage between the parallel-moving electrodes, and the selective bonding on the base material A vertical movement lower electrode disposed immediately below the site where the reactive substance is immobilized, and a vertical movement upper electrode disposed at a position facing the selective binding material immobilized surface via the test sample solution, The vertical moving upper electrode and the vertical moving Hybridization apparatus selective binding substance having a DC-AC application means for applying a DC voltage and / or AC voltage between the part electrodes. The material of the electrode for parallel movement and the upper electrode for vertical movement are each selected from platinum, titanium, gold, silver, aluminum, copper, palladium alone or an alloy thereof, carbon or a carbon compound, or a conductive polymer. The hybridization device for a selective binding substance according to claim 8, which is at least one kind. Immobilizing a selective binding substance on a substrate, bringing a test sample solution containing a corresponding selective binding substance that selectively binds to the selective binding substance into contact with the immobilized selective binding substance, A substrate for causing a binding reaction between the binding substance and the corresponding selective binding substance, wherein the selective binding substance on the substrate is immobilized. Two opposing parallel displacement electrodes disposed outside both ends of the region, a vertical displacement lower electrode disposed immediately below the selective binding substance fixing portion, and a conductive pattern connected to the vertical displacement lower electrode A substrate for fixing a selective binding substance having a wiring. 11. The selective binding substance immobilization site is provided in a plurality, a selective binding substance arrangement region in which they are arranged is present, and the electrodes are arranged outside both ends of the selective binding substance arrangement region. 2. The substrate for fixing a selective binding substance according to item 1. The substrate for fixing a selective binding substance according to claim 10 or 11, wherein the site for fixing a selective binding substance is a convex portion or a concave portion provided on a substrate. The base material for fixing a selective binding substance according to claim 12, wherein the site for fixing a selective binding substance is a fiber or a fiber bundle inserted into a hole provided in the base material. The substrate for immobilizing a selective binding substance according to any one of claims 10 to 13, wherein the selective binding substance is at least one selected from nucleic acids, proteins, saccharides, antibodies, and antigenic compounds. The substrate for immobilizing a selective binding substance according to claim 14, wherein the selective binding substance and the corresponding selective binding substance are single-stranded nucleic acids, and the binding reaction is hybridization between nucleic acids. The material of the parallel movement electrode and / or the vertical movement lower electrode is made of platinum, titanium, gold, silver, aluminum, copper, palladium alone or an alloy thereof, carbon or a carbon compound, or a conductive polymer. The substrate for fixing a selectively binding substance according to any one of claims 10 to 15, which is at least one member selected from the group consisting of:

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