JP4039201B2 - Hybridization detection unit, sensor chip, and hybridization method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a technique related to a hybridization detection unit that can be suitably used for a sensor chip such as a DNA chip. More specifically, the present invention relates to a technique for improving hybridization efficiency by aligning and fixing an extended detection nucleotide chain to a predetermined electrode site in a reaction region that provides a hybridization field.
[0002]
[Prior art]
The main prior art of the present invention will be described below. Currently, integrated substrates for bioassays called DNA chips or DNA microarrays (hereinafter collectively referred to as “DNA chips”), in which predetermined DNA is finely arranged by microarray technology, are used for gene mutation analysis, SNPs (single nucleotides). Polymorphism) analysis, gene expression frequency analysis, etc., and has begun to be widely used in drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and other fields.
[0003]
Since this DNA chip has a large number of DNA oligo chains and cDNA (complementary DNA) integrated on a glass substrate or silicon substrate, comprehensive analysis of intermolecular interactions such as hybridization becomes possible. It is characterized by dots.
[0004]
To briefly explain an analysis method using a DNA chip, a fluorescent probe dNTP is obtained by reverse transcription PCR reaction or the like by extracting mRNA extracted from cells, tissues, etc. with respect to a DNA probe solid-phased on a glass substrate or a silicon substrate. In this method, PCR amplification is carried out while incorporating, hybridization is performed on the substrate, and fluorescence is measured with a predetermined detector.
[0005]
Here, DNA chips can be classified into two types. The first type is one in which oligonucleotides are directly synthesized on a predetermined substrate using a photolithographic technique applying a semiconductor exposure technique, and a typical one is by Affymetrix (Affymetrix). (For example, refer to Patent Document 1). Although this type of chip has a high degree of integration, there is a limit to DNA synthesis on the substrate, and the length is about several tens of bases.
[0006]
The second type is also referred to as “Stanford method”, and is prepared by dispensing and solidifying a prepared DNA on a substrate using a tip-breaking pin ( For example, see Patent Document 2). This type of chip is less integrated than the former, but has the advantage that a DNA fragment of about 1 kb can be immobilized.
[0007]
[Patent Document 1]
Japanese National Patent Publication No. 4-505863
[Patent Document 2]
Japanese National Patent Publication No. 10-503841
[0008]
[Problems to be solved by the invention]
However, in the above-described conventional DNA chip technology, the nucleotide chain for detection such as a DNA probe immobilized on the detection surface site (spot site) is tangled or rounded in a random coil shape due to the action of Brownian motion. In addition, the integration density was uneven on the detection surface.
[0009]
For this reason, steric hindrance occurs during hybridization with the target nucleotide chain, so that the efficiency of hybridization is poor, the reaction takes a long time, and further, it may show false positive or false negative. There was also a technical problem.
[0010]
Therefore, the present invention mainly aims to improve hybridization efficiency by devising to align and fix the extended detection nucleotide chain in the reaction region that provides a hybridization field at the detection surface site. Objective.
[0011]
[Means for Solving the Problems]
  In order to solve the above technical problem,The present inventionIn, a reaction region that becomes a hybridization field between the detection nucleotide chain and a target nucleotide chain having a base sequence complementary to the detection nucleotide chain extends the detection nucleotide chain by an electric field, A “hybridization detection unit” and a “sensor chip” including the detection unit, which are configured to be fixed to scan electrode sites arranged in the reaction region by the action of dielectrophoresis, can be implemented using these. Hybridization methods "are provided.
[0012]
In the present invention, the detection nucleotide chain can be extended by the electric field formed in the reaction region, and in addition, the region around the edge of the scan electrode arranged in the reaction region is locally unevenly distributed ( An electric field in which electric lines of force concentrate in part) is formed, and the extended detection nucleotide chain existing in the reaction region is dielectrophoresed toward the end of the scanning electrode to be in the extended state. It is possible to obtain an action and an effect that the detection nucleotide chain can be fixed so as to be bridged between the electrodes constituting the scanning electrode.
[0013]
The above-described effects can be obtained by the configuration of an electrode (including a counter electrode and a common electrode) for forming an electric field for extending the nucleotide chain and a scanning electrode for fixing the extended nucleotide chain for detection. It can be appropriately determined within the range. In a scanning electrode with an appropriate number of electrodes, by switching a switch provided at a predetermined position on the chip, the electrodes to which a voltage is applied are selected in order, and the electrodes are close to the applied electrodes (electrodes in the applied state). Existing (extended) nucleotide chains for detection can be attracted to the electrode side by the action of dielectrophoresis and fixed one after another so as to be bridged between the ends of adjacent scanning electrodes. .
[0014]
By this means or method, the detection nucleotide chain that is dropped and dispersed in the reaction region and entangled in a random coil shape by Brownian motion (in a form not suitable for hybridization) is extended and hybridized. The density of the nucleotide chains for detection in the reaction region can be averaged by adjusting to a linear form that can be easily performed, and by aligning and fixing the nucleotide chains for detection to the scanning electrode site in an elongated state.
[0015]
The target nucleotide chain added after the detection nucleotide chain aligned and fixed in the reaction region can be extended by the same means, attracted to the scanning electrode site, and hybridized. In the extended nucleotide chains, the bases do not overlap each other. As a result, there is no steric hindrance during the reaction, and the hybridization reaction can be carried out smoothly.
[0016]
The elongation of the nucleotide chain is determined by applying a high-frequency electric field of about 1 MV / m from a number of polarization vectors composed of the nucleotide chain (negative charge of the nucleotide chain comprising phosphate ions and the like and the positive charge of ionized hydrogen atoms). This is based on the principle that a dielectric polarization occurs in the nucleotide chain, which results in the nucleotide molecule being stretched linearly parallel to the electric field.
[0017]
The present invention relates to a hybridization detection unit capable of efficiently performing hybridization detection essential for gene mutation analysis, SNPs (single nucleotide polymorphism) analysis, gene expression frequency analysis, and the like, a DNA chip including the detection unit, and the like This technology has the technical significance of providing such sensor chips to drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and other related industries.
