Patent Application
20100213056
The present invention relates to a technique for detecting an interaction between materials. More specifically, the invention relates to a technique for detecting an interaction between materials by utilizing an electrodynamic action.
In recent years, an integrated substrate for bioassay so-called a DNA chip or a DNA microarray (hereinafter generally referred to as “a DNA chip” in this application) in which predetermined DNAs are finely arranged by utilizing a microarray technique has been utilized for an analysis of mutation of a gene, an analysis of SNPs (single nucleotide polymorphisms), an analysis of a frequency of genomic expression, and the like. Also, the DNA chip begins to be generally used in the fields of a drug discovery, a clinical diagnosis, a pharmacogenomics, a research of evolution, a forensic medicine, and the others.
The feature of this “DNA chip” is that an exhaustive analysis of a hybridization becomes possible because the various and multiple DNA oligo chains or nucleotide chains such as a complementary DNA (cDNA) are integrated on a glass substrate or a silicon substrate. In addition thereto, a sensor chip (for example, a protein chip) for detecting an interaction between biologic molecules other than nucleic acid molecules or a detector is variously developed.
Here, a technique utilizing an electrodynamic effect such as an electrophoresis or a dielectrophoresis in an assay system for causing an interaction between materials to progress, and detecting the interaction in a predetermined reaction region has recently began to be proposed. Hereinafter, this technique will be exemplified.
Firstly, Japanese Patent Laid-Open No. 2001-330608 discloses a technique that a nucleic acid probe template chain immobilized on a template substrate is used to synthesize a nucleic acid probe chain along the template chain, and the synthesized probe chain is immobilized on another array substrate by utilizing an electric field, thereby simply manufacturing a nucleic acid chain immobilized array at a low cost.
Japanese Patent Laid-Open No. 2001-242135 discloses a technique that a chip for detection is composed of a main body portion and a frame which are detachable from each other, a large number of pin electrodes are protruded in matrix inside the main body portion, oligonucleotide composed of different gene sequences is immobilized on the pin electrodes, a common electrode is disposed in a hollow of the frame so as not to contact any of the pin electrodes, a voltage is applied across the common electrode and each of the pin electrodes, thereby detecting a double-strained DNA obtained through a hybridization by detecting a current.
Japanese Patent Laid-Open No. 2004-135512 discloses a hybridization detecting portion structured such that a reaction region becoming a field for a hybridization between a nucleotide chain for detection and a target nucleotide chain having a base sequence complementary to the nucleotide chain for detection can be fixedly fastened to an end portion of a scanning electrode through an action of a dielectrophoresis while the nucleotide chain for detection is elongated by application of an electric field.
Each of the prior arts described above utilizes the electrode or a group of electrodes which is disposed so as to face the reaction region. Therefore, each of the prior arts described above involves a technical problem that it is difficult to exert the desired electrodynamic effect on the entire reaction region because the electric field (electric lines of force) is generated across the counter electrodes.
In addition, the reaction region in the sensor chip such as the DNA chip or the protein chip is generally limited. Hence, when the electrode or the group of electrodes showing the counter disposition relationship with the reaction region are provided, there are encountered technical problems: (1) The structure of the reaction region becomes more complicated, (2) the morphologic restriction to the reaction region occurs because of the formation of the counter electrode, (3) the number of processes for manufacturing the chip increases, (4) the distribution of the electric supply line to the electrodes is also complicated, and so forth.
In the light of the foregoing, it is a principal object of the present invention to provide an interaction detecting portion which is capable of enabling an electrodynamic effect to be obtained in an entire reaction region, and which includes a reaction region having a simpler structure, and the like.
The present invention firstly provides an interaction detecting portion including at least a reaction region for providing a field for an interaction between materials, and an electrode or a group of electrodes, having the same potential, which is provided so as to face the reaction region, and a sensor chip including at least one interaction detecting portion concerned. An A.C. electric field, for example, is adopted as an electric field applied to the electrode or the group of electrodes, thereby obtaining a desired electrodynamic effect such as a dielectrophoresis.
In addition, adoption of a structure in which the electrode or the group of electrodes has an irregular shape in its surface makes it possible to form a nonuniform electric field in the irregular shape, especially, in the vicinity of a stepped portion. An action of the dielectrophoresis can be efficiently obtained in a region in which the nonuniform electric field is formed. In addition thereto, the surface of the electrode or the group of electrodes is covered with an insulating layer, thereby making it possible to prevent an electrochemical reaction from occurring due to an ion solution which is accumulated in the reaction region in some cases.
