JP2003344398A - Method of bioassay and device for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To heighten the efficiency of mutual reaction by controlling the formation of an electric field in a reaction domain wherein the mutual reactions between substances such as hybridization are performed. <P>SOLUTION: A method of bioassay is that a detection part 1 (10) provided with: at least a detecting surface S (S') finished so as to fix a detection substance D thereon; a reaction domain R (R') which provides a mutual reaction field between the substance D for detection in a state fixed to the detection surface S (S') and a target substance T added thereto; an electric field forming means E forming the electric field in the reaction domain R (R'), by generating an electric potential difference therein; and at least a process turning on/off the electric field with a prescribed timing by the electric field impressing means E is included. A device for bioassay capable of preferably performing the method of bioassay is provided. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a bioassay method and a bioassay substrate which are particularly useful in the field of bioinformatics.

[0002]

[Prior art]

The main prior art of the present invention will be described below.
Currently, an integrated substrate for bioassay called a so-called DNA chip or a DNA microarray (hereinafter collectively referred to as “DNA chip”) in which predetermined DNAs are finely arrayed by the microarray technology is used for gene mutation analysis, S
It is used for NPs (single nucleotide polymorphism) analysis, gene expression frequency analysis, and the like, and has begun to be widely used in the fields of drug discovery, clinical diagnosis, pharmacogenomics, forensics, and others.

This DNA chip has various types and many kinds of DNA oligo chains and cDNA (co
It is characterized by the fact that it is possible to comprehensively analyze intermolecular interactions such as hybridization because of the accumulation of complementary DNA).

Briefly explaining an example of an analysis method using a DNA chip, a DNA probe immobilized on a glass substrate or a silicon substrate is subjected to reverse transcription PCR reaction of mRNA extracted from cells, tissues or the like. This is a method of performing PCR amplification while incorporating a fluorescent probe dNTP, performing hybridization on the substrate, and performing fluorescence measurement with a predetermined detector.

Here, DNA chips can be classified into two types. The first type is a method of directly synthesizing an oligonucleotide on a predetermined substrate by using a photolithography technique to which a semiconductor exposure technique is applied, and is typically produced by Affymetrix. (See, for example, Japanese Patent Publication No. 4-505763). Although this type of chip has a high degree of integration, it has a limit in DNA synthesis on a substrate and has a length of about several tens of bases. The second type is also called the "Stanford method", and is prepared by dispensing and immobilizing DNA prepared in advance on a substrate using a tip pin ( For example, Japanese Patent No. 3272365
(See Japanese Patent Publication). Although this type of chip has a lower degree of integration than the former, it has the advantage that it can immobilize a DNA fragment of about 1 kb.

[0007] At present, a predetermined detection substance is immobilized on a minute detection surface portion provided on a thin flat plate, which is called a biosensor chip including a protein chip. A biosensor technology is in progress to observe and analyze the mutual reaction of both substances by using the surface plasmon resonance principle and the principle of a crystal oscillator, etc., by passing a small amount of a solution containing the target substance, antibody-antigen reaction and hormone. It has become a useful technique in the analysis of interaction reactions between substances such as response reactions.

[0008]

However, in the above-mentioned conventional DNA chip technology and biosensor technology,
In the two-dimensional narrow detection part of the substrate, nucleotide chains for detection such as DNA probes and proteins are immobilized (immobilized) to promote mutual reaction between substances such as hybridization reaction and light source antibody reaction. Because of the technique of performing the reaction, the reaction product is limited in freedom under the reaction condition that the degree of freedom of the reaction product is spatially limited, and steric hindrance may occur during the reaction. The structure was such that the mutual reaction proceeds based on the Brownian motion of the product. Therefore, the conventional DNA chip technology or biosensor technology has a technical problem that the efficiency of interaction is low and the reaction time is long.

Further, in the existing DNA chip or the like, the sample solution is merely dropped on a predetermined spot portion (detection section) on the substrate, and the target substance contained in the sample solution and the spot are included. No measures have been taken to perform relative positioning with respect to the detection substance fixed to the site.

Therefore, in the present invention, formation of an electric field in a reaction region where interactions such as hybridization occur between substances is controlled, and relative positioning between the substance for detection and the target substance and adjustment of the substance structure are performed. The main object of the present invention is to provide a bioassay method and a bioassay apparatus capable of increasing the efficiency of interaction by carrying out.

[0011]

In order to solve the above technical problems, the present invention firstly provides the following "bioassay method". In the present application, the term “bioassay” broadly means biochemical analysis based on hybridization or other mutual reaction between substances.

