KR101162365B1 - Bioassay Method, Bioassay Device, and Bioassay Substrate - Google Patents

Abstract

By controlling the formation of the electric field in the reaction zone in which the interaction reaction between materials such as hybridization is performed, the efficiency of the interaction reaction can be increased.

A bioassay method and a bioassay device capable of carrying out the method suitably. The detection surface S (S ') surface-treated so that the detection substance D can be fixed, and fixed to the detection surface S (S'). A potential difference is generated between the reaction zone R and R 'providing a place for interaction between the substance D for detection of the state and the target material T purged, and the reaction zone R and R'. In the detecting section (1) (10) having at least an electric field forming means (E) for forming an electric field in the reaction region (R) (R '), a method for detecting the interaction reaction between materials is provided. At least a step of turning on / off the electric field formation by the means E at a predetermined timing.

Bioassay methods, bioassay devices, hybridization, DNA, nucleotide chains, detection materials, target materials, substrates

Description

Bioassay Method, Bioassay Device, and Bioassay Substrate

The present invention relates to a bioassay method, a bioassay device and a substrate for bioassay, which are particularly useful in the field of bioinformatics (bioinformatics).

The main prior art of the present invention is explained below.

Currently, an integrated substrate for bioassay called a DNA chip or a DNA microarray (hereinafter, collectively referred to as a "DNA chip") in which predetermined DNA is microarrayed by microarray technology is used for analysis of gene mutation, SNPs ( It is used for monobasic polymorphism analysis, gene expression analysis, etc., and is widely used in the fields of new drug development, clinical diagnosis, pharmacological genomics, and forensic medicine.

This DNA chip is characterized in that a comprehensive analysis of intermolecular interactions such as hybridization is possible because a large number of DNA oligo chains and cDNA (complementary DNA), etc. are integrated on a glass substrate or a silicon substrate. .

An example of an analysis method using a DNA chip will be described briefly. For a DNA probe solidified on a glass substrate or a silicon substrate, mRNA amplified from cells and tissues is PCR amplified by incorporating a fluorescent probe dNTP by reverse transcription PCR. In this method, hybridization is performed on the substrate and fluorescence measurement is performed with a predetermined detector.

Here, DNA chips can be classified into two types. The first type is to synthesize oligonucleotides directly on a predetermined substrate using a photolithography technique using a semiconductor exposure technique, and is typically made by Affymetrix (USA). , US Pat. No. 5,445,934. This type of chip has a high degree of integration, but there is a limit to the synthesis of DNA on a substrate, which is about tens of bases long.

The second type, also referred to as a "stamped method", is produced by dispensing and solidifying DNA prepared in advance on a substrate using an end split pin (see, for example, US Patent No. 5,807,522). . This type of chip has an advantage that the degree of integration is lower than that of the former, but it is possible to solidify a DNA fragment of about 1 kb.

In addition, at present, a predetermined detection material is solidified on a minute detection surface provided on a thin plate called a biosensor chip containing a protein chip, and a solution containing a target material is added to the detection material. Advances in biosensor technology for monitoring dose and volumetric interactions between the two substances using principles such as surface plasmon resonance and crystal oscillators have been developed, and analysis of interactions between substances such as antibody antigen response and hormonal response. It is a useful technique in.

However, in the above-described conventional DNA chip technology and biosensor technology, in a narrow detection portion of a two-dimensional substrate, the nucleotide chains or proteins for detection such as DNA probes are solidified (fixed), and hybridization reactions and antigen antibodies. Because it is a technology that advances the interaction reaction between substances such as a reaction, the reaction is limited only under the reaction conditions that are not necessarily suitable, in which the degree of freedom of the reaction product is limited spatially and steric hindrance may occur during the reaction. The interaction was based on the Brownian motion of the product. For this reason, in the conventional DNA chip technology or biosensor technology, there existed a technical problem that the efficiency of interaction reaction is bad and reaction time is long.

In a conventional DNA chip or the like, the sample solution is only dropped to a predetermined spot portion (detection portion) on the substrate, and the relative relation between the target substance contained in the sample solution and the detection substance fixed to the spot portion is determined. No research has been done to locate them.

Therefore, in the present invention, the interaction is carried out by controlling the electric field formation of the reaction region in which the interaction between the substances such as hybridization is performed, and adjusting the relative positioning and the structure of the substance between the detection substance and the target substance. It is a main object to provide a bioassay method, a bioassay device and a bioassay substrate well suited for use in these bioassay methods and bioassay devices, which make it possible to improve the efficiency of the.

In order to solve the said technical subject, the following "bioassay method" is first provided in this application. In addition, in this application, "bioassay" means the biochemical analysis based on the mutual reaction between substances, such as hybridization, widely.

The "bioassay method" according to the present invention provides a detection surface surface-treated so that a detection substance can be fixed, and a place for interaction between a detection substance and a target substance in a state fixed to the detection surface. A field on the premise of a method for detecting interaction between materials in a detection section having at least a field forming means for generating a potential difference in the reaction zone by generating a potential difference in the reaction zone. And turning on / off electric field formation at a predetermined timing.