[0018]
  here,The present inventionDefinition of main technical terms in. In the present application, the term “nucleotide chain” means a polymer of a phosphate ester of a nucleoside in which a purine or pyrimidine base and a sugar are glycosidically bonded, and an oligonucleotide, a polynucleotide, a purine nucleotide and a pyrimidine nucleotide are polymerized including a DNA probe. Widely includes DNA (full length or a fragment thereof), cDNA obtained by reverse transcription (cDNA probe), RNA, polyamide nucleotide derivative (PNA) and the like.
[0019]
The “detection nucleotide chain” is a nucleotide chain immobilized directly or indirectly on the detection surface, and the “target nucleotide chain” is a nucleotide chain having a base sequence complementary to the detection nucleotide chain. In some cases, it is labeled with a fluorescent substance or the like.
[0020]
“Hybridization” means a complementary strand (duplex) formation reaction between nucleotide strands having a complementary base sequence structure.
[0021]
The “reaction region” is a sample solution storage region that can provide a place for a hybridization reaction in a liquid phase. In this reaction region, in addition to the interaction between single-stranded nucleotides, that is, hybridization, a desired double-stranded nucleotide is formed from the detection nucleotide strand, and the double-stranded nucleotide and peptide (or protein) interact with each other. Enzymatic response reactions and other intermolecular interactions can also be performed. For example, when the double-stranded nucleotide is used, the binding of a receptor molecule such as a hormone receptor, which is a transcription factor, and a response element DNA portion can be analyzed.
[0022]
“Steric hindrance” makes it difficult to access the reaction partner molecule due to the presence of bulky substituents near the reaction center in the molecule, the posture of the reaction molecule, and the three-dimensional structure (higher order structure). This means a phenomenon in which a desired reaction (hybridization in the present application) hardly occurs.
[0023]
“Dielectrophoresis” is a phenomenon in which molecules are driven to a stronger electric field in a field where the electric field is not uniform. Even when an AC voltage is applied, the polarity of the polarization is reversed as the polarity of the applied voltage is reversed. 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 to P46, Chapter 5 Manipulation of cells and DNA).
[0024]
“Counter electrodes” means a pair of electrodes to which a voltage can be applied. “Common electrode” means an electrode to which a voltage can be applied between a plurality of electrodes. “Scanning electrode” means an electrode group in which a plurality of electrodes to which voltage can be sequentially applied by turning on / off a switch are arranged.
[0025]
“Sensor chip” means a substrate formed of quartz glass, synthetic resin or the like provided with a detection surface and a reaction region capable of detecting the interaction between substances, and a typical example is a DNA chip. it can.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings.
[0027]
<First Embodiment>
First, FIG. 1 is a diagram illustrating a configuration of a main part of a first embodiment (reference numeral 1a) of a hybridization detection unit (hereinafter abbreviated as “detection unit”) and a sensor chip according to the present invention.
[0028]
First, a reaction region R is provided in the detection unit 1a according to the first embodiment. The reaction region R is a storage region to which a sample solution containing the detection nucleotide chain X or the target nucleotide chain Y is added, and is a region or space that provides a field for hybridization reaction.
[0029]
In this reaction region R, counter electrodes A and B are arranged. The counter electrodes A and B are preferably arranged in parallel, and both power sources V₁It is connected to the. The illustrated switch s is turned on and the power source V₁When a high frequency voltage is applied, a uniform electric field (electric field where electric lines of force are not concentrated in part) is formed in the reaction region R between the electrodes A and B (reference symbol L in FIG. 1).₁(See the line marked with).
[0030]
The condition of the electric field of the counter electrodes A and B is about 1 × 10⁶The conditions of V / m and about 1 MHz are preferable (Masao Washizu and Osamu Kurosawa: “Electrostatic Manipulation of DNA in Microfabricated Structures”, IEEE Transaction on Industrial Application Vol. 26, No. 26, p. 1165-1172 (1990 )reference). Since all the electric field conditions described below are the same as this, description thereof will be omitted.
[0031]
Uniform electric field L₁As a result, the detection nucleotide chain X present in a dispersed form in the form of a random coil or the like in the reaction region R is converted into the uniform electric field L.₁It can be extended in a direction along the line to form a straight chain (the principle is already described).
[0032]
Subsequently, between the counter electrodes A and B, the second counter electrodes G and C (C₁, C₂, C₃,... Cx, Cy, Cz) are arranged, and the respective power sources V illustrated₂It is configured to be connectable to or connectable to.
[0033]
In the detection unit 1a, a single rectangular electrode is adopted as the electrode G, and a scanning electrode in which each electrode is arranged to face the electrode G with a predetermined distance is adopted as one electrode C group. Has been. That is, the scan electrode group C is connected to the switch S.₁, S₂, S₃... Sx, Sy, Sz are sequentially turned on / off to sequentially apply a voltage between a pair of adjacent scanning electrodes D. In the conductive state, between each scanning electrode (for example, G and C_x, C_y) (Particularly around the end of the scanning electrode C), the electric field lines concentrate the non-uniform electric field L.₂Is formed (see FIG. 3).
[0034]
Each electrode C of the scan electrode C group₁, C₂... The arrangement interval of Cz is not more than the molecular length of the detection nucleotide chain X 'for the purpose of fixing the extended detection nucleotide chain X' so as to bridge (hereinafter, in all embodiments). The same).
[0035]
Next, FIG. 2A is a cross-sectional view taken along the line II in FIG. 1, and FIG. 2B is a modified form (reference numeral 1) of the detection unit 1a shown in FIG. It is II sectional view taken on the line which represents' a).
[0036]
As shown in FIG. 2A, the detection unit 1a and its modified form 1'a are formed of a substrate M formed of a synthetic resin such as quartz glass, silicon, polycarbonate, or polystyrene.₁, M₂It is provided in a narrow gap between. In the detection unit 1a, the thicknesses of the electrodes A, B, etc. and the depth (or width) of the reaction region R are the same.