The "bioassay method" according to the present invention is
A detection surface that has been surface-treated so that the detection substance can be immobilized, a reaction region that provides a field of interaction between the detection substance immobilized on the detection surface and the target substance, and the reaction region An electric field forming means for forming an electric field in the reaction region by generating a potential difference therein, and a method premised on a method of detecting an interaction reaction between substances in a detection part, the electric field applying means Is a method including at least a procedure of turning on / off the electric field formation by a predetermined timing.

The electric field formed in the reaction region is mainly
(1) a substance for detection such as a nucleotide chain, an action of elongating the target substance, (2) an action of moving the target substance existing apart from the substance for detection in the front-back direction along the lines of electric force,
(3) The higher-order structure of the reaction product (the conjugate of the detection substance and the target substance) obtained by the mutual reaction of the detection substance and the target substance is specific to the fluorescently labeled target substance and the reaction product. It exhibits the action of arranging into a structure capable of more accurately reading the fluorescence emitted from the fluorescence intercalator bound to the.

That is, by forming a desired electric field (applying on) or not forming an electric field (applying off) at a suitable predetermined timing with respect to the reaction region of the detection portion, the above (1) ) To (3) can be sequentially exerted.

More specifically, by applying the high frequency and high voltage, the above-mentioned action (1) is obtained to facilitate the reaction between the substance for detection and the target substance, for example, a straight chain in which the nucleotide sequences of nucleotide chains are not overlaid. It can be arranged in a striped structure (extended structure).

Subsequently, by applying a rectangular wave voltage (pulse-shaped DC voltage), more specifically, by alternately applying a rectangular wave voltage (that is, +/-), the above (2) Upon obtaining the action, the target substance existing in the reaction region in a free state is brought close to the detection substance immobilized on the detection surface. As a result, the reaction efficiency (reaction machine) is increased, and the reaction time can be shortened.

Also in the detection stage, by applying a high frequency and high voltage to the reaction region, the action of (3) is obtained, and fluorescence or the like from the reaction product present in the reaction region can be detected by optical means or the like. Can be accurately detected by the detecting means.

Here, in order to surely proceed the mutual reaction such as the hybridization of the detection substance and the target substance, it is considered preferable to form the reaction region in which the Brownian motion of the substance can be exclusively committed. Therefore, in the present invention, in a series of electric field forming procedures, a time during which no electric field is formed in the reaction region is positively provided. That is, by providing the rectangular wave voltage OFF time after the rectangular wave voltage ON, mutual reaction between substances can be promoted.

Here, in the "bioassay method" of the present invention, the types of the substance for detection and the target substance are not particularly limited. In addition, the “target substance” includes both those labeled with a fluorescent label and other labels and those not labeled. The “detection substance” is a small molecule, a high molecule, which is directly immobilized on the detection surface or indirectly immobilized via a linker and exhibits a specific interaction with a target substance labeled with a fluorescent substance or the like. It includes a wide range of molecules and biological substances, and is not interpreted narrowly.

The "detection surface" means a surface portion which has been subjected to a suitable surface treatment capable of immobilizing the ends of nucleotide chains and the like. For example, when the surface is treated with streptavidin, the detection surface is suitable for immobilizing a biotinylated nucleotide chain.

The present method is within a range consistent with the objects and effects of the present invention, and is protein-protein, nucleotide chain-nucleotide chain (DNA-DNA, DNA-R).
Including both NA. ), Protein-nucleotide chains (including double strands) and other interactions between polymers, interactions between polymers and low molecules, and interactions between low molecules.

As an example, when the substance for detection and the target substance are nucleotide chains and the mutual reaction is hybridization, the hybridization efficiency can be ensured even in a narrow reaction region (spot region). Therefore, it is possible to newly provide a DNA chip or microarray method which has a short reaction time and high reading accuracy.

Here, as a preferred bioassay method in the case where the interaction between substances in the reaction region is hybridization, (a) the detection surface is extended with a nucleotide chain for detection in which the terminal site is fixed. So as to form an electric field, (b) a step of adding a target nucleotide chain to the reaction region after the above procedure, and (c) a potential difference in the reaction region after the above procedure to generate a potential difference in the reaction region. And (d) a step of relatively moving the detection nucleotide chain and the target nucleotide chain of
And a step of carrying out hybridization with the application turned off after the above step. Below (a)
Each step of (d) to (d) will be specifically described.

Here, in the present application, "nucleotide chain"
The term, means a polymer of a nucleoside phosphate ester in which a purine or pyrimidine base and a sugar are glycosidically linked, and an oligonucleotide including a DNA probe, a polynucleotide, a DNA in which a purine nucleotide and a pyrimidine nucleothiode are polymerized (full length or a fragment thereof ), CDNA (cDNA probe) obtained by reverse transcription, R
Widely includes NA, etc.

