JP2006329763A - Detection part of interaction between substances, sensor chip using it and bioassay method using electric field - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently and effectively apply an electric field to a reaction region by a simple configuration and to especially concentrate the electric field to the specific region of the electrode fronted to the reaction region. <P>SOLUTION: In the detection part having the reaction region R becoming the field of the interaction between substances, electrode layers 12 and 13 used when the electric field E is applied to the reaction field R and the insulating layers 14 for covering the surfaces of electrodes. Specific insulating parts 141 high in dielectric constant are provided to the insulating layers 14 to concentrate the electric field to the specific insulating parts 141. For example, the specific insulating parts 141 are set to a region formed of a material of which the dielectric constant is higher than that of a peripheral insulating layer 142. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a technique useful when detecting an interaction between substances. More specifically, the present invention relates to a technique useful for achieving immobilization of a probe substance and promotion of interaction by effectively using an electrodynamic action.

  In recent years, an integrated substrate for bioassay called a so-called DNA chip or DNA microarray (hereinafter collectively referred to as “DNA chip” in the present application), in which predetermined DNA is finely arranged by microarray technology, is used for gene mutation analysis, Single nucleotide polymorphism) analysis, gene expression frequency analysis, etc., have begun to be widely used in fields such as drug discovery, clinical diagnosis, pharmacogenomics, evolutionary research, forensic medicine and others.

  Since this “DNA chip” has a large number of nucleotide chains such as DNA oligo strands and cDNA (complementary DNA) accumulated on a glass substrate or silicon substrate, comprehensive analysis of hybridization is possible. Is characterized. In addition, various sensor chips (for example, protein chips) and detection devices that detect interactions between biomolecules other than nucleic acid molecules have been developed.

  Here, in recent years, a technique using an electrodynamic effect such as electrophoresis or dielectrophoresis has begun to be proposed in an assay system that detects and detects an interaction between substances in a predetermined reaction region. Hereinafter, this technique will be exemplified.

  First, in Patent Document 1, a nucleic acid probe template strand fixed on a template substrate is used, a nucleic acid probe strand is synthesized along the template strand, and the synthesized probe is separated into another array using an electric field. A technique for producing a nucleic acid chain-immobilized array simply and at low cost by fixing on a substrate is disclosed.

  Patent Document 2 is composed of a body part and a frame that can be attached to and detached from each other. Inside the body part, a large number of pin electrodes protrude in a matrix, and oligonucleotides having different gene sequences are fixed to the pin electrodes. In addition, a common electrode is disposed in the recess of the frame so as not to contact the pin electrode, a voltage is applied between the common electrode and the pin electrode, current is detected, and the two obtained by hybridization of the oligonucleotide Techniques for detecting strand DNA have been disclosed.

In Patent Document 3, a reaction region serving as a hybridization field between a detection nucleotide chain and a target nucleotide chain having a base sequence complementary to the detection nucleotide chain is formed by applying an electric field to the detection nucleotide chain. A hybridization detection unit is disclosed that is configured to be fixed to the end of a scan electrode by the action of dielectrophoresis while being elongated.
JP 2001-330608 A. Japanese Patent Application Laid-Open No. 2001-242135. Japanese Patent Application Laid-Open No. 2004-135512.

  In the above-mentioned prior art, all use an electrode or a group of electrodes arranged opposite to the reaction region. However, in such a technology, an electrochemical solution using an ionic solution that may be stored in the reaction region is used. The electrode surface facing the reaction region is covered with an insulating layer (insulating film) because it is necessary to prevent unwanted reactions, and it is also necessary to prevent corrosion of the electrode by preventing electron transfer between the electrode and the solvent. It is desirable. Furthermore, the insulating layer can also prevent peeling of the electrode that may occur due to generation of bubbles due to electrolysis.

  However, if an insulating layer is provided on the electrode, the electric field strength that can actually be applied to the reaction region is reduced due to the electric storage function (capacitor function) of the insulating layer. It needs to be high. For this reason, there has been a technical problem that the energy efficiency is deteriorated and a device-like device for applying a high voltage is required. Also, in the reaction region, for example, assuming the case of using an electrodynamic effect called dielectrophoresis that uses the driving force of a non-uniform electric field with respect to a dipole of a substance, an electrode shape is formed in order to form a non-uniform electric field in the vicinity of the electrode. A method of forming an edge portion where the electric field tends to concentrate is effective, but such fine processing of the electrode is complicated.

