JP4367441B2 - Hybridization method - Google Patents

Description

  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.

  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.

  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.

  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.

  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.

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 solid-phased.
Japanese National Patent Publication No. 4-505863 Japanese National Patent Publication No. 10-503841

  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.

  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.

  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.

  In order to solve the above technical problem, in the present invention, 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 provided. The “hybridization detection unit” and the “sensor chip” including the detection unit are configured to extend the nucleotide chain for detection by an electric field and fix it to a scanning electrode site arranged in the reaction region by the action of dielectrophoresis. And a “hybridization method” that can be carried out using them.

  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.

  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 elongated detection nucleotide chain. 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. .

  By this means or method, hybridization is performed by extending a 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). 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.

  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.

  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.

  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.

  Here, main technical terms in the present invention are defined. 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.

  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.

  “Hybridization” means a complementary strand (duplex) formation reaction between nucleotide strands having a complementary base sequence structure.

  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.

  “Steric hindrance” makes it difficult to access the target molecule due to the presence of bulky substituents in the vicinity of 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.

  “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).

  “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.

  “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. A typical example is a DNA chip. it can.

  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.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings.

<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.

  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.

In this reaction region R, counter electrodes A and B are arranged. The counter electrode A, B are preferably arranged parallel to the, it is connected both to the power source V _1. When the illustrated switch s is turned on and a high frequency voltage is applied by the power source V ₁ , a uniform electric field (an 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 (see FIG. 1 (see the line indicated by L ₁ in FIG. ₁ ).

The electric field conditions of the counter electrodes A and B are preferably about 1 × 10 ⁶ V / m and about 1 MHz (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)). Since all the electric field conditions described below are the same as this, description thereof will be omitted.

By the action of the uniform electric field L _1, 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 elongated in the direction along the uniform electric field L ₁ , It can be a chain (as described above for the principle).

Subsequently, between the counter electrodes A and B, the second counter electrodes G and C (C ₁ , C ₂ , C ₃ ,... Cx, Cy, Cz are formed so as to be orthogonal to the counter electrodes A and B. ) is arranged, which is connected or connectable to the power source V ₂ illustrated respectively.

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, in the scan electrode group C, a voltage is successively applied between a pair of adjacent scan electrodes D by sequentially turning on / off the switches S ₁ , S ₂ , S ₃ ... Sx, Sy, Sz. It has a structure to go. In the conductive state, between the scan electrodes (e.g., G and C _x, C between _y) (the peripheral edge portion of the particular scanning electrode C) to the respective regions of non-uniform electric field L ₂ lines of electric force are concentrated is formed (See FIG. 3).

In addition, the arrangement interval of the electrodes C ₁ , C ₂ ... Cz of the scanning electrode C group is set such that the detection nucleotide chain X ′ is fixed so as to bridge the extended detection nucleotide chain X ′. (Hereinafter, the same applies to all embodiments).

  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).

As shown in FIG. 2 (A), the detection unit 1a and the deformation mode 1′a are formed between substrates M ₁ and M ₂ formed of synthetic resin such as quartz glass, silicon, polycarbonate, and polystyrene. It is provided in a narrow gap. 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.

In some cases, as in the detection unit 1′a shown in FIG. 2B, each electrode A, B, etc. is sandwiched by the dielectric U, thereby widening the distance between the substrates M ₁ and M _2. 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.

Here, FIG. 3 shows that the voltage is applied between the counter electrodes A-B and the voltage between the second counter electrodes G-Cx and G-Cy in the detection unit 1a (or 1′a). There is applied, the scanning electrodes Cx, shows a state where non-uniform electric field L ₂ is formed in the vicinity of Cy. FIG. 3 shows a state in which the target nucleotide chain Y has already been added to the reaction region R, and the electric field (L ₁ ) formed between the counter electrodes AB is omitted and shown. Yes.

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 present in the adjacent scan electrodes C ₁ -C ₂ , C ₂ -C ₃ ... Cx-Cy. Are driven by dielectrophoresis and are fixed in a state where they are bridged between the end portions dd of each electrode D.

A symbol X ′ in FIG. 3 indicates a detection nucleotide chain fixed between adjacent scanning electrodes C (the same applies to other drawings). Incidentally, FIG. 3, switch Sx, Sy is turned on, shows the steps between the electrodes G and Cx-Cy inhomogeneous electric field L ₂ is formed.