Since the detection nucleotide chain (single strand) whose terminal site is to be immobilized on the detection surface does not necessarily have a linear conformation, the detection nucleotide chain is detected by the procedure (a) above. The nucleotide chain is linearly extended along the line of electric force. This exposes the base sequence of the detection nucleotide chain to the reaction region,
It is possible to form a state in which a hydrogen bonding reaction with a complementary base sequence easily proceeds.

More specifically, a nucleotide chain composed of a large number of polarization vectors composed of a negative charge of a nucleotide chain having a phosphate ion and a positive charge of an ionized hydrogen atom is elongated by application of an electric field, and therefore, The bases do not overlap with each other, and as a result, steric hindrance is eliminated, and the hybridization reaction with the nearby target nucleotide can be smoothly performed.

The principle of elongation or movement of a nucleotide chain will be described in detail. An ion cloud is formed by a phosphate ion (negative charge) forming the skeleton of the nucleotide chain and a hydrogen atom (positive charge) around which water is ionized. It is considered that the polarization vector (dipole) generated by these negative and positive charges is directed in one direction as a whole by the application of high frequency and high voltage, and as a result, the nucleotide chain is elongated. In addition, when an inhomogeneous electric field in which electric lines of force are concentrated is applied, the nucleotide chains move toward the site where electric lines of force are concentrated (Seiichi Suzuki,
Takeshi Yamanashi, Shin-ichi Tazawa, Osamu Kurosaw
a and Masao Washizu: ”Quantitative analysis on el
ectrostatic orientation of DNA in stationary AC el
ectric field using fluorescence anisotropy ”, IEEE
Transaction on Industrial Applications, Vol.34, No.
1, P75-83 (1998)).

Subsequently, after the step (a), a sample solution containing the target nucleotide chain in the liquid phase of the reaction region is kept in the liquid phase of the reaction region while the voltage application is kept on or the voltage application is once turned off. Perform the adding (b) procedure.

Following this step (b), an electric field is formed in the reaction region to relatively move the detection nucleotide chain and the target nucleotide chain in the reaction region, and the above step (c) is carried out. It forms a reaction environment in which hybridization easily proceeds. That is, according to the procedure (c), by forming an electric field in the reaction region, it is possible to remarkably increase the reaction opportunity between both nucleotide chains, which has been conventionally solely based on Brownian motion. And the detection accuracy can be improved.

Next, in the step (d), after the step (c) is completed, the application is turned off, and hybridization based on Brownian motion is performed. The reason why the application is turned off in the step (d) is that the hybridization is a reaction that is exclusively subject to hydrogen bonds between complementary base pairs.

Next, the present application provides a "bioassay device" having the following configuration, as a tool capable of suitably carrying out the above bioassay method.

That is, the bioassay device according to the present invention comprises a detection surface which is surface-treated so that the detection substance can be immobilized, and the detection substance and the target substance which are immobilized on the detection surface. An electric field can be formed in the reaction region by generating a potential difference in the reaction region, which provides a field for interaction reaction, and the electric field formation can be turned on / off at a predetermined timing. The electric field forming means described above is used.

The above-mentioned "detection section" has the above-mentioned three essential components, that is, the detection surface, the reaction region, and the electric field forming means, and the other configurations and structures are not particularly limited. For example, a minute cell structure, a channel structure, or the like partitioned from the periphery may be formed on the substrate, and the above-mentioned essential configuration may be provided in these structural portions to serve as the detection unit. The number of detection units on the substrate can be determined according to the purpose and application, and the form of the substrate can be appropriately selected and determined from rectangular, disk-shaped, and other flat plate-shaped forms.

As described above, the present invention has the technical significance of providing a novel technique related to a DNA chip or a biosensor chip.

[0035]

Preferred embodiments of the present invention will be described below with reference to the accompanying drawings.

FIGS. 1A to 1F are flow charts for explaining a preferred procedure of the bioassay method according to the present invention and a preferred embodiment of the bioassay device according to the present invention, and FIG. FIG. 7 is a waveform diagram for explaining an example of an on / off procedure of voltage application performed in the method or the apparatus.

In the following, the present invention will be described by way of a representative example in which both the substance for detection and the target substance are single-stranded nucleotide chains and the mutual reaction action is hybridization. Based on this explanation, the detection substance and the target substance are not limited to nucleotide chains, and the interaction reaction which is the subject of the present invention is not limited to hybridization.