  Therefore, the present invention provides an interaction detector that can efficiently and effectively apply an electric field to the reaction region with a simple configuration, and in particular, concentrates the electric field effectively on a specific portion of the electrode facing the reaction region. The main object is to provide an interaction detection unit capable of

  The present invention first comprises a reaction region serving as an interaction field between substances, an electrode used for applying an electric field to the reaction region, and an insulating layer covering the surface of the electrode. Provided are an interaction detector provided with a specific insulating part having a high dielectric constant (relative dielectric constant), and a sensor chip provided with the interaction detector. In the present invention, the “specific insulating portion” means a predetermined portion of an insulating layer formed for the purpose of increasing the electric field strength or concentrating the electric field as compared with the surroundings.

  Here, the specific insulating portion is preferably a portion formed of a material having a dielectric constant higher than that of the surrounding insulating layer or a portion formed of a thinner film than the surrounding insulating layer. In this specific insulating portion, the electric field strength can be increased as compared with the surrounding insulating layer region, or the non-uniform electric field can be locally formed by concentrating the electric field.

  Therefore, in the present invention, by using a detection unit including a reaction region serving as an interaction field between substances, an electrode used for applying an electric field to the reaction region, and an insulating layer covering the surface of the electrode, Provided is a bioassay method for concentrating an electric field on a specific insulating portion having a high dielectric constant provided in an insulating layer to promote the immobilization of a probe substance or the interaction.

  According to this method, the specific insulating portion where the electric field is likely to concentrate is electrodynamically attracted by the migration of the probe material that forms charges and dipoles from the periphery, or the target material is present where the probe material exists. It is possible to increase the concentration by attracting and pulling. Alternatively, the higher-order structure and orientation (transformation) of the substance can be adjusted using electrodynamic action.

  Here, main technical terms related to the present invention will be described.

  “Interaction” broadly means non-covalent bonds, covalent bonds, chemical bonds or dissociations including hydrogen bonds, such as hybridization between nucleic acid molecules, interaction between proteins, antigen-antibody reaction, etc. Widely includes chemical bonds or dissociation between these substances. “Hybridization” means a complementary strand (double strand) formation reaction between complementary base sequence structures.

  “Nucleic acid chain” means a polymer (nucleotide chain) of a phosphate ester of a nucleoside in which a purine or pyrimidine base and a sugar are glycosidically linked. The oligonucleotide, polynucleotide, purine nucleotide and pyrimidine nucleotide containing probe DNA Polymerized DNA (full length or a fragment thereof), cDNA obtained by reverse transcription (c probe DNA), RNA, polyamide nucleotide derivative (PNA) and the like are widely included.

  The “reaction region” is a region that can provide an interaction field such as hybridization, and, for example, a reaction field having a well shape that can store a liquid phase, a gel, or the like.

  “Dielectrophoresis” is a phenomenon in which molecules are driven toward a stronger electric field in a field where the electric field is not uniform. When an AC voltage is applied, the polarity of the polarization is also reversed as the polarity of the applied voltage is reversed, so that the driving effect can be obtained in the same way as in the case of direct current (supervised by Teru Hayashi, “Micromachine and Material Technology (issued by CMC) ) ", P37-P46, Chapter 5, Cell and DNA manipulation). In particular, in a high-frequency alternating electric field, force acts on the dipole in proportion to the gradient of the square of the temporal average electric field, causing migration.

  For example, nucleic acid molecules are known to stretch or move when subjected to the action of an electric field in the liquid phase. The principle is thought to be that ion turbidity is formed by phosphate ions (negative charge) forming the skeleton and hydrogen atoms (positive charge) formed by ionization of water around them, and are generated by these negative charges and positive charges. The polarization vector (dipole) is oriented in one direction as a whole by the application of a high frequency high voltage, and as a result, expands. In addition, when a non-uniform electric field is applied, it moves toward the part where the electric lines of force are concentrated. (Seiichi Suzuki, Takeshi Yamanashi, Shin-ichi Tazawa, Osamu Kurosawa and Masao Washizu: “Quantitative analysis on electrostatic orientation of DNA in stationary AC electric field using fluorescence anisotropy”, IEEE Transaction on Industrial Applications, Vol. 34, No. 1 , P75-83 (1998)).

  “Sensor chip” means a substrate for detecting the interaction by causing an interaction between substances in a predetermined reaction region on the substrate, and widely includes regardless of the type of the substance. The detection principle of action does not matter. This sensor chip includes a DNA chip (DNA microarray) in which nucleic acid chains such as DNA probes are immobilized and in a finely arranged state, a protein chip suitable for detecting interactions between proteins, antigen-antibody reactions, and the like. Including at least.