The interaction which is made to progress in the reaction region is not construed in a limiting sense. However, the hybridization between the nucleic acid chains with which it is expected that the action of the dielectrophoresis gives the efficiency and precision of the interaction the better effect can be given as an example of the interaction described above.
Next, the present invention provides an interaction detector including at least a reaction region for providing a field for an interaction between materials, an electrode or a group of electrodes, having the same potential, which is provided so as to face the reaction region, and means for applying an electric field to the electrode or the group of electrodes.
Means for applying an A.C. electric field across the electrode or the group of electrodes, and a grounding portion, for example, can be adopted as the electric field applying means constituting the interlayer detector. In addition, the electric field applying means can also apply a high frequency electric field to the electrode or the group of electrodes through an impedance matching circuit.
With the interaction detector, the electrodynamic action such as the dielectrophoresis is given to the material existing in the reaction region by application of the electric field.
Here, the main technical terms relating to the present invention will now be described.
“The interaction” generally means a chemical bond or dissociation containing a noncovalent bond, a covalent bond and a hydrogen bond between materials. For example, “the interaction” generally contains the hybridization between nucleic acid molecules, the interaction between protein materials, and the chemical bond or dissociation, between materials, such as an antigen-antibody complex reaction. It is noted that “the hybridization” means a complementary strand (duplex) forming reaction, between materials, having a complementary base sequence structure.
“The nucleic acid chain” means a polymer (nucleotide chain) of phosphate ester of nucleoside obtained by glycosidically bonding a purine or pyrimidine base and sugar to each other. Thus, “the nucleic acid chain” generally contains a DNA (an entire length or a fragment) obtained by polymerizing oligonucleotide containing a prove DNA, a polynucleotide, purinenucleotide, and pyrimidinenucleotide, a cDNA (c prove DNA) obtained through reverse transcription, an RNA, a polyamidenucleotide derivative (PNA), and the like.
“The reaction region” is a region in which the field for the interaction such as the hybridization can be provided. A reaction field, having a well shape, in which a liquid phase material or a gel can be accumulated is given as an example of “the reaction region”.
“The dielectrophoresis” is a phenomenon that molecules are driven to a stronger electric field in a field having a nonuniform electric field. Even when the A.C. voltage is applied to the molecules, the driving effect can be obtained similarly to the case of application of the D.C. voltage because the polarity of the polarization is also inversed along with inversion of the polarity of the applied voltage (refer to a literature of “Micro Machine and Materials (published by CMC Publishing Co., Ltd.)” complied under the super vision of Teru Hayashi, Manipulation of Cells and DNAs (Fifth Chapter), pp. 37 to 46). In particular, the force acts on the dipoles of the molecules in proportion to a gradient of a square of a time average electric field within the high frequency A.C. electric field, so that the molecules migrate.
For example, it is known that when receiving the action of the electric field exerted thereon in the liquid phase, the nucleic acid molecules elongate or move. With regard to the principles thereof, it is thought that phosphoric acid ions (negative electric charges) constituting a skeleton, and hydrogen atoms (positive electric charges) into which the water lying in the vicinity of the phosphoric acid ions are ionized form an ionic cloud together with each other. Also, the polarization vectors (dipoles) generated from the negative electric charges and the positive electric charges are oriented to one direction as a whole by application of the high voltage having a high frequency to elongate. In addition thereto, when the nonuniform electric field is applied to the nucleic acid molecules, the polarization vectors move toward the portion on which the electric lines of force concentrate (refer to a literature of Seiichi Suzuki, Takeshi Yamanashi, Shinichi 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 Application, Vol. 34, No. 1, pp. 75 to 83 (1998)).
“The sensor chip” means a substrate for causing an interaction between materials to progress, and detecting the interaction between the materials in a predetermined reaction region on the substrate. Thus, the sensor chip generally includes such substrates irrespective of the kind of material, and the principles for detecting the interaction is not subject matter in the sensor chip. The sensor chip at least includes the DNA chip (DNA microarray) in which the nucleic acid chains such as the DNA probe is immobilized on the substrate to become a state of being finely arranged, the protein chip which is suitable for detecting the interaction between the protein materials, and the antigen-antibody complex reaction or the like, and the like.
The present invention adopts the structure that the electrode (that is, the counter electrode) disposed so as to face the reaction region is not provided, but the electrode or the group of electrodes having the same potential is provided. As a result, the applied electric field (electric lines of force) can be made to diverge in all the directions in the vicinity of the electrode. That is to say, since the electric field (electric lines of force) can be made to diverge far and wide toward the entire reaction region, the electrical gradient, or the nonuniform electric field can be formed in the entire reaction region. As a result, the materials dispersed in the reaction region can be efficiently collected up in larger numbers to the electrode side through the electrodynamic migration action.