First, in the bioassay method or apparatus according to the present invention, a detection surface S which is surface-treated so that the substance for detection shown by reference symbol D in FIG. 1 can be immobilized, and the detection surface S. Reaction region R that provides a field of interaction between the detection substance D fixed in the state and the added target substance T
And the detection unit 1 including at least a positive electrode E ₁ and a negative electrode E ₂ that function as an electric field forming means E that generates a potential difference in the reaction region R and generates a potential difference in the reaction region R. It is assumed that. Note that reference numeral 2
Represents a part of the substrate provided with the detection unit 1, and reference numeral V represents a power supply for energizing the positive electrode E ₁ and the negative electrode E ₂ .

FIG. 1A shows that, with respect to the detection surface S,
The state in which the terminal portion of the single-stranded detection nucleotide chain (for example, DNA probe) D ₁ is fixed is briefly shown. In this state, the higher-order structure of the detection nucleotide chain D ₁ is often not linear, but has a multilayered structure in which the chains are entangled as shown in FIG. 1 (A), for example. For this reason, not all the base sequences are exposed in the reaction region R, and it is considered that efficient hybridization with the target nucleotide chain T added later to the reaction region R is difficult to obtain. .

Therefore, in the present invention, in the liquid phase (salt solution or the like) of the reaction region R between the positive electrode E ₁ and the negative electrode E ₂ ,
A high frequency high voltage is applied to generate a potential difference in the reaction region R, and an electric field (electric field) is formed. This electric field exerts an action of extending the detection nucleotide chain D ₁ linearly (linearly) along the electric field. As a result, the base sequence of the detection nucleotide chain D ₁ is exposed in the liquid phase (see FIG. 1 (B)).

Next, the target substance T (target nucleotide chain T ₁ ) is accurately applied to the reaction region R from the nozzle 3 having a predetermined structure with the application of the high frequency high voltage once turned off or turned on.
A sample solution containing is added. Target nucleotide chain T
₁ is not necessarily linear at the beginning of the addition as shown in FIG. 1 (C), but in the liquid phase to which a high frequency voltage is applied, as shown in FIG. 1 (D), It becomes a linearly extended state (indicated by symbol T ₂ ), and it becomes a state in which it is easy to form a complementary chain with the detection nucleotide chain D _{2 which} is also linearly formed.

Then, the application of the rectangular wave voltage is turned on / off a few times to obtain both nucleotide chains D ₂ and T ₂
Are moved stepwise, the target nucleotide chain is moved back and forth, and the timing of the reaction is adjusted.

The reason why the application of the rectangular wave voltage is turned off is that the complementary strand forming reaction between the linear target nucleotide chain T ₂ and the linear detection nucleotide chain D ₂ , that is, the hybridization is performed exclusively by the Brownian reaction. This is because it is entrusted to exercise to proceed. In addition, FIG. 1 (E) simply shows a reaction product (D ₂ + T ₂ ) in a state where the complementary base sequence portions of both chains D ₂ and T ₂ are hydrogen-bonded to form a double-stranded chain. ing.

In the present invention, in addition to the purpose of detecting the target nucleotide chain T ₁ (T ₂ ) having a base sequence complementary to the detecting nucleotide chain D ₁ (D ₂ ), the above procedure is used. , The target double-stranded nucleotide chain (DN
A), a method for detecting an interaction between a specific protein such as a transcription factor and a specific response sequence part in the double-stranded nucleotide chain (DNA), and a search for an endocrine disrupting substance using this method , May be applied to certification, etc.

FIG. 1 (F) is based on the optical means 4,
Labeled (eg, fluorescently labeled) target nucleotide chain T
₂ represents a procedure for detecting and reading ₂ , and the labeling method is not narrowly limited in the present invention. For example, a fluorescent intercalator may be used. The fluorescent intercalator comprises a detection nucleotide chain D and a target nucleotide chain T.
It is incorporated into the hybridized double-stranded nucleotide chain so that it is inserted into a hydrogen bond between bases of and. As a result, the fluorescence wavelength shifts to the longer wavelength side, and quantitative detection becomes possible based on the correlation between the fluorescence intensity and the amount of the fluorescent intercalator incorporated into the double-stranded DNA. As the fluorescent dye used in the fluorescent intercalator, POPO-1 and TOTO-3 are conceivable.

In the detection step, by applying a high-frequency high voltage, the higher-order structure of the reaction product (double-stranded nucleotide chain) formed in the reaction region R is adjusted to a structure not present in the overlay, and It can be accurately detected by the automatic means 4.