  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 part 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.

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.

The detection unit 1b is different from the detection unit 1a (1′a) according to the first embodiment described above, and the electrodes (Cx, Cy, Cz) facing the scanning electrode groups C ₁ , C ₂ , C _3. The electrode corresponding to the symbol G in FIGS. 1 and 3 is not provided. Between adjacent scanning electrodes _{C 1 -C 2, C 2 -C} 3, ··· Cx-Cy, for a ..., based on the procedure of the on / off predetermined switch S group shown, is shown voltage from the power source V ₃ has a configuration in which is sequentially applied a.

In FIG. 4, the counter electrodes A-B, a voltage to the scanning electrodes Cx-Cy is applied, the scanning electrodes Cx-Cy between the non-uniform electric field L ₂ is formed, for detection fixed between the scanning electrodes Cx-Cy A state where the target nucleotide chain Y is attracted toward the nucleotide chain X ′ (see the arrow portion) is illustrated.

<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.

Detector 1c represented in Figure 5, the counter electrodes A, in the region between the B, so as to face the scan electrodes D group to be applied by the scan electrode group C and the power supply V ₄ is applied by the power supply V ₃ It is arranged in. Specifically, the scanning electrodes D ₁ , D ₂ , D ₃ ... Are opposed to the scanning electrodes C ₁ , C ₂ , C _3. Dx, Dy, Dz are arranged.

In FIG. 5, the counter electrodes A-B, the scan electrodes Cx-Cy, is applied to Dx-Dy, target nucleotide chain Y is, by the action of the non-uniform electric field L _2, between the scan electrodes Cx-Cy, inter Dx-Dy The state of being attracted to the detection nucleotide chain X ′ immobilized on each (see the arrow portion) is illustrated.

<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.

Like the detection unit 1c according to the third embodiment, the detection unit 1d faces the scan electrodes C ₁ , C ₂ , C ₃ ... Cx, Cy, Cz arranged in parallel. Scan electrodes D ₁ , D ₂ , D ₃ ... Dx, Dy, Dz are arranged. Scan electrode group C and group D has a structure which is applied by the common power supply V _5.

In FIG. 6, the detection nucleotide chain X ′ to which the target nucleotide chain Y is fixed is applied to the counter electrode AB and the scanning electrodes Cx-Dx, Cy− and Dy, respectively, by the action of the non-uniform electric field L _2. The state of being attracted (see the arrow portion) is illustrated.

  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.

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- C ₁ · C ₂ → G-C ₂ · C ₃ →...

  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.

  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.

The detection unit 1b of 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 (C ₁ −C ₂ → C ₂ −C ₃ →...

  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.

  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.

  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.

  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.

  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.

  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.

<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.

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 (C _{1 to} Cz) is disposed.

Common electrode G is connected to the power supply V _1, each electrode C ₁ ～Cz scan electrode group C arranged in a row are connected to the power supply V ₁ by sequentially going to turn on the switch S ₁ ~Sz It becomes the composition which is done. 9, switch Sy is turned on, a voltage is applied between the electrodes G-Cy, and shows a state in which the peripheral edge portion of the scanning electrodes Cy inhomogeneous electric field L ₂ is formed (step).

When the switches S _{1 to} Sz are switched between the common electrode G and the electrodes C _{1 to} Cz constituting the scanning electrode group C, and the high frequency voltage is sequentially applied, the electrode G and the electrodes C ₁ to Cz are applied. the reaction area R between cz, non-uniform electric field L ₂ is formed.

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, the nucleotide chain for detection that has been extended in the reaction region R by the non-uniform electric field L ₂ formed around the end E of each of the electrodes C _{1 to} Cz to which a voltage is applied. X is dielectrophoresed toward each end E of each of the electrodes C _{1 to} Cz, and the detection nucleotide chain X is between the electrodes (C ₁ -C ₂ , C ₂ -C ₃ ,... Cy-Cz). ) To be bridged.