  In the present invention, in the insulating layer in the reaction region, a portion or region in which the electric field (lines of electric force) is particularly concentrated can be formed in the reaction region by a simple substrate configuration in which a specific insulating layer having a higher dielectric constant than the surroundings is formed. Therefore, it is possible to easily obtain an electrodynamic effect such as formation of a non-uniform electric field and dielectrophoresis using the non-uniform electric field.

  By this effect, the probe material that forms electric charges and dipoles can be attracted and fixed toward the specific insulating part where the electric field is particularly concentrated, and further, the target material is located where the probe material exists. To enhance interaction efficiency such as hybridization. Alternatively, the higher-order structure and orientation of the substance can be adjusted using electrodynamic action to reduce the influence of steric hindrance during interaction such as hybridization. As a result, the accuracy of interaction such as hybridization can be improved.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the accompanying drawings. Each embodiment shown in the attached drawings shows an example of a typical embodiment of the thing and method concerning the present invention, and, thereby, the scope of the present invention is not interpreted narrowly.

  FIG. 1 is a cross-sectional view illustrating a basic configuration of a first embodiment suitable as an interaction detection unit (hereinafter, abbreviated as “detection unit”) according to the present invention.

  First, reference numeral 1a shown in FIG. 1 represents a first preferred embodiment of the detection unit according to the present invention. The detection unit 1a includes a support substrate 11, a first electrode layer 12 made of a conductive material stacked on the support substrate 11, a second electrode layer 13 made of a conductive material disposed opposite to the first electrode layer 12, In a place (region) sandwiched between the first electrode layer 12 and the second electrode layer 13, a well-shaped (recessed shape) reaction region R capable of storing or holding a medium such as a solution or gel is provided. It has been.

  This reaction region 2 functions as a region or space that provides a field for interaction between various substances such as hybridization, protein-protein interaction, and antigen-antibody reaction. An electric field is applied to the reaction region 2 via the electrode layers 12-13 that are disposed so as to face the reaction region 2. 1 is a power source (voltage generation source), and G is a switch for operating on / off of electric field application (the same applies to other drawings).

  Here, the first electrode layer 12 is formed of a metal such as aluminum or gold, or a transparent conductor such as ITO (indium-tin-oxide). In the example of this embodiment, the first electrode layer 12 is made of glass or light-transmitting synthetic material. It is laminated on a support substrate 11 made of a resin material.

  If the first electrode layer 12 is made of light-transmitting ITO or the like, the fluorescence excitation light P having a predetermined wavelength traveling from below the support substrate 11 is guided into the reaction region R and excited in the reaction region R. The fluorescence F generated in this way can be led out to the lower side of the support substrate 11 (see FIG. 1).

  The fluorescence excitation light P referred to here excites a fluorescent substance labeled with a substance existing in the reaction region R or a fluorescent intercalator that is inserted and bonded to a complementary strand generated by hybridization (an example of interaction). The light. By further forming a reflective layer (not shown) on the second electrode layer 13, the fluorescence F can be reflected and led out downward, so that the amount of fluorescence that can be measured can be increased.

The electrode surface 12a facing the reaction region R of the first electrode layer 12 is covered with an insulating layer 14 formed of a material selected from SiO ₂ , SiC, SiN, SiOC, SiOF, TiO _{2 and the} like. The insulating layer 14 plays a role of preventing an electrochemical reaction by an ionic solution that may be stored in the reaction region R, a role of preventing corrosion and peeling of the electrode, and the like. Although not particularly illustrated, it is desirable to provide an insulating layer on the electrode surface facing the reaction region R of the second electrode layer 13.

  The insulating layer 14 is provided with an insulating portion (referred to as a “specific insulating portion”) 141 having a dielectric constant higher than that of the surrounding area and an insulating portion 142 around the insulating portion 141 at a predetermined interval. The number, density, shape, and arrangement pattern of the specific insulating portions 141 are not particularly limited, and a plurality of specific insulating portions 141 may be formed in a single reaction region R or may be formed independently depending on the purpose. Good (the same applies to other embodiments).

  For example, as shown in FIG. 2, a specific insulating portion 141a having a circular shape when viewed from above is arranged in a predetermined interval on the insulating layer 14, and a specific shape having a rectangular shape when viewed from above is shown in the insulating layer 14 as shown in FIG. It is also free to adopt a configuration in which the insulating portions 141b are arranged at predetermined intervals according to the purpose.

The material of the specific insulating part 141 (141a, 141b) is formed of a material having a high dielectric constant such as titanium dioxide (TiO ₂ ) having a dielectric constant (ε) of 40, and the other insulating layer 142 part is For example, it can be formed of silicon dioxide (SiO ₂ ) having a dielectric constant (ε) of 4.