Next, an embodiment of the application on / off procedure will be described with reference to FIG. In addition, this embodiment corresponds to the procedure after FIG.

<Step a> By placing the nucleotide chain under an electric field formed by applying a high frequency and high voltage, induced polarization of the nucleotide chain is achieved, thereby extending the nucleotide chain. Therefore, in this step a, by applying a high frequency and high voltage, the target nucleotide chain T ₁ is extended, and the detection nucleotide chain D ₁ is similarly extracted.
₂ is also extended (see the state shown in FIG. 1 (C) and FIG. 1 (D)).

The high frequency high voltage is 1 × 10 ⁶ V
/ M, about 1MHz is considered to be a suitable condition (Masao
Washizu and Osamu Kurosawa: ”Electrostatic Manipul
ation of DNA in Microfabricated Structures ”, IEEE
Transaction on Industrial Application Vol.26, No.2
6, p.1165-1172 (1990)).

<Step b> Both nucleotide chains D ₂ , T
_{When 2} seems to move relatively and approach, the voltage application is turned off, and the hybridization proceeds based solely on Brownian motion (see the state shown in FIG. 1E).

<Step c> By turning on the rectangular wave voltage in the + direction and moving the target nucleotide chain T ₂ which is still in an inaccessible state by Coulomb force,
The position is set to be suitable for hybridization with the detection nucleotide chain D ₂ (from the state of FIG. 1D to the state of FIG. 1E).
See the process of transitioning to).

<Step d> The voltage application is turned off again,
Hybridization proceeds exclusively based on Brownian motion (see the state in FIG. 1E).

<Step e> Next, the application of the rectangular wave voltage to the − direction is turned on to move the target nucleotide chain T ₂ which is still in an unapproximated state (from the state of FIG. 1D to FIG. See the process of transition to the state of E)).

<Step f> The voltage application is turned off again,
Hybridization proceeds exclusively based on Brownian motion (see the state shown in FIG. 1 (E)).

<Step g> The application of the high rectangular wave voltage to the + direction is turned on again to move the target nucleotide chain T ₂ which is still in an inaccessible state (from the state of FIG. 1D to the state of FIG. 1E). ) See the process of transitioning to the state).

<Step h> The voltage application is turned off again,
Hybridization proceeds exclusively based on Brownian motion (see the state shown in FIG. 1 (E)).

<Step i> The application of the rectangular wave voltage to the − direction is turned on again to move the target nucleotide chain T ₂ which is still in the inaccessible state (from the state of FIG. 1D to the state of FIG. 1E). See the process of transitioning to).

<Step j> The voltage application is turned off again,
Hybridization proceeds exclusively based on Brownian motion (see the state shown in FIG. 1 (E)).

<Step k> Double-stranded nucleotide chain (D ₂ + T) which is a reaction product of hybridization.
₂ ) is extended under high frequency and high voltage and subjected to fluorescence reading (see FIG. 1 (F)).

In the procedure for turning on / off the application of the rectangular wave voltage or the like described above, a suitable procedure and timing can be selected and determined according to the type of reaction product to be handled. Not limited. Further, the frequency and the magnitude of the voltage can be appropriately determined according to the purpose.

Next, the bioassay substrate that can be used in the present invention and the bioassay device related to the substrate will be described.

FIG. 3 is an external perspective view of a bioassay substrate on which a large number of detection units of a predetermined structure are arranged, and FIG. 4 is an enlarged external view of a preferred embodiment of the “detection unit” provided on the substrate. Is. The substrate that can be used in the present invention is not limited to the illustrated form.

First, the bioassay substrate 2 (hereinafter abbreviated as “substrate 2”) shown in FIG.
It is formed of a base material used for a disk substrate (disk) used for an optical information recording medium such as D.

The base material is made of quartz glass, silicon, polycarbonate, polystyrene, or other disk-shaped synthetic resin, preferably injection-moldable synthetic resin, in the disk shape. By using an inexpensive synthetic resin substrate, it is possible to realize a low running cost as compared with the conventionally used glass chips. In the center of the substrate 2, a hole (not shown) for fixing a spindle, which is used when the substrate is rotated, may be formed.

On one surface of the substrate 2, an aluminum vapor deposition layer having a thickness of about 40 nm, which is a reflection film, is formed.
The layer functions as a reflective film. This reflective film has a surface reflection of 4% or more from a single substrate having a refractive index of 1.5 or more. On the upper layer of this reflective film, a light transmission layer made of transparent glass, transparent resin, or the like is formed. When the base material is a material having a high reflectance, the surface of the base material itself functions as a reflecting surface, and thus the reflective film may not be formed. If a high reflectance film such as a metal film is formed, the fluorescence intensity of the fluorescently labeled target substance can be detected with high sensitivity.