[0037]
In some cases, each electrode A, B or the like is sandwiched by a dielectric U as in the detection unit 1′a shown in FIG.₁, M₂And the volume of the reaction region R may be increased. Although not shown in the figure, in the reaction region R, a plurality of scan electrodes C may be arranged in a row in the depth direction of the reaction region R.
[0038]
Here, FIG. 3 shows a case where a voltage is applied between the counter electrodes A and B and the voltage between the second counter electrodes G and Cx and between the G and Cy in the detection unit 1a (or 1′a). Is applied, and the non-uniform electric field L is generated near the scan electrodes Cx and Cy.₂Represents a state in which is formed. FIG. 3 shows a state in which the target nucleotide chain Y has already been added to the reaction region R, and an electric field (L₁) Is abbreviated.
[0039]
As shown in FIG. 3, the detection nucleotide chain X extended by the action of the uniform electric field by the counter electrodes A and B is located near the scanning electrode C.₁-C₂, C₂-C₃... Are driven by dielectrophoresis between Cx-Cy... And fixed between the ends dd of each electrode D.
[0040]
The symbol X ′ in FIG. 3 indicates a detection nucleotide chain fixed between adjacent scanning electrodes C (the same applies to other drawings). In FIG. 3, the switches Sx and Sy are turned on, and the non-uniform electric field L between the electrodes G and Cx-Cy is shown.₂Indicates the stage in which is formed.
[0041]
Here, the target nucleotide chain Y added later to the reaction region R is elongated in the same manner as the detection nucleotide chain X under the action of the uniform electric field formed by the counter electrodes A and B. When this extended target nucleotide chain Y (complementary portion thereof) is attracted to the (extended) detection nucleotide chain X ′ immobilized between the electrodes C, it is not affected by steric hindrance, and the efficiency Good hybridization proceeds.
[0042]
Second Embodiment
Next, FIG. 4 shows the principal part structure of 2nd Embodiment (code | symbol 1b) of the detection part and sensor chip based on this invention.
[0043]
The detection unit 1b is different from the detection unit 1a (1′a) of the first embodiment described above, and each scanning electrode group C₁, C₂, C₃... Electrodes (electrodes corresponding to symbol G in FIGS. 1 and 3) facing Cx, Cy, and Cz are not provided. Between adjacent scan electrodes C₁-C₂, C₂-C₃,... For Cx-Cy,..., The power supply V shown in the figure is based on the ON / OFF procedure of the predetermined switch S group shown in the figure.₃The voltage is applied sequentially.
[0044]
In FIG. 4, a voltage is applied to the counter electrode AB and the scan electrodes Cx-Cy, and a non-uniform electric field L is generated between the scan electrodes Cx-Cy.₂Is formed, and the target nucleotide chain Y is attracted toward the detection nucleotide chain X ′ fixed between the scan electrodes Cx and Cy (see the arrow portion).
[0045]
<Third Embodiment>
FIG. 5 shows the configuration of the third embodiment (reference numeral 1c) of the detection unit and sensor chip according to the present invention.
[0046]
The detection unit 1c shown in FIG. 5 includes a power supply V in a region between the counter electrodes A and B.₃Scan electrode group C and power supply V applied by₄Is arranged so as to face the scanning electrode D group applied by the above. Specifically, the scanning electrodes C arranged side by side₁, C₂, C₃... Scanning electrodes D so as to face Cx, Cy, Cz, respectively₁, D₂, D₃... Dx, Dy, Dz are arranged.
[0047]
In FIG. 5, the target nucleotide chain Y is applied to the counter electrode AB, the scan electrodes Cx-Cy, Dx-Dy, and the non-uniform electric field L₂The state of being attracted to the detection nucleotide chain X ′ fixed between the scan electrodes Cx-Cy and between Dx-Dy by the action of (see the arrow portion) is illustrated.
[0048]
<Fourth embodiment>
Subsequently, FIG. 6 shows a main part configuration of the fourth embodiment (reference numeral 1d) of the detection unit and the sensor chip according to the present invention.
[0049]
This detection part 1d is arranged in parallel with the scanning electrode C arranged in the same manner as the detection part 1c of the third embodiment.₁, C₂, C₃... Scanning electrodes D so as to face Cx, Cy, Cz, respectively₁, D₂, D₃... Dx, Dy, Dz are arranged. The scan electrode C group and the D group have a common power source V₅Is applied.
[0050]
In FIG. 6, the non-uniform electric field L is applied to the counter electrode AB and the scan electrodes Cx-Dx, Cy- and Dy, respectively.₂The state (see the arrow portion) in which the target nucleotide chain Y is attracted to the detection nucleotide chain X ′ by the action of is illustrated.
[0051]
Next, an example of voltage application operation will be described with reference to FIGS. 7 illustrates a voltage application operation example (three examples) corresponding to the detection unit 1a, and FIG. 8 illustrates a voltage application operation example (three examples) corresponding to the detection unit 1b.
[0052]
The detection unit 1a in FIG. 1 will be described as an example. As shown in FIG. 7A, the voltage applied between the counter electrodes AB is changed when the scanning electrodes are sequentially turned on (G- C₁・ C₂→ GC₂・ C₃→ ...), the application may be always on.
[0053]
Further, as shown in FIG. 7B, the voltage applied between the counter electrodes A and B may be turned off each time the scanning electrodes are turned on in order.
[0054]
Furthermore, as shown in FIG. 7C, the voltage applied between the counter electrodes A and B may be turned off when applied to the scan electrodes. By repeating these voltage application operations, the detection nucleotide chain X can be reliably fixed to the scanning electrode.
[0055]
The detection unit 1b in FIG. 4 will be described as an example. As shown in FIG. 8A, the voltage applied between the counter electrodes AB is changed when the scanning electrodes are sequentially turned on (C₁-C₂→ C₂-C₃→ ...), the application may be always on.