  Here, referring to FIG. 1 again, FIG. 1 schematically shows a state in which the electric field indicated by the symbol E is concentrated on the specific insulating layers 141 and 141. In addition, the specific insulating layers 141 and 141 show a state in which the probe substance X such as oligo DNA is aligned and fixed.

  Immediately after being introduced into the reaction region R, these probe substances X are scattered and scattered in the reaction region R due to Brownian motion or the like, but when an electric field E such as a high-frequency AC electric field is applied, the specific insulating layer 141 , 141 are migrated and gathered together, and the end portions thereof are chemically bonded and fixed to the surfaces of the specific insulating layers 141, 141.

  The probe substance X can be fixed, for example, by a reaction such as a coupling reaction between the surface of the electrode layer 12 covered with the specific insulating layers 141 and 141 and the end of the probe substance. For example, when the insulating layer is surface-treated with streptavidin, it is suitable for fixing the end of the biotinylated probe substance X. Alternatively, when the surface is treated with a thiol (SH) group, it is suitable for immobilizing a probe substance having a thiol group modified at the terminal with a disulfide bond (-SS-bond).

  FIG. 4 is a cross-sectional view illustrating the basic configuration of the second embodiment of the interaction detection unit according to the present invention.

  In the second embodiment shown in FIG. 4 and the first embodiment shown in FIG. 1, only the configuration of the insulating layer 14 is different, and the insulation of the detector 1b according to the second embodiment is different. The layer 14 is characterized in that a specific insulating portion 143 having a thickness smaller than that of the surrounding insulating layer 144 is provided.

  Since the specific insulating portions 143 and 143 having such a small thickness have a higher dielectric constant than that of the surrounding insulating layer 144, the electric field E is concentrated similarly to the specific insulating portion 141 of the detecting portion 1a and is not present near the surface. A uniform electric field is easily formed. For this reason, it is suitable as a fixed location of the probe substance X.

  When the electric field E is applied after or simultaneously with the introduction of the target substance Y such as a nucleic acid chain into the reaction region R, the specific insulating parts 143 and 143 in which the probe substance X is already aligned and fixed Since the target material Y migrates toward the surface and the concentration of the target material Y near the specific insulating portions 143 and 143 increases, the interaction efficiency increases as a result (the same applies to the specific insulating portion 141 of the detecting portion 1a). . Alternatively, the higher order structures of the probe substance X and the target substance Y are adjusted by an electric field, and steric hindrance during interaction is reduced. For example, the nucleic acid strands entangled in a random coil shape are extended by the action of the electric field E, and the bases that are complementarily bonded are exposed to promote hybridization (the same applies to the detection unit 1a).

  Next, FIG. 5 is a diagram showing a third embodiment of the detection unit according to the present invention.

The detection part 1c which is 3rd embodiment shown by FIG. 5 has a total of two pairs of counter electrodes. Specifically, the upper and lower counter electrodes 12-13, a counter electrode 15-16 distributed to the leftward right reaction regions R, includes a power supply _{G 1} as the electric field applying means to the counter electrode 12-13 Metropolitan A power source G ₂ and a switch S ₂ are provided as means for applying an electric field to the switch S ₁ and the counter electrode 15-16.

  Part of the insulating layer 14 covering the electrode layer 12 is provided with a specific insulating layer 141 (or 143), which functions as a fixed portion of the probe substance X or a place for promoting interaction (see FIG. 5). . Specific insulating layers 171 and 181 having a high dielectric constant are formed in part of the insulating layers 17 and 18 that respectively cover the electrode layers 15 and 16.

  Here, the counter electrode 12-13 fixes the probe material X to the electrode part covered with the specific insulating layer 141 (or 143), or causes the target material Y to migrate toward the fixed probe material X. Used for.

  One counter electrode 15-16 is present in the reaction region R and can be a harmful substance when detecting the interaction, for example, a target material released in the reaction region R without interaction. It can be used when removing the probe substance X from the periphery of the fixed portion.

  The insulating layers 17 and 18 that respectively cover the electrode layers 15 and 16 are formed with the specific insulating layers 171 and 181 having a high dielectric constant, and an electric field is concentrated on the specific insulating layers 171 and 181 so as to be nonuniform. An electric field is formed, and a substance can be moved using an electrodynamic action such as dielectrophoresis.