A large number of detecting portions 1 shown in an enlarged scale in FIG. 4 are arranged at predetermined locations on the light transmitting layer. In the following description of the detection unit 1, the case where both the detection substance and the target substance are single-stranded nucleotide chains is taken as a representative example. However, for the purpose of limiting the target reaction substance of the detection unit 1 to this. Absent.

Here, in the detection part 1, a detection surface S which has been surface-treated so that the end portion of the nucleotide chain for detection indicated by the symbol D can be fixed, and the above-mentioned fixed surface S previously fixed on the detection surface S. A reaction region R serving as a field for a hybridization reaction between the positive electrode E _{1 and the} negative electrode E ₂ for forming an electric field for extending the detection nucleotide chain D and the detection nucleotide chain D and the target nucleotide chain T. It has and. Here, the reaction region R is a cell having a rectangular shape as viewed from above, which opens upward, for example, a depth of 1 μm and a length of 100.
Although it is formed as a cell having a width of 50 μm and a width of 50 μm, it is not limited to the illustrated shape and size.

The detection surface S is a reaction region R on the negative electrode E2 side.
It is formed on the inner wall surface portion facing the side. The detection surface S is surface-treated so that the end of the detection nucleotide chain D is fixed by a chemical bond such as a coupling reaction. That is, the detection surface S is not particularly limited as long as it has been subjected to a surface treatment suitable for immobilizing the pre-processed end portion of the detection nucleotide chain D such as a DNA probe. As an example, the detection surface S surface-treated with streptavidin is suitable for immobilizing a biotinylated nucleotide chain end.

Now, a preferred method for immobilizing the detection nucleotide chain on the detection surface S will be described. By applying a non-uniform electric field to the reaction region R, it was obtained by the action of the electric field (reaction region). (R is free)
The nucleotide chain for detection moves toward the detection surface S portion where the lines of electric force are concentrated, and the end portion thereof collides with the detection surface S. As a result, the nucleotide chain can be reliably immobilized on the detection surface S. To perform this method favorably, the negative electrode E ₂ is comb-shaped as shown in FIG. The symbol V in FIG. 4 is a power supply for energizing the positive electrode E ₁ and the negative electrode E _2.

Here, reference numeral 4 in FIG. 4 indicates the tip of the nozzle for dropping the sample solution 5. The nozzle 4 places the sample solution 5 containing the detecting nucleotide chain D and the sample solution 5 ′ containing the target nucleotide chain T on the reaction region R position based on the position information and the rotation synchronization information provided from the substrate 2. It is configured to accurately follow and drop.

As the dropping means, an inkjet printing method can be preferably adopted. The reason is that it is possible to accurately follow the predetermined reaction region R and precisely drop the minute droplets (the same applies to the substrate 20 described later).

The "ink jet printing method" is
It is a method of applying a nozzle used in an inkjet printer, and is a method of ejecting and fixing a detection substance from a printer head onto a substrate using electricity, like an inkjet printer. This method includes a piezoelectric inkjet method, a bubble jet (registered trademark) (registered trademark) (registered trademark) (registered trademark) method, and an ultrasonic jet method.

The piezoelectric ink jet method is a method in which a droplet is ejected by a displacement pressure generated by applying a pulse to a piezoelectric body. Bubble Jet (registered trademark)
The (registered trademark) (registered trademark) (registered trademark) method is a thermal method in which droplets are ejected by the pressure of bubbles generated by heating a heater in a nozzle. A silicon substrate, which serves as a heater, is embedded in the nozzle, and uniform bubbles are created by controlling at about 300 ° C./s to extrude droplets. However, since the liquid is exposed to high temperatures, caution is required when using it for biological material samples. The ultrasonic jet method is a method in which an ultrasonic beam is applied to the free surface of a liquid and a high pressure is locally applied to eject a droplet from that position. Droplets with a diameter of about 1 μm can be formed at high speed without the need for nozzles.

In the present invention, the "piezoelectric ink jetting method" can be preferably adopted as the "ink jet printing method". By changing the shape of the applied pulse, it is possible to control the size of the droplet (microdroplet), which is suitable for improving the analysis accuracy. When the radius of curvature of the droplet surface is small, the droplet can be made small, and when the radius of curvature of the droplet is large, the droplet can be made large.
It is also possible to reduce the radius of curvature by pulling the droplet surface inward by abruptly changing the pulse in the negative direction.