[0056]
Further, as shown in FIG. 8B, the voltage applied between the counter electrodes A and B may be turned off each time the scanning electrodes are turned on in order.
[0057]
Further, as shown in FIG. 8C, the voltage applied between the counter electrodes A and B may be turned off when applied to the scan electrodes. By repeating these voltage application operations, the detection nucleotide chain X can be reliably fixed to the scanning electrode. It should be noted that the same voltage application operation as described above can be performed for the detection units 1c and 1d described above.
[0058]
As shown in FIGS. 7B, 7C, 8B, and 8C, the voltage between the counter electrodes A and B is turned on / off and applied intermittently. The target nucleotide chain Y in the reaction region R is made to approach the detection nucleotide chain X ′ stepwise, the target nucleotide chain Y is moved back and forth, and the reaction timing is adjusted. There is an advantage that you can.
[0059]
Further, by turning off the voltage application between the counter electrodes A and B, a complementary strand formation reaction between the linear target nucleotide chain Y and the linear detection nucleotide chain X ′, that is, hybridization. Can be carried out solely by the Brownian movement.
[0060]
By the above voltage application operation, complementarity between the detection nucleotide chain X ′ that is linearly extended and fixed to the scanning electrode D group or the like and the target nucleotide chain Y that is linearly extended similarly to this. The formation of hydrogen bonds between certain bases has less steric hindrance and proceeds efficiently.
[0061]
That is, a result is obtained that the hybridization reaction between the detection nucleotide chain X ′ and the target nucleotide chain Y proceeds efficiently. As a result, it is possible to obtain a favorable result that the hybridization reaction time is shortened and the probability of showing false positive or false negative is also reduced.
[0062]
<Fifth Embodiment>
FIG. 9 shows a main part configuration of the fifth embodiment (reference numeral 1e) of the detection unit and sensor chip according to the present invention.
[0063]
The detection section indicated by reference numeral 1e is provided with a reaction region R. In this reaction area R, a common electrode indicated by reference numeral G and a group of scan electrodes arranged in parallel with the common electrode G, respectively. C (C₁To Cz).
[0064]
  The common electrode G is a power source V₁Each electrode C of the scan electrode group C arranged in a row.₁~ Cz is the switch S₁Power supply V by turning on ~ Sz in turn₁It is configured to be connected to.FIG.The switch Sy is turned on, a voltage is applied between the electrodes G-Cy, and a non-uniform electric field L is generated around the end of the scan electrode Cy.₂The state (stage) in which is formed is shown.
[0065]
Each electrode C constituting the common electrode G and the scanning electrode group C₁Switch S to ~ Cz₁When the high frequency voltage is applied in order by switching to Sz, the electrode G and each electrode C₁In the reaction region R between ˜Cz, the non-uniform electric field L₂Is formed.
[0066]
Due to the action of the electric field, the detection nucleotide chains X dispersed and present in the form of a random coil or the like in the reaction region R are elongated in the direction along the electric field to be linear. Simultaneously with the elongation action of the nucleotide chain, each electrode C to which a voltage is applied₁~ Uneven electric field L formed around edge E of Cz₂As a result, the detection nucleotide chain X in the reaction region R is extended to each electrode C.₁Dielectric migration toward each end E of ~ Cz, and the detection nucleotide chain X is connected between the electrodes (C₁-C₂, C₂-C₃,... Cy-Cz).
[0067]
More specifically, one end of the detection nucleotide chain X that is extended in the reaction region R has a non-uniform electric field L₂Electrode C by dielectrophoresis with₁End E of₁And then the other end of the nucleotide chain X is connected to the next electrode C to which voltage is applied next.₂End E of₂Is attracted and fixed by adhesion. In this way, the detection nucleotide chain X is transferred between the electrodes (C₁-C₂, C₂-C₃,... Cy-Cz) to be bridged. In addition, the code | symbol X 'in FIG. 9 represents the nucleotide chain for a detection of the state fixed to the electrode edge part.
[0068]
<Sixth Embodiment>
Subsequently, FIG. 10 is a diagram illustrating a configuration of main parts of a sixth embodiment (reference numeral 1f) of the detection unit and the sensor chip according to the present invention.
[0069]
  Similar to the detection unit 1e, the detection unit 1f is provided with a common electrode G in the reaction region R. This detection section 1f is provided with two rows of scanning electrodes C and D, and each electrode C constituting the scanning electrode C group.₁, C₂, C₃... Each electrode end of Cx, Cy ... CzEAre each electrode D which comprises the scanning electrode D group.₁, D₂, D₃... Each electrode end of Dx, Dy ... DzeAre arranged so as to face each other.
[0070]
  Here, each electrode C of the scanning electrode C group₁~ Cz is the switch S₁Power supply V by turning on ~ Sz in turn₁To each electrode D of the scanning electrode D group.₁~ Dz is switch s₁By turning on ~ sz in order, power supply V₁It is the structure connected to. That is, switch S₁And s₁Turns on the common electrode GC₁, D₁A voltage is applied between the switches S₂And s₂Turns on the common electrode GC₂, D₂A voltage is applied between them. In FIG. 2, the switches Sy and sy are turned on and the electrodes G-Cy,DyA state is shown in which a voltage is applied between them.
[0071]
<Seventh embodiment>
Next, FIG. 11 is a diagram illustrating a configuration of main parts of a seventh embodiment (reference numeral 1g) of the detection unit and the sensor chip according to the present invention.
[0072]
As in the detection units 1e and 1f, the detection unit 1g shown in FIG. 3 is provided with a common electrode G in the reaction region R and two rows of scanning electrodes D and F. ing. Each electrode D constituting the scanning electrode D group₁, D₂, D₃... Each electrode end E of Dx, Dy,... Dz corresponds to each electrode F constituting the scanning electrode F group.₁, F₂, F₃... Are arranged so as to face the electrode ends e of Fx, Fy,.