Here, a detection unit having a substrate configuration similar to that of the detection unit 1a as shown in FIG. 1 and in which the electrode layers 12-13 are arranged with an interval of 5 μm in the vertical direction (vertical direction) of the reaction region R Assuming that an AC voltage of 1 MHz and 20 Vpp is applied, the dielectric constant (ε) of the insulating layer 14 is 1.0, 4.0 (for example, an insulating layer of SiO ₂ ), 10.0, 40.0 ( For example, FIG. 6 shows the result of simulation calculation of the upper field strength of each of the TiO ₂ insulating layers. As shown in FIG. 6, it can be seen that the higher the dielectric constant of the insulating layer, the higher the electric field strength at the upper portion of the insulating layer.

For example, an insulating layer (specific insulating portion) formed of TiO ₂ having a dielectric constant ε = 40 is slightly less than twice that of a general insulating layer (specific insulating portion) formed of SiO ₂ having a dielectric constant ε = 4.0. The electric field strength of is obtained. During dielectrophoresis, a driving force is generated in the target substance in proportion to the gradient of the square of the electric field. Therefore, when an electric field is applied, substances such as nucleic acid chains are concentrated in the vicinity of the insulating layer (specific insulating part) formed of TiO _2. Will move.

Next, FIG. 7 is a diagram (drawing substitute graph) showing the results of calculating the electric field strength when the electrodes are covered with insulating layers formed of SiO ₂ having different film thicknesses. The film thickness W of the insulating layer 14 was assumed to be two types, 250 nm and 20 nm. Further, in this calculation simulation, as shown in FIG. 8, the insulating layer is formed on the central axis C (D = 0) of the specific insulating portion 141 and at a position (D = 5) 5 μm apart from the central axis in the horizontal direction. The electric field strength was calculated in the height (Z) direction from the surface.

  As a result of this calculation, it is clear that the insulating layer having a thin film thickness of 20 nm has a larger electric field strength. Further, such an effect is not much depending on the height from the surface of the insulating layer and the distance in the horizontal direction. I found no difference. Thus, it was verified that an electrode surface having a larger electric field strength than the surroundings can be formed by reducing the thickness of the insulating layer. It was also verified that a constant electric field strength can be obtained in the vicinity of the thin insulating layer (specific different insulating portion) regardless of the horizontal and vertical directions.

  INDUSTRIAL APPLICABILITY The present invention can be used as a technique for detecting an interaction between substances efficiently and in a short time with high accuracy by utilizing an electrodynamic action. For example, it can be used as a sensor chip technique represented by a DNA chip or a protein chip, or a technique for detecting the interaction.

It is sectional drawing for demonstrating the basic composition of suitable 1st embodiment of the interaction detection part which concerns on this invention. It is a top view which shows an example of the arrangement configuration of the specific insulation part (141a) in an insulating layer (14). It is a top view which shows an example of the other arrangement structure of the specific insulation part (141a) in an insulating layer (14). It is sectional drawing for demonstrating the basic composition of suitable 2nd embodiment of the interaction detection part which concerns on this invention. It is sectional drawing for demonstrating the basic composition of suitable 3rd embodiment of the interaction detection part which concerns on this invention. It is a drawing substitute graph which shows the result of the simulation calculation which verified the effect of the present invention (when an insulating layer is formed with materials with different dielectric constants). It is a drawing substitute graph which shows the result of the simulation calculation (in the case of the insulating layer from which film thickness differs) which verified the effect of this invention. It is reinforcement explanatory drawing of the implementation method of the simulation calculation of FIG.

Explanation of symbols

1a, 1b, 1c Interaction detector (detector)
12, 13, 15, 16 Electrode layers 14, 17, 18, 131 Insulating layer 141 (141a, 141b), 143 Specific insulating part E Electric field G Power supply R Reaction region S Switch X Probe material Y Target material

Claims (7)

A reaction region serving as a field of interaction between substances, an electrode used for applying an electric field to the reaction region, and an insulating layer covering the surface of the electrode,
The interaction detecting unit, wherein the insulating layer is provided with a specific insulating unit having a high dielectric constant.   The interaction detecting unit according to claim 1, wherein the specific insulating unit is a part formed of a material having a higher dielectric constant than a surrounding insulating layer.   The interaction detecting unit according to claim 1, wherein the specific insulating unit is a portion formed in a thinner film than a surrounding insulating layer.   The interaction detecting unit according to claim 1, wherein a probe substance is fixed to the specific insulating unit.   The interaction detection unit according to claim 1, wherein the interaction is hybridization between nucleic acid strands.   A sensor chip provided with the interaction detection unit according to claim 1. Using a detection unit comprising a reaction region serving as an interaction field between substances, an electrode used for applying an electric field to the reaction region, and an insulating layer covering the surface of the electrode,
A bioassay method characterized by promoting the immobilization of a probe substance or the interaction by concentrating an electric field on a specific insulating part having a high dielectric constant provided in the insulating layer.

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