The electric field formed in the reaction zone is mainly used for (1) detecting a substance such as nucleotide chain, extending the target substance, and (2) moving the target substance which is separated from the detection substance in the forward and backward directions along the electric line of force. (3) fluorescence that specifically binds the higher-order structure of the reaction product (combination of the detection material and the target material) obtained by the interaction between the detection material and the target material with the fluorescently labeled target material or the reaction product. The present invention functions to adjust the structure in which the fluorescence emitted from the interlocker can be read more accurately.

In other words, by forming a desired electric field (applying on) or not generating (applying off) the electric field at a predetermined timing which is well suited for the reaction region of the detection unit, the action of (1) to (3) can be exerted in sequence. Can be.

More specifically, the structure of the said (1) is acquired by application of a high frequency high voltage, and the structure which is easy to react a detection substance and a target substance, for example, the linear structure in which the nucleotide sequence of a nucleotide chain is not layered (extension | stretching) Can be adjusted).

Subsequently, by applying a rectangular wave voltage (pulse direct current voltage), more specifically, by applying an alternating rectangular wave voltage (that is, +/-), the action of (2) is obtained, and it is advantageous in the reaction region. The target substance present is thereby approached to the detection substance which is fixed on the detection surface. As a result, the reaction efficiency (reaction opportunity) is improved, so that the reaction time can be shortened.

Also in the detection step, by applying the high frequency high voltage to the reaction region, the fluorescence or the like from the reaction product present in the reaction region can be accurately detected by the detection means such as optical means.

Here, in order to reliably advance the interaction between the detection material and the target material, such as hybridization, it is considered that it is better to form the reaction region that can be left only to the Brownian movement of the material. Therefore, in the present invention, in a series of electric field forming steps, it is to actively create a time that does not form an electric field in the reaction region. In other words, by allowing the rectangular wave voltage to be turned off after the rectangular wave voltage on, the interaction between materials can be promoted.

Here, in the "bioassay method" according to the present invention, the types of the detection substance and the target substance are not particularly limited. In addition, a "target substance" includes both the thing which has a mark, such as a fluorescent mark, and the thing which is not. The "detection material" broadly includes low molecules, polymers and biomaterials which solidify directly on the detection surface or indirectly through a linker and exhibit specific interactions with target substances labeled by fluorescent materials or the like. It is not interpreted narrowly.

"Detection surface" means the surface part which carried out the surface treatment suitable for the surface which can fix | immobilize terminal, such as nucleotide chain. For example, when surface-treated with strapavidin, it is a detection surface suitable for immobilization of biotinylated nucleotide chains.

The method comprises, between the protein-protein, between the nucleotide chain and the nucleotide chain (including both DNA-DNA and DNA-RNA) and protein-nucleotide chains (in a range consistent with the object and effect of the present invention). It is applicable to the interaction between polymers, the interaction between polymers and low molecules, and the interaction between low molecules.

For example, the detection substance and the target substance are nucleotide chains, and when the interaction reaction is hybridization, hybridization efficiency can be reliably improved even in a narrow reaction region (spot region). In addition, it is possible to provide a novel DNA chip or microarray technique with a short reaction time and high read accuracy.

Here, as a very suitable bioassay method in the case where the interaction between substances in the reaction region is hybridization, (a) detecting a nucleotide chain for detection in which the terminal is fixed to the detection surface Forming an electric field to extend, (b) adding a target nucleotide chain to said reaction zone after said step, and (c) generating a potential difference in said reaction zone after said step, thereby detecting Relatively moving the nucleotide chain and the target nucleotide chain, and (d) performing hybridization with the application off after the step. Hereinafter, each step of (a)-(d) is demonstrated concretely.

As used herein, the term "nucleotide chain" refers to a polymer of a phosphate ester of a nucleotide, in which a purine or pyrimidine base and a sugar are glycosylated, and an oligonucleotide, a polynucleotide, a purine nucleotide, and a DNA probe. Pyrimidine nucleotides include polymerized DNA (full length or fragment thereof), cDNA (cDNA probe) obtainable by reverse transcription, RNA, and the like.

Since the detection nucleotide chain (single strand) to which the end portion is to be fixed to the detection surface does not necessarily have a straight chain composition, according to step (a) above, the detection nucleotide chain is directly chained along the electric line of force. Elongate. As a result, the nucleotide sequence of the detection nucleotide chain is exposed to the reaction region, whereby a state in which the hydrogen bond reaction with the complementary nucleotide sequence is easily proceeded can be formed.

More specifically, a nucleotide chain composed of a plurality of polarization vectors composed of negative charges of nucleotide chains containing phosphate ions and positive charges of ionized hydrogen atoms is elongated by application of an electric field, and thus the bases are not layered. As a result, steric hindrance is eliminated and the hybridization reaction with the existing target nucleotide is performed smoothly.