[0073]
Each electrode D of the scanning electrode D group₁~ Dz is switch S₁Power supply V by turning on ~ Sz in turn₁To each electrode F of the scan electrode F group.₁~ Fz is switch s₁By turning on ~ sz in order, power supply V₁It is the structure connected to. That is, switch S₁And s₁Turns on the common electrode GD₁, F₁When a voltage is applied between them and the switches s2 and S2 are turned on, the common electrode GD₂, F₂A voltage is applied between them. FIG. 11 shows a state in which the switches Sy and sy are turned on and a voltage is applied between the electrodes G-Dy and Fy.
[0074]
Here, in the seventh embodiment of FIG.₁And F₁, D₂And F₂, D₃And F₃..., the distance H between Dz and Fz is gradually increased. A voltage was applied in the reaction region R by adopting a configuration in which the distance H between the counter electrodes constituting the scanning electrodes D group and F group was gradually increased and the reaction field between the electrodes was gradually expanded. Desirably, the nucleotide chain for detection X that dielectrophoreses toward (the end of) the electrode can move freely without being physically disturbed by the adjacent electrode (the electrode to which no voltage is applied). An effect is obtained. The same effect can be obtained during dielectrophoresis of the target nucleotide chain Y.
[0075]
Next, FIG. 12A is a cross-sectional view taken along line II-II in FIG. 9, and FIG. 12B is a line II-II showing a modified form (reference numeral 1'e) of the detection unit 1e. It is arrow sectional drawing.
[0076]
As shown in FIG. 12, the detection units 1e and 1'e are substrates M made of synthetic resin such as quartz glass, silicon, polycarbonate, polystyrene, or the like.₁, M₂In the detection unit 1e, a configuration in which the thicknesses of the common electrode G and the scanning electrode group C and the depth (or width) of the reaction region R coincide with each other is employed.
[0077]
In some cases, a configuration in which the common electrode G and the scanning electrode group C are sandwiched by the dielectric U as in the detection unit 1′e in FIG. As a result, the substrate M₁, M₂And the volume of the reaction region R can be increased. Although not shown, in the reaction region R, a plurality of scan electrodes C may be arranged in the depth direction of the reaction region R (the same applies to the detection units 1f and 1g).
[0078]
In the detection units 1e to 1g having the above-described configuration, the target nucleotide chain Y added to the reaction region R is the electrode GC (C₁~ Cz), G-D (D₁To Dz), G-F (F₁~ Fz) non-uniform electric field L formed between each of₂In the same manner as the detection nucleotide chain X, the detection nucleotide chain X ′ is extended (extended) while being immobilized to the scanning electrodes C group, D group, and F group. Thus, efficient hybridization proceeds without being affected by steric hindrance.
[0079]
FIG. 9 shows a state in which hybridization proceeds between the detection nucleotide chain X ′ fixed between the scan electrodes Cx and Cy and the target nucleotide chain Y subjected to the elongation action, and FIG. 10 shows the scan electrode Cx. FIG. 11 shows the state in which hybridization proceeds between the detection nucleotide chain X ′ immobilized between −Cy and between Dx and Dy and the target nucleotide chain Y subjected to the extension action. It should be noted that the state of hybridization proceeding between the detection nucleotide chain X ′ fixed between Fx and Fy and the target nucleotide chain Y subjected to the extension action is schematically shown.
[0080]
Next, an example of voltage application operation will be described with reference to FIG. 13A is a voltage application operation example corresponding to the detection unit 1e, FIG. 13B is a voltage application operation example corresponding to the detection unit 1f, and FIG. 13C corresponds to the detection unit 1g. Each of the voltage application operation examples is shown.
[0081]
  In the detection unit 1e of FIG. 9, as shown in FIG.₁, Electrode G-C₂In the meantime, voltages are applied in order, and in the detection unit 1f of FIG. 10, as shown in FIG.₁・D ₁ , Electrode G-C₂・D ₂ In the meantime, voltages are applied in order, and in the detection unit 1g of FIG. 11, as shown in FIG.₁・ F₁, Electrode GD₂・ F₂... In the meantime, voltage is applied in order. In addition, each voltage application time can be determined suitably.
[0082]
Further, the series of voltage application operations in the above order may be repeated a plurality of times as necessary. By repeating a series of voltage application operations, an effect is obtained that the detection nucleotide chain X can be reliably fixed by the scanning electrode.
[0083]
Furthermore, as shown in FIG. 14, when applying voltage between the electrodes in each of the detection units 1e to 1g, detection is performed by applying the voltage intermittently by repeating on / off of the voltage a desired number of times. The timing of fixing the nucleotide chain X for use is adjusted, or the target nucleotide chain Y in the reaction region R is made to approach the target nucleotide chain X ′ stepwise with respect to the already-fixed detection nucleotide chain X ′, or the target nucleotide The chain Y can be moved back and forth, and further, the reaction timing can be adjusted.
[0084]
Further, by ensuring a time T (see FIG. 14) during which the voltage is turned off when a voltage is applied between the electrodes, the linear target nucleotide chain Y and the linear detection nucleotide chain X ′ The complementary strand formation reaction, i.e., hybridization, can be allowed to proceed exclusively by Brownian motion.
[0085]
By the above voltage application operation, the detection nucleotide chain X ′ and the detection nucleotide fixed in the scanning electrodes C group, D group, and F group of the detection units 1e to 1g in a linearly stretched state. The formation of a hydrogen bond between bases complementary to the target nucleotide chain Y extended in a straight line like the chain X ′ proceeds efficiently without any steric hindrance problem. That is, a result is obtained that the hybridization reaction between the detection nucleotide chain X ′ and the target nucleotide chain Y proceeds efficiently. As a result, it is possible to obtain a favorable result that the hybridization reaction time is shortened and the probability of showing false positive or false negative is also reduced.