More specifically, one end of the detection nucleotide chain X extended in the reaction region R is attracted to and adhered to the end E ₁ of the electrode C ₁ by dielectrophoresis by the non-uniform electric field L ₂ , Then, the remaining other end of the nucleotide chain X is attracted to the end E ₂ of the adjacent electrode C ₂ to which a voltage is applied next, and is bonded and fixed. In this way, the detection nucleotide chain X is fixed so as to be bridged between the electrodes (C ₁ -C ₂ , C ₂ -C ₃ ,... Cy-Cz). 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.

<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.

Similar to the detection unit 1e, the detection unit 1f is provided with a common electrode G in the reaction region R. Two rows of scanning electrodes C and D are disposed in the detection unit 1f, and each of the electrodes C ₁ , C ₂ , C ₃ ... Cx, Cy,. Each electrode end E is disposed so as to face each electrode end e of each of the electrodes D ₁ , D ₂ , D ₃ ... Dx, Dy,.

Here, the electrodes C _{1 to} Cz of the scan electrode C group are electrically connected to the power source V ₁ by sequentially turning on the switches S _{1 to} Sz, and the electrodes D _{1 to} Dz of the scan electrode D group are switched on. By sequentially turning on s _{1 to} sz, the power supply V ₁ is electrically connected. That is, when the switches S ₁ and s ₁ are turned on, a voltage is applied between the common electrodes G-C ₁ and D _1, and when the switches S ₂ and s ₂ are turned on, a voltage is applied between the common electrodes G-C ₂ and D _2. Is applied. FIG. 2 shows a state in which the switches Sy and sy are turned on and a voltage is applied between the electrodes G-Cy and Dy.

<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.

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. The electrode ends E of the electrodes D ₁ , D ₂ , D ₃ ... Dx, Dy,... Dz that constitute the scanning electrode D group are the electrodes F ₁ , F ₂ , F that constitute the scanning electrode F group. ₃ ... Are arranged so as to face the respective electrode end portions e of Fx, Fy,.

The electrodes D _{1 to} Dz of the scan electrode D group are electrically connected to the power source V ₁ by sequentially turning on the switches S _{1 to} Sz, and the electrodes F _{1 to} Fz of the scan electrode F group are switched to the switches s ₁ to It is configured to be electrically connected to the power supply V ₁ by sequentially going to turn on sz. That is, when turning on the switch _{S 1} and _{s 1,} a voltage is applied between the common electrode _{G-D 1, F} 1, when turning on the switch s2 and S2, the voltage between the common electrode _{G-D 2, F} 2 is applied Is done. 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.

Here, in the seventh embodiment of FIG. 11, the distance H between the electrodes D ₁ and F ₁ , D ₂ and F ₂ , D ₃ and F ₃ ... Dz and Fz is gradually increased. It is configured. 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.

  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.

As shown in FIG. 12, the detection units 1e and 1'e are arranged in a narrow gap between the substrates M ₁ and M ₂ formed of synthetic resin such as quartz glass, silicon, polycarbonate, polystyrene, or the like. In 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.

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. Thus widening the spacing of the substrate M _1, M _2, 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).

In the detection unit 1e~1g the above configuration, the target nucleotide chain Y, which is added to the reaction region R, the electrodes _{G-C (C 1 ~Cz)} , G-D (D 1 ~Dz), G-F (F 1 Are fixed to the scanning electrodes C group, D group, and F group while being elongated by the action of the electric field in the same manner as the detection nucleotide chain X under the action of the non-uniform electric field L ₂ formed between each of the. Are attracted to the detection (stretched) nucleotide chain X ′ in an activated state, and efficient hybridization proceeds without the influence of steric hindrance.

  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.

  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.

9, as shown in FIG. 13A, voltages are sequentially applied between the electrodes G-C ₁ , the electrodes G-C ₂ ..., And the detection unit 1 f of FIG. in, as shown in FIG. 13 (B), the electrodes _{G-C 1} · _D 1, between the electrodes _{G-C 2 · D 2 ···} , continue to apply a voltage in order, the detection unit 1g of FIG. 11 Then, as shown in FIG. 13C, voltages are sequentially applied between the electrodes GD ₁ , F ₁ , electrodes GD ₂ , F ₂ . In addition, each voltage application time can be determined suitably.

  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.

  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.

  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.

  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.

<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.

  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. .

  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.

Incidentally, FIG. 15 represents a voltage is applied to the scanning electrodes Cx-Cy by the power supply V _4, is a voltage applied to the scanning electrodes Dx-Dy by the power V _5, a state where the non-uniform electric field L ₂ is formed ing.