Here, most of the detection nucleotide chains D dropped into the reaction region R are fixed on the detection surface S in a state in which bases are layered (see FIG. 1 (A)). During the hybridization with the dropped target nucleotide chain T, a problem such as steric hindrance occurs and the reaction efficiency is not good.

Therefore, based on the bioassay method according to the present invention, the positive and negative electrodes E ₁ and E arranged in the detection unit 1 are arranged.
₂ is energized to generate a potential difference in the reaction region R, thereby forming an electric field in the liquid phase (salt solution) stored in the reaction region R, and detecting nucleotide chain D along the direction of the electric field of the electric field. Also, the target nucleotide chain can be extended linearly (linearly).

The hydrogen bond (complementary bond) between the bases of the linear detection nucleotide chain D and the target nucleotide chain T labeled with, for example, a fluorescent dye reduces steric hindrance, etc. It becomes easy for the nucleotide sequences having a difference to approach each other, and the sequence proceeds efficiently.

That is, it is possible to obtain the result that the hybridization reaction between the detection nucleotide chain D and the target nucleotide chain T proceeds efficiently. As a result, the reaction time of hybridization is shortened and
The preferred result is that the probability of false positives or false negatives on reading is also reduced.

To read the substrate information, the substrate 2 is irradiated with laser light (for example, blue laser light) to excite each reaction region R, and the intensity of fluorescence is detected by a detector (not shown). The state of binding reaction between the detecting nucleotide chain D and the labeled target nucleotide chain is detected. Finally, the fluorescence intensity for each reaction region R is calculated by A / D
It is visualized by converting and displaying the binding reaction ratio distributed on the screen of the computer C (the same applies to the substrate 20 described later).

Next, with reference to FIG. 5, a second embodiment of the substrate that can be used in the present invention will be described. Figure 5
(A) is a plan view of the substrate 20 according to the second embodiment as viewed from above,
FIG. 5 (B) is an enlarged plan view of the X portion in FIG. 5 (A).

The substrate designated by the reference numeral 20 in FIG. 5 is provided with the groove detecting portion 10. In this detection unit 10, a reaction region R ′ is formed in the shape of a groove extending radially on a disk-shaped substrate, and the inner wall surface Y on one side in the longitudinal direction forming the groove is detected by the detection unit 1. Detecting surface S ′ having the same structure as the surface S
Are arranged at predetermined intervals. In addition, positive and negative electrodes E ₃ and E ₄ are provided opposite to each other on each part where the detection surface S ′ is formed so as to sandwich the reaction region R ′ (see FIG. 5B).
The groove detection unit 10 can also be said to be a configuration in which the cell-shaped detection units 1 are arranged in the groove 21.

Positive electrode E_Three, E_Three..Use as common electrodes
And likewise the electrode E_Four, E _FourAlso ...
Can be used as a common electrode. That is, they are opposed to each other with the reaction region R ′ in between.
So that the common electrode E_ThreeAnd common electrode E_FourJuxtaposing
You can In this configuration, the needle-shaped probe is connected to the common electrode E.
_ThreeAnd common electrode E_FourBy pressing on the
Can be energized.

The reaction region R'may be formed in pits (not shown) arranged in each groove 21. By dropping a minute droplet in the reaction area in the pit, almost the same spot size can be realized, and fluorescence intensity detection with good reproducibility can be realized.

Further, when the groove detecting section 10 having the above-mentioned structure is adopted, the liquid feeding means utilizing the capillary phenomenon or the liquid feeding means utilizing the centrifugal force generated by rotating the disk-shaped substrate by a predetermined method. Can also be used.

Specifically, the liquid reservoir 2 is provided at the center of the substrate 20.
1, the sample solution 5 (5 ′) (see FIG. 4) and a cleaning liquid for removing an excess target substance that has not been actively bound after the reaction are injected into the liquid reservoir 21.
By rotating the substrate 21 from the central region of the substrate.
The liquid can be smoothly and reliably fed into the inside (that is, the reaction region R ′).

In either case of the detection unit 1 of the substrate 2 or the groove detection unit 10 provided on the substrate 20, different detection substances D are fixed for each detection unit or for each of a plurality of grouped detection units. be able to.

Here, the position information and rotation synchronization information of the substrate 2 (20) will be briefly described. A large number of address pits formed in advance by an optical disc mastering process are formed in the rotation direction of the substrate 2 (20). When the substrate 2 (20) is considered as an optical disk, the reaction area R (R ′), which is the drop detection position, is considered as the user data area, and the other areas are arranged with synchronization pits by the sample servo method and the tracking servo is performed. Further, the position information is given by inserting the address portion (the geographical address on the disc) immediately after.