[0086]
<Eighth Embodiment>
FIG. 15 is a diagram illustrating a configuration of a main part of an eighth embodiment (reference numeral 1h) of the detection unit and the sensor chip according to the present invention.
[0087]
The detection unit 1h includes a reaction region R that serves as a hybridization field between the detection nucleotide chain X and the target nucleotide chain Y having a base sequence complementary to the detection nucleotide chain X. The first scanning electrode C group arranged in the reaction region R, and the second scanning electrode D group arranged so that the ends of the first scanning electrode C group and each electrode face each other are provided. .
[0088]
An electric field is formed by sequentially applying a voltage between adjacent electrodes of the first scanning electrode group C and between adjacent electrodes of the second scanning electrode group D, and the nucleotide chain for detection is converted into the electric field. Dielectric migration toward the applied scanning electrode while extending by means of (1), and the elongated detection nucleotide chain X ′ can be fixed so as to be bridged between the scanning electrodes.
[0089]
FIG. 15 shows the power supply V₄A voltage is applied to the scan electrodes Cx-Cy by the power source V₅The voltage is applied to the scan electrodes Dx-Dy by the non-uniform electric field L₂Represents a state in which is formed.
[0090]
<Ninth Embodiment>
FIGS. 16 and 17 are diagrams showing the configuration of the main parts of the ninth embodiment (reference numeral 1i) of the detection unit and sensor chip according to the present invention.
[0091]
The detection unit 1i is first provided with a reaction region R. The reaction region R includes counter electrodes G and C (C₁, C₂, C₃,... Cx, Cy, Cz) are arranged and the illustrated power supply V₁And V₂It is configured to be able to connect to.
[0092]
In this detection unit 1i, one rectangular electrode is adopted as the electrode G, and one of the electrodes C group is a scanning electrode arranged to face the electrode G with a predetermined interval. Switches S and S₁, S₂, S₃..., Sx, Sy, Sz and switch W₁, W₂, W₃..,... By sequentially turning on / off Wx, Wy, Wz₁, C₂, C₃... A configuration in which voltages are successively applied between Cx, Cy, and Cz.
[0093]
Further, the scan electrode group C includes a switch S.₁, S₂, S₃..., Sx, Sy, Sz and switch W₁, W₂, W₃,..., Wx, Wy, and Wz are sequentially turned on / off to sequentially apply a voltage between a pair of adjacent scanning electrodes C. In the conductive state between the electrode G and the scan electrode C group, a non-uniform electric field L is formed at the end of the scan electrode C group.₂Are formed (see FIG. 16), and between the scan electrodes constituting the scan electrode C group (for example, C₁And C₂In the conductive state between the non-uniform electric field L at each scanning electrode end.₂Is formed (see FIG. 17).
[0094]
In the ninth embodiment shown in FIGS. 16 and 17, one selected scanning electrode (for example, C as shown in FIG. 16) is selected.₁) To fix one end of the elongated detection nucleotide chain X ′, and to the adjacent scanning electrode (for example, C₂) Is fixed between the adjacent scanning electrodes (for example, C₁-C₂And the procedure of immobilizing the detection nucleotide chain X ′ so as to cross-link, and the (extended) target nucleotide chain can be hybridized to the cross-linked and immobilized detection nucleotide chain X ′. . This method can be implemented by an arbitrary configuration including the scanning electrode group and the common electrode G facing the scanning electrode group.
[0095]
18A is a cross-sectional view taken along the line III-III in FIGS. 16 and 17, and FIG. 18B is a modified form of the detection unit 1i shown in FIG. 18A. It is a III-III arrow directional cross-sectional view which shows (code | symbol 1'i).
[0096]
As shown in FIG. 18A, the detection units 1i, 1'i are substrates M formed of synthetic resin such as quartz glass, silicon, polycarbonate, polystyrene, or the like.₁, M₂It is provided in a narrow gap between. In the detection unit 1i, the thickness of the electrode C and the like and the depth (or width) of the reaction region R are the same.
[0097]
In some cases, each electrode C group is sandwiched by the dielectric U as in the detection unit 1′i shown in FIG.₁, M₂And the capacity of the reaction region R may be increased. Although not shown, the reaction region R may be arranged in a plurality of rows of scan electrodes C.
[0098]
Here, FIG. 16 illustrates the switches S and S illustrated in the detection unit 1i (or 1′i).₁Turn on the power supply V₁Counter electrode GC₁A state in which a high frequency voltage is applied between them is shown.
[0099]
Electrode G-C₁When a high frequency voltage is applied between them, the reaction region R has a non-uniform electric field L₂Is formed. The non-uniform electric field L₂As a result, the detection nucleotide chain X that is randomly dispersed in the reaction region R is converted into the non-uniform electric field L.₂It can be made to extend in the direction along the straight line. Furthermore, the non-uniform electric field L₂Among them, the detection nucleotide chain X is driven by dielectrophoresis and has an electric field strength C.₁The one end is fixed in the state extended to the end of the.
[0100]
Next, by turning off the switch S, the electrode G-C₁Turn off the high-frequency voltage applied between them. At the same time or after that, switch S₂And W₂By turning on ~ Wz, power supply V₂Electrode C₁-C₂A high frequency voltage is applied between them. C₁The other end of the detection nucleotide chain X ′ having one end fixed to the electrode C₁-C₂The electrode C moves in the direction along the electric field lines of the non-uniform electric field generated between the electrodes₂It is fixed to the end.
[0101]
Then switch S₂, W₁ON, S₁, W₂Is turned off, and similarly, one end of the detection nucleotide chain X is connected to the scanning electrode C.₂After fixing to the end, switch S, W₂Off, switch S₃And W₁And W₃~ Wz is turned on, scan electrode C₂-C₃In the meantime, the nucleotide X ′ for detection in an extended state is fixed.
[0102]
As described above, by sequentially scanning the voltage applied between the electrodes, the detection nucleotide chain X ′ can be fixed between the electrode ends of the scanning electrode B group. FIG. 17 shows a state in which the detection nucleotide X ′ is bridged and fixed between the scan electrodes Cx-Cy.