<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.

First, a reaction region R is provided in the detection unit 1i, and counter electrodes G and C (C ₁ , C ₂ , C ₃ ,... Cx, Cy, Cz) are arranged in the reaction region R. And is configured to be connectable to the illustrated power supplies V ₁ and V ₂ .

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. By sequentially turning on / off the switches S and S ₁ , S ₂ , S ₃ ,... Sx, Sy, Sz and the switches W ₁ , W ₂ , W ₃ ,... Wx, Wy, Wz, the electrode G And the electrodes C ₁ , C ₂ , C ₃ ... Cx, Cy, Cz are successively applied with a voltage.

Further, the scanning electrode C group sequentially turns on / off the switches S ₁ , S ₂ , S ₃ ,... Sx, Sy, Sz and the switches W ₁ , W ₂ , W ₃ ,... Wx, Wy, Wz. By doing so, a voltage is successively applied between a pair of adjacent scanning electrodes C. In the conductive state between the electrode G scan electrode group C, the end of the scan electrode group C inhomogeneous electric field L ₂ is formed (see FIG. 16), between the scanning electrodes constituting the scan electrode group C (e.g., C ₁ and C ₂ ), a non-uniform electric field L ₂ is formed at each scanning electrode end (see FIG. 17).

In the ninth embodiment shown in FIGS. 16 and 17, one end of the detection nucleotide chain X ′ in an extended state is applied to one selected scan electrode (for example, C ₁ as shown in FIG. 16). And, following the procedure, by fixing the other end of the detection nucleotide chain to the adjacent scan electrode (for example, C ₂ ), the adjacent scan electrodes (for example, C ₁ -C ₂₎ And 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.

  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).

As shown in FIG. 18A, the detection units 1i and 1′i are narrow gaps between the substrates M ₁ and M ₂ formed of synthetic resin such as quartz glass, silicon, polycarbonate, and polystyrene. Is provided. 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.

In some cases, as in the detection unit 1′i shown in FIG. 18B, each electrode C group is sandwiched by the dielectric U, thereby increasing the distance between the substrates M ₁ and M ₂ and reacting. The capacity of the region R may be increased. Although not shown, the reaction region R may be arranged in a plurality of rows of scan electrodes C.

Here, FIG. 16 shows a state in which the illustrated switches S and S ₁ are turned on and a high-frequency voltage is applied between the counter electrodes G-C ₁ by the power source V _{1 in the} detection unit 1 i (or 1′i). Represents.

When a high frequency voltage is applied between the electrodes G-C ₁ , a non-uniform electric field L ₂ is formed in the reaction region R. Due to the action of the non-uniform electric field L _2, the detection nucleotide chains X that are randomly dispersed in the reaction region R are elongated in the direction along the non-uniform electric field L ₂ , can do. Further, the detection nucleotide chain X is driven by dielectrophoresis in the non-uniform electric field L ₂ , and one end thereof is fixed in a state of extending to the end portion of the electrode C _{1 having} high electric field strength.

Then by turning off the switch S, turn off the high-frequency voltage applied between the electrodes G-C _1. Simultaneously or subsequently thereto, by turning on the switch _{S 2} and _W 2 _~Wz, a high frequency voltage is applied between the electrodes _C 1 -C ₂ by the power supply _{V 2.} The other end of which one end to the C ₁ is not fixed in the nucleotide chain X 'for detection is fixed, moves in a direction along the electric force lines of the non-uniform electric field generated between the electrodes C ₁ -C _2, the electric field It is fixed to the end of the larger electrode C ₂ intensity.

Subsequently, the switches S ₂ and W ₁ are turned on, the S ₁ and W ₂ are turned off, and similarly, one end of the detection nucleotide chain X is fixed to the end of the scanning electrode C ₂ , and then the switches S and W ₂ are turned off and the switch S ₃ and W ₁ and W _{3 to} Wz are turned on, and the detection nucleotide X ′ in an extended state is fixed between the scan electrodes C _{2 to} C ₃ .

  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.

  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.

  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.

In order to the dielectrophoretic to the target nucleotide chain electrode group B, to the electric field may be sequentially scanned to be applied as between the common electrode G-C ₁ between ~G-Cx, a plurality of electrodes C group May be applied at the same potential, and an electric field may be applied between the electrodes GC at once.