Since it is unnecessary to form the counter electrode in the reaction region, the number of morphologic restrictions to the reaction region is less, and thus the structure of the detection portion can be simplified. For example, in the electric field applying means, with respect to the power feeding wiring itself, the grounding portion has to be previously connected to the system side, and the wiring for the detection portion has to be connected to only the electrode in the hybridization region. As a result, the structure of the detector can also be simplified.
Hereinafter, preferred embodiments for carrying out the present invention will be described with reference to the accompanying drawings. It is noted that each of embodiments shown in the accompanying drawings merely shows an example of a typical embodiment of a product or a method relating to the present invention, and thus the scope of the present invention is not intended to be construed in a limiting sense.
Firstly,
The reaction region 2 functions as a region or a space for providing a field for an interaction, such as a hybridization, between materials. An A.C. electric field, for example, is applied to the medium accumulated or held in the reaction region 2 through an electrode E1 which is formed so as to face the reaction region 2.
Here, the electrode E1 is made of either a metal such as aluminum or gold, or a transparent conductor such as ITO (indium-tin-oxide). In an example of this embodiment, the electrode E1 is disposed at a central position of a bottom surface 21 of the reaction region 2. It is noted that the place where the electrode E1 is formed is not limited to the bottom surface 21. For example, the electrode E1 may also be formed either in an upper side substrate 5 or in a position of a spacer 6 which will be described later so as to face the reaction region 2.
As shown in
A surface of the electrode E1 can be operated as a detection surface on which a material D to be detected such as a DNA probe is immobilized. More specifically, surface processing for allowing the end of the material D to be detected such as the probe DNA to be immobilized is previously executed for the electrode E1.
The material D to be detected can be immobilized on the surface of the electrode E1 based on a coupling reaction or the like occurring between the surface of the electrode E1 and the end of the probe DNA (an example of the material D to be detected). For example, in the case of the electrode surface for which the surface processing is executed by using streptavidin, it is suitable for immobilizing the end of the biotinylated material D to be detected. Or, in the case of the electrode surface for which the surface processing is executed by using a thiol (SH) group, it is suitable for immobilizing the material D to be detected, such as the probe DNA, having the end modified with the thiol group by using a disulfide bond (—S—S-bond).
It is noted that reference symbols D and T shown in each of
Reference numerals 4 and 5 shown in each of
Reference numeral 6 shown in each of
Subsequently, a structure is adopted in which a voltage can be applied from a power source V such as an A.C. power source across the electrode E1 and a grounding portion 7 provided outside the reaction region 2 in accordance with an operation for turning ON/OFF a switch S (electric field applying means).
With such a structure of the electric field applying means, since it is unnecessary to form a counter electrode corresponding to the electrode E1 within the limited reaction region 2, the structure of the reaction region 2 can be simplified. Moreover, when the electric field is applied thereacross, with respect to the wiring itself, the grounding portion has to be previously connected to the system side, and the wiring to the detection portion has to be connected to only the electrode E1 within the reaction region 2. As a result, the structure of the detector (for detecting the hybridization and a signal) can also be simplified.
When the switch S is turned ON, the electric field concentrates on the vicinity of the electrode E1, so that the nonuniform electric field can be formed. With regard to the electrode structure suitable for generating the nonuniform electric field, while not especially illustrated, it is expected that the surface of the electrode E1, for example, is subjected to rough surfacing processing so as to obtain an irregular shape, or is patterned so as to obtain an island-like shape, thereby causing the electric field to be easy to concentrate on a convex portion (angled portion) of the surface of the electrode E1 concerned.
With such a structure, the electric field can be especially caused to concentrate on a corner portion of the convex portion, a bend portion of a recess portion, or the like. As a result, the action and effect of the dielectrophoresis can be more reliably utilized in the region in the vicinity of the electrode E1. It is noted that although the method of subjecting the electrode surface to the rough surfacing processing can be carried out by, for example, utilizing the known sputter depositing technique, epitaxy depositing technique or etching technique, it is not especially limited.
Based on the action of the nonuniform electric field, the material which dispersively exists in the reaction region 2 described above at random can be drawn near the electrode E1 side or can be elongated in a direction along the electric field due to the electrodynamic action such as the dielectrophoresis.
The strength, frequency, and an application period of time of the applied electric field are not especially limited. Thus, the proper electric field strength, frequency, and application period of time of the applied electric field are desirably selected in accordance with the kind, the molecular length and the like of the object to which the electric field is applied. Also, the waveform of the applied electric field is not also limited to the sine wave, and thus for example, may be a chopping wave or the like.