[0103]
Although not shown, a high frequency voltage is applied to the electrode GC group at one time, and a nucleotide chain for detection is extended and arranged at each end of the scan electrode C group, and then between adjacent electrodes of the scan electrode C group. The detection nucleotide X ′ may be bridged and fixed between the electrodes of the scan electrode group C by scanning the electric field applied to the electrode.
[0104]
Here, the target nucleotide chain added later to the reaction region R is elongated in the same manner as the detection nucleotide chain X under the action of the heterogeneous electric field formed by the common electrodes G and C, and the scanning electrode B It is attracted to the end of the group by dielectrophoresis. The target nucleotide chain attracted to the scan electrode B group is efficiently hybridized with the detection nucleotide chain X ′ fixed to each end of the scan electrode B group without being affected by steric hindrance. To do.
[0105]
In order to perform dielectrophoresis of the target nucleotide chain to the electrode B group, the common electrode GC₁The electric field to be applied may be sequentially scanned such as between the gap and G-Cx, or the electric field may be applied between the electrodes GC at a time with the plurality of electrode C groups having the same potential.
[0106]
FIG. 19 shows a typical example of the end E of the scan electrode with the scan electrodes Cx to Cy as representative examples among all the scan electrodes described above (the same applies to the other scan electrodes D and F). . 19A shows a scan electrode having a rectangular end Ea, FIG. 19B shows a scan electrode having a triangular end Eb, and FIG. 19C shows a scan electrode having an arcuate end Ec. Represents. Electric field lines are concentrated and non-uniform electric field L₂It is also considered that a scan electrode having an arcuate end Ec is particularly suitable because it is also easy to form and to fix the detection nucleotide chain X.
[0107]
The surface of the scanning electrode end E (Ea, Eb, Ec) may be surface-treated so that the end of the detection nucleotide chain X is fixed by a reaction such as a coupling reaction. For example, in the case of a detection surface that has been surface-treated with streptavidin, it is suitable for immobilizing the end of a biotinylated nucleotide chain.
[0108]
Hybridization can be detected by a conventional method, such as a fluorescent dye labeled on the target nucleotide chain Y or a POPO-1 or TOTO-3 that specifically binds between bases of double-stranded nucleotides. The fluorescence intercalator can be irradiated with excitation light, and the resulting fluorescence can be detected using a conventional detector.
[0109]
  More specifically, the reaction region R is excited by irradiating laser light (for example, blue laser light), the magnitude of the fluorescence intensity is detected by a detector (not shown), and the detection nucleotide chain X ′ And the state of hybridization between the target nucleotide chain Y and the target nucleotide chain Y. Finally, the fluorescence intensity for each reaction region R is A / D converted, and the binding reaction rate is calculated.ComputerVisualization is possible by displaying the distribution on the screen.
[0110]
【The invention's effect】
In the present invention, a detection nucleotide chain adjusted to an extended state is aligned and fixed in a reaction region (between scanning electrodes) in a reaction region that provides a hybridization field on a surface portion of a sensor chip such as a DNA chip. Thus, it is possible to reliably achieve improvement in hybridization efficiency, reduction in reaction time, prevention of false positives or false negatives, and the like.
[Brief description of the drawings]
FIG. 1 is a diagram showing a main configuration of a hybridization detection unit (1a) and a sensor chip according to the present invention.
2A is a cross-sectional view taken along the line I-I of the detection unit 1a shown in FIG.
(B) II representing a variation (1′a) of the detection unit (1a) shown in FIG.
FIG. 3 shows a scanning electrode in which a voltage is applied between the counter electrodes A and B and a voltage is applied between the second counter electrodes G and Cx and between G and Cy in the detection unit 1a (or 1′a). Non-uniform electric field L near Cx and Cy₂The figure showing the state that was formed
FIG. 4 is a diagram illustrating a configuration of a main part of a second embodiment (reference numeral 1b) of a detection unit and a sensor chip according to the present invention.
FIG. 5 is a diagram showing a main configuration of a detection unit and a sensor chip according to a third embodiment (reference numeral 1c) according to the present invention.
FIG. 6 is a diagram showing a main configuration of a detection unit and a sensor chip according to a fourth embodiment (reference numeral 1d) according to the present invention.
FIG. 7 is a diagram illustrating voltage application operations (three examples) for the detection unit (1a).
FIG. 8 is a diagram illustrating voltage application operation examples (three examples) corresponding to the detection unit (1b).
FIG. 9 is a diagram illustrating a configuration of main parts of a fifth embodiment (reference numeral 1e) of a detection unit and a sensor chip according to the present invention.
FIG. 10 is a diagram showing the main configuration of a detection unit and a sensor chip according to a sixth embodiment (reference numeral 1f) according to the present invention.
FIG. 11 is a diagram showing a main configuration of a detection unit and a sensor chip according to a seventh embodiment (reference numeral 1g) according to the present invention.
12A is a cross-sectional view taken along line II-II in FIG.
(B) II-II arrow sectional drawing showing the modification (code | symbol 1'e) of a detection part (1e).
FIG. 13A is a diagram illustrating an example of a voltage application operation corresponding to the detection unit (1e).
(B) The figure showing the example of voltage application operation corresponding to a detection part (1f).
(C) The figure showing the voltage application operation example corresponding to a detection part (1g).
FIG. 14 is a diagram illustrating another voltage application operation example.
FIG. 15 is a diagram illustrating a configuration of main parts of an eighth embodiment (reference numeral 1h) of a detection unit and a sensor chip according to the present invention.
FIG. 16 is a diagram illustrating a configuration of main parts of a ninth embodiment (reference numeral 1i) of a detection unit and a sensor chip according to the present invention.
FIG. 17 is a diagram showing the main configuration of the ninth embodiment (reference numeral 1i).
18A is a cross-sectional view taken along the line III-III in FIG. 16 and FIG.