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. Easy to form a non-uniform electric field L ₂ by concentrating the electric flux lines, and the detecting nucleotide chain X because it is also a fixed easily shaped, end portion Ec is arcuate scan electrode be particularly suitable.

  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.

  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.

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 ratio can be visualized by being distributed on a computer screen.

The figure showing the principal part structure of the hybridization detection part (1a) and sensor chip based on this invention. (A) II-I arrow sectional drawing of the detection part (1a) represented by FIG. (B) The II sectional view taken on the line of the arrow which represents the deformation | transformation form (1'a) of the detection part (1a) shown by the (A) figure. In the detection unit 1a (or 1'a), 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 the G and Cy, and the vicinity of the scan electrodes Cx and Cy. diagram illustrating a state in which non-uniform electric field L ₂ is formed. The figure showing the principal part structure of 2nd Embodiment (code | symbol 1b) of the detection part and sensor chip which concern on this invention. The figure showing the principal part structure of 3rd Embodiment (code | symbol 1c) of the detection part and sensor chip which concern on this invention. The figure showing the principal part structure of 4th Embodiment (code | symbol 1d) of the detection part and sensor chip which concern on this invention. The figure showing voltage application operation (3 examples) with respect to a detection part (1a). The figure showing the voltage application operation example (3 examples) corresponding to a detection part (1b). The figure showing the principal part structure of 5th Embodiment (code | symbol 1e) of the detection part and sensor chip which concern on this invention. The figure showing the principal part structure of 6th Embodiment (code | symbol 1f) of the detection part and sensor chip which concern on this invention. The figure showing the principal part structure of 7th Embodiment (code | symbol 1g) of the detection part and sensor chip which concern on this invention. (A) 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). (A) The figure showing the voltage application operation example corresponding to a detection part (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). The figure showing the other example of voltage application operation. The figure showing the principal part structure of 8th Embodiment (code | symbol 1h) of the detection part and sensor chip which concern on this invention. The figure showing the principal part structure of 9th Embodiment (code | symbol 1i) of the detection part and sensor chip which concern on this invention. The figure showing the principal part structure of the 9th embodiment (code | symbol 1i). (A) III-III arrow sectional drawing in FIG. 16, FIG. (B) III-III arrow directional cross-sectional view which shows the deformation | transformation form (code | symbol 1'i) of the detection part 1i shown by the front figure (A). (A) The figure showing the rectangular-shaped edge part (Ea) of a scanning electrode. (B) The figure showing the triangular-shaped edge part (Eb) of a scanning electrode. (C) The figure showing the circular-arc-shaped edge part (Ec) of a scanning electrode.

Explanation of symbols

1 (1a-1i) hybridization detection unit A, B counter electrode _{C (C 1, C 2,} C 3, ··· Cx, Cy, Cz) scan electrode (s)
D (D ₁ , D ₂ , D ₃ ,... Dx, Dy, Dz) Scan electrode (group)
F (F ₁ , F ₂ , F ₃ ,... Fx, Fy, Fz) Scan electrode (group)
E, e electrode end G common electrode R reaction region S, s, W switch V power supply X detection nucleotide chain X ′ immobilized detection nucleotide chain Y target nucleotide chain

Claims (3)

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;
Using a sensor chip including a hybridization detection unit including at least a scanning electrode in which a plurality of electrodes are arranged in parallel in the reaction region,
By sequentially applying a voltage between each electrode constituting the common electrode and the scanning electrode ,
A procedure for fixing one end of the detection nucleotide chain to one selected electrode of the scanning electrodes;
Following this procedure, among the scanning electrodes, the other end of the detection nucleotide chain is fixed to the electrode adjacent to the electrode, thereby fixing the detection nucleotide chain between adjacent scanning electrodes. hybridization method for performing a procedure, the. The hybridization method according to claim 1, further comprising a step of extending the detection nucleotide chain in the reaction region by an alternating electric field . By applying a voltage to the scan electrode after the detection nucleotide chain is fixed, the target nucleotide chain is moved toward the scan electrode and hybridized with the detection nucleotide chain fixed to the scan electrode. The hybridization method according to claim 2, further comprising

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