In the case where the A.C. electric field is actually applied to the electrode E1, it is preferable that as shown in
When the electric field is applied, the two variable capacitors C1 and C2 play a role for adjusting a capacitance, and the inductance neutralizing circuit Ln plays a role for adjusting an inductance. With such a structure, there is performed the impedance matching between an internal impedance outputted from the power source (signal generating source) V, and a load impedance of the power source.
Moreover, a more detailed constitution of a preferred embodiment of the electric field applying means is shown in the form of a block diagram in
A signal outputted from the grounded power source (that is, the signal generating source) V is amplified by an amplifier 81 connected to the grounded power source V and is taken out in the form of an output signal. Also, a transmission wave Wt about the resulting output signal is inputted and detected by a signal detector (sensor) 83 through a directional coupler designated with reference numeral 82. Also, an output power of the output signal thus detected is measured by a measuring instrument 84. A control portion 85 monitors this measured value.
In addition, a reflected wave Wr from the electrode E1 located at a termination is detected by the signal detector (sensor) 86 through the directional coupler 82, and a reflected power thereof is measured by a measuring instrument 87. The control portion 85 monitors this measured value.
The impedance matching circuit 8 adjusts a magnitude of the internal impedance Zv of the power source, and a magnitude of the load impedance Ze of the counter electrode E1 in accordance with a control signal C which the control portion 85 outputs based on the measured value described above (refer to
By the impedance matching means described above, the loss of the power supplied from the power source V to the counter electrode E1 is reduced, so that the supplied power becomes maximum by utilizing the technique for exerting the electrodynamic action on the material existing in the reaction region 2 of the detection portion 1, for example, the nucleic acid molecules.
In addition, it is possible to reliably carry out the uniformity (stabilization) of the applied power (supplied power) to the reaction region 2, and moreover the suppression of a disturbance in a waveform of a pulse signal which occurs when the high frequency electric field or the A.C. electric field, especially, the high frequency A.C. electric field is utilized, the erasing of the reflected wave Wr from the electrode E1 causing a problem about a phase delay of the applied power or the like, and the like.
Giving a description by showing the case where the nucleic acid chain as the target material T exists in the reaction region 2 as an example, the nucleic acid chain concerned receives the electrodynamic action of the dielectrophoresis to migrate toward the electrode E1 having the stronger electric field strength. As a result, the target nucleic acid chains are collected on the surface of the electrode E1 on which the nucleic acid chains such as the probe DNA functioning as the material D to be detected are previously immobilized, so that the hybridization efficiently progresses.
That is to say, the target nucleic acid chains are moved to the surface of the electrode E1 for a short period of time through the dielectrophosresis described above to increase the concentration thereof, which results in that a period of time required for the hybridization with the nucleic acid chains immobilized on the surface of the electrode E1 can be greatly shortened.
Moreover, when the single stranded nucleic acid chain elongates resulting from the action of the dielectrophoresis, the steric hindrance in the phase of the hybridization is reduced as compared with the case of being curled up into a random coil state. As a result, it is possible to enhance the efficiency of the hybridization.
It is noted that with respect to a signal about the hybridization, a quantity of light emitted from an intercalate which is specifically bonded to the double strand (double stranded nucleic acid) may be measured, a quantity of light emitted from a fluorescence dye which is previously bonded to the target DNA may be measured after the excessive DNAs are removed after completion of the hybridization. Or, a quantity of emitted light following the hybridization reaction may be measured by utilizing a molecular beacon.
Here,
In the detection portion 1 according to the present invention, as shown in
That is to say, since the electric field (electric lines of force) can be made to diverge extensively toward the entire reaction region, the electrical gradient or the nonuniform electric field can be formed throughout the reaction region 2. As a result, the material dispersed in the reaction region 2 can be efficiently collected up in larger numbers on the electrode E1 side based on the electrodynamic migration action.
Here,
It is understood from the results represented in these drawings that the electric field is formed within the reaction region, the electric field strength is larger in the vicinity of the electrode (E1), and thus the nonuniform electric field is generated. Note that, when the A.C. electric field is actually applied, as described above, it is preferable that the impedance matching is obtained, thereby allowing the power to be efficiently applied.
The present invention can be utilized as the technique for detecting efficiently and precisely the interaction between the materials for a short period of time by utilizing the electrodynamic action. Also, the present invention can be utilized as the technique for the sensor chip typified by the DNA chip or the protein chip, or as the apparatus for detecting the interaction.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-164591 | Jun 2005 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/309571 | 5/12/2006 | WO | 00 | 5/14/2010 |