(B) III-III arrow sectional drawing which shows the deformation | transformation form (code | symbol 1'i) of the detection part 1i shown by the front figure (A).
FIG. 19A is a diagram illustrating a rectangular end (Ea) of a scan electrode.
(B) The figure showing the triangular end part (Eb) of a scanning electrode
(C) The figure showing the arcuate end (Ec) of the scanning electrode
[Explanation of symbols]
1 (1a-1i) Hybridization detector
A, B Counter electrode
C (C₁, C₂, C₃, ... Cx, Cy, Cz) Scan electrode (group)
D (D₁, D₂, D₃,... Dx, Dy, Dz) Scan electrode (group)
F (F₁, F₂, F₃, ... Fx, Fy, Fz) Scan electrode (group)
E, e Electrode end
G Common electrode
R reaction zone
S, s, W switch
V power supply
X Nucleotide chain for detection
X ′ immobilized nucleotide chain for detection
Y target nucleotide chain

Claims (19)

A reaction region serving as a hybridization field between the detection nucleotide chain and a target nucleotide chain having a base sequence complementary to the detection nucleotide chain;
A counter electrode for applying an electric field to the detecting nucleotide chain in the reaction region,
Scanning electrodes arranged in the reaction region, each configured to be able to apply a voltage,
By applying a voltage between a pair of adjacent electrodes of the scan electrode, the detection nucleotide chain to which an electric field is applied by the counter electrode is moved to the scan electrode and bridged between the electrodes of the scan electrode. A sensor chip comprising means for fixing in such a manner. The target nucleotide chain to which an electric field is applied by the counter electrode is moved toward the scan electrode by applying a voltage to the scan electrode , and the detection nucleotide chain is fixed between the electrodes of the scan electrode. The sensor chip according to claim 1 , wherein the sensor chip is hybridized with the sensor chip. Edge shape of the scanning electrodes, arc-shaped or claim 1, wherein the sensor chip, characterized in that it comprises a polygonal shape. The counter electrode, according to claim 1, wherein the sensor chip is characterized in that facing arranged in parallel. The counter electrode and the electric field formed by the scan electrodes, according to claim 1, wherein the sensor chip, characterized in that an alternating current. A reaction region serving as a hybridization field between the detection nucleotide chain and a target nucleotide chain having a base sequence complementary to the detection nucleotide chain;
A common electrode disposed in the reaction region;
A plurality of electrodes arranged in parallel, and a scanning electrode,
An electric field is formed by sequentially applying a voltage between the common electrode and each electrode constituting the scanning electrode, and the detection nucleotide chain existing in the reaction region is moved toward the scanning electrode by the electric field. And a sensor chip comprising means for fixing the scanning electrodes so as to be bridged between the electrodes . The sensor chip according to claim 6 , comprising: the common electrode; and two rows of the scanning electrodes arranged so that ends of the electrodes face each other. The sensor chip according to claim 6, wherein the distance between the electrodes facing each other of the scanning electrodes is increased stepwise in the order in which the voltage is applied. By sequentially applying a voltage between the common electrode and the electrodes constituting the scan electrode, the target nucleotide chain existing in the reaction region is moved to the scan electrode and fixed between the electrodes of the scan electrode. The sensor chip according to claim 6 , wherein the sensor chip is hybridized with the nucleotide chain for detection in a formed state. The sensor chip according to claim 6, wherein the end shape of the scanning electrode has an arc shape or a polygonal shape. The sensor chip according to claim 6, wherein an electric field formed by the common electrode and the scanning electrode is an alternating current. A reaction region serving as a hybridization field between the detection nucleotide chain and a target nucleotide chain having a base sequence complementary to the detection nucleotide chain;
A first scan electrode arranged in the reaction region;
A first scan electrode and a second scan electrode arranged so that ends of the electrodes face each other;
An electric field is formed by sequentially applying a voltage between adjacent electrodes of the first scan-through electrode and between adjacent electrodes of the second scan electrode, and a nucleotide chain for detection is applied to the scan electrode by the electric field. A sensor chip comprising means for moving toward and fixed so as to bridge between the electrodes of the scanning electrode. An electric field is formed by sequentially applying a voltage between adjacent electrodes of the first scan electrode and between adjacent electrodes of the second scan electrode, and the target nucleotide chain is transferred to the scan electrode by the electric field. Move towards
The sensor chip according to claim 12 , wherein the sensor chip is hybridized with the detection nucleotide chain fixed between the electrodes of the scanning electrode. The sensor chip according to claim 12, wherein end shapes of the first scan electrode and the second scan electrode have an arc shape or a polygonal shape. The sensor chip according to claim 12, wherein the electric field formed by the first scan electrode or the second scan electrode is alternating current. A reaction region serving as a hybridization field between the detection nucleotide chain and a target nucleotide chain having a base sequence complementary to the detection nucleotide chain;
A common electrode disposed in the reaction region;
A scanning electrode arranged so that the common electrode and the end of each electrode face each other, and
A means for forming an electric field by sequentially applying a voltage between the common electrode and the constituent electrodes of the scan electrode, and moving the detection nucleotide chain toward the scan electrode by the electric field;
Between adjacent electrodes of said scanning electrodes, by sequentially applying the voltages, respectively, a sensor chip provided with means for fixing to bridging the detecting nucleotide chain between the electrodes of the scan electrodes. An electric field is formed by sequentially applying a voltage between the common electrode and the constituent electrodes of the scan electrode, and the target nucleotide chain is moved toward the scan electrode by the electric field, and the scan electrode 17. The sensor chip according to claim 16 , wherein the sensor chip is hybridized with the detection nucleotide chain fixed between the electrodes . The sensor chip according to claim 16, wherein an end shape of the scanning electrode has an arc shape or a polygonal shape. 17. The sensor chip according to claim 16 , wherein an electric field formed between the common electrode and the scan electrode and between the electrodes of the scan electrode is an alternating current.

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