The address portion starts from the sector mark which is the top pattern, and gives a VFO (Variable Frequency Oscillato) which gives the rotation phase of the actually rotating disk.
r), an address mark that gives the start position of the address data, and an ID (Identi
fer) etc. are combined.

In order to perform a bioassay using the substrate 2 (20) having the above structure, it is preferable to use an apparatus having at least the following means and mechanism. That is, the substrate 2 (20) is rotatably held, and the substrate 2 (20) is rotated by the substrate rotating means to detect the nucleotide chain-containing solution 5 for detection and the target nucleotide chain-containing solution 5 '. Dropping means for dropping the reaction area R (R ′) in a predetermined order and timing, and a focus servo for maintaining a constant distance between (the nozzle of the dropping means) and the substrate 2 (20). Mechanism and substrate 2
At least a tracking servo mechanism for making the dropping of the solutions 5 and 5 ′ follow the reaction region R (R ′) of the substrate 2 (20) based on the position information and the rotation synchronization information provided from (20). A device (not shown).

If the substrates 2 and 20 described above and the bioassay apparatus using them are adopted, the bioassay method according to the present invention described above can be suitably implemented.

[0091]

(1) In the bioassay method or bioassay apparatus according to the present invention, the detection substance and the target substance in the reaction region are controlled by controlling the timing of electric field formation in the reaction region. It is possible to increase the reaction efficiency of the interaction reaction of and to control the timing of the reaction.

(2) It is particularly useful for a bioassay method based on a DNA chip or a biosensor chip and can be used for gene mutation analysis, SNPs (single nucleotide polymorphism) analysis, gene expression frequency analysis, etc. It can be used in a wide range of fields such as diagnosis, pharmacogenomics, forensic medicine and the like, and further can be used for examination of antigen-antibody reaction, assay of endocrine disrupting substances and the like.

[Brief description of drawings]

FIG. 1 is a flow chart for explaining a preferred procedure of a bioassay method according to the present invention and a preferred embodiment of a bioassay device according to the present invention.

FIG. 2 is a waveform diagram for explaining an example of a procedure of turning on / off voltage application of the bioassay method or apparatus.

FIG. 3 is an external perspective view of a bioassay substrate (2) provided with a large number of detection units (1).

FIG. 4 is an enlarged external view of a preferred embodiment of a detection unit (1) provided on the substrate (2).

5A is a plan view of a substrate (20) which is a second embodiment of the substrate used in the bioassay device according to the present invention, seen from above, and (B) is an enlarged plan view of a portion X in FIG.

[Explanation of symbols]

1, 10 Detecting Part 3 Nozzle 2, 20 Bioassay Substrate E (E ₁ , E ₂ , E ₃ , E ₄ ) Electric Field Forming Means D Detecting Substance T Targeting Substance R, R ′ Reaction Area S, S ′ Detecting Surface

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 102 C12N 15/00 AF term (reference) 4B024 AA11 CA04 CA05 CA07 CA09 HA14 4B029 AA07 AA23 BB20 CC03 FA12 FA15 4B063 QA01 QA18 QQ03 QQ42 QQ52 QR32 QR35 QR38 QR55 QR62 QR66 QR82 QS12 QS25 QS34 QS39 QX02 QX04

Claims (4)

[Claims] 1. A detection surface that has been surface-treated so that a detection substance can be immobilized, and a reaction that provides a field of interaction between the detection substance immobilized on the detection surface and the target substance. An electric field forming means for forming an electric field in the reaction area by generating a potential difference in the reaction area, and a method for detecting an interaction reaction between substances in a detection unit, comprising: A bioassay method comprising at least a procedure of turning on / off the electric field formation by the forming means at a predetermined timing. 2. The bioassay method according to claim 1, wherein the detection substance and the target substance are nucleotide chains, and the mutual reaction is hybridization. 3. A step of forming an electric field so as to extend the detection nucleotide chain in a state where the terminal site is fixed on the detection surface, and a step of adding a target nucleotide chain to the reaction region after the step, After the procedure, an electric field is formed in the reaction region, a step of relatively moving the detection nucleotide chain and the target nucleotide chain in the reaction area, and a step of performing hybridization with the application turned off after the procedure. The bioassay method according to claim 2, which comprises: 4. A detection surface that has been surface-treated so that the detection substance can be immobilized, and a reaction that provides a field for interaction between the detection substance immobilized on the detection surface and the target substance. An electric field forming means capable of forming an electric field in the reaction area by generating a potential difference in the reaction area, and at least turning on / off the electric field formation at a predetermined timing; A bioassay device comprising a detection unit provided.