When the principle of nucleotide chain elongation or movement is explained in detail, it is thought that the phosphate ions (negative charge) forming the skeleton of the nucleotide chain and the surrounding water form an ion cloud by ionizing hydrogen atoms (positive charges). And a polarization vector (dipole) generated by positive charges is generally directed in one direction by application of a high frequency high voltage, and as a result, the nucleotide chain is elongated. In addition, when a non-uniform electric field in which the electric line is concentrated in part is applied, the nucleotide chain moves toward the area where the electric line is concentrated (Seiichi Suzuki, Takeshi Yamanashi, Shin-ichi Tazawa, Osamu Kurosawa and Masao Washizu: "Quantitative analysis on electrostatic orientation of DNA in stationary AC electric field using fluorescence anisotropy ", IEEE Transaction on industrial Applications, Vol. 34, No. 1, P75-83 (1988)).

Subsequently, after the step (a), the step of (b) adding the sample solution containing the target nucleotide chain to the liquid phase of the reaction zone while the voltage application is turned on or the voltage application is turned off once. Run

Subsequent to step (a), an electric field is formed in the reaction zone, and step (c) of relatively moving the detection nucleotide chain and the nucleotide chain in the reaction zone is performed to create a reaction environment in which hybridization is likely to proceed. Form. That is, according to the step (c), by forming an electric field in the reaction region, the reactor circuit between both nucleotide chains, which was conventionally based solely on the Brownian motion, can be significantly increased, thereby increasing the reaction efficiency and improving the detection accuracy. .

Next, in step (d), after completion of step (c), the application is turned off, and hybridization based on the Brownian motion is performed. The reason why application (d) is turned off in this step (d) is that hybridization is a reaction that is entirely left to hydrogen bonding between complementary base pairs.

Next, in this invention, the "hybridization apparatus" provided with the following structures is provided as a tool which can implement the said bioassay method suitably.

In other words, the bioassay device according to the present invention is a reaction that provides a place for interaction between a detection surface that is surface-treated so that a detection material can be fixed, and a detection material that is fixed to the detection surface and a target material. By generating a potential difference in the region and the reaction region, a detection section having at least electric field forming means configured to enable electric field formation in the reaction region and to turn on / off the electric field formation at a predetermined timing is used.

The said "detection part" has three essential structures, a detection surface, a reaction area, an electric field formation means, and is not specifically limited about other structure and a structure. For example, a fine cell structure, a channel structure, or the like partitioned around the substrate may be formed on the substrate, and the above-described essential structures may be formed in these structure portions to form a detection unit. The number of detection parts on a board | substrate can be determined according to an objective and a use, and the form of a board | substrate can select suitably the form of flat form, such as rectangular shape and disk shape.

Moreover, in this invention, the "bioassay board | substrate" provided with the following structures is provided as a tool which can implement the said bioassay method suitably.

The "bioassay substrate" according to the present invention is a detection surface on which at least (1) a terminal portion of a nucleotide chain for detection is surface-fixed on a disc-shaped substrate on which optical recording information can be read, (2 ) An electrode forming an electric field which extends the detection nucleotide chain in a fixed state on the detection surface, and (3) a reaction region serving as a site for the hybridization reaction between the detection nucleotide chain and the target nucleotide chain. Detector ”is provided.

In the bioassay substrate which has this "detection part," the sample solution containing a target nucleotide chain is extended after extending | stretching the nucleotide chain linearly by applying the electric field to the detection nucleotide fixed to the detection surface, The reaction region of the detection unit can be accurately dropped to advance the hybridization reaction between the detection nucleotide chain and the target nucleotide chain. As a result, the advantageous effect that hybridization reaction can be performed efficiently in a short time can be acquired.

As described above, the nucleotide chain composed of a plurality of polarization vectors composed of a negative charge of a nucleotide chain having phosphate ions or the like and a positive charge of ionized hydrogen atoms is elongated by application of an electric field, and the bases are not laminated. As a result, the steric hindrance during the reaction is eliminated, and the hybridization reaction between the target nucleotide and the detection nucleotide in the vicinity is performed smoothly.

Here, the principle of nucleotide chain elongation or movement will be explained again. It is thought that the phosphate ions (negative charge) forming the skeleton of the nucleotide chain and the surrounding water form an ion cloud by ionized hydrogen atoms (positive charges). The polarization vectors (dipoles) generated by these negative and positive charges are generally directed in one direction by application of a high frequency high voltage, and as a result, the nucleotide chains are elongated. In addition, when a non-uniform electric field in which the electric field lines concentrate on a portion is applied, the nucleotide chain moves toward the portion where the electric field lines concentrate.

Here, when an electric field in which electric force lines are concentrated on the detection surface is applied, as a result, nucleotide chains which have been elongated (described above) by the electric field move toward the detection surface, and the terminal portions thereof collide with the detection surface, thereby detecting. The nucleotide chain of the dragon can be reliably fixed to the detection surface.

A "reaction zone" is a partitioned area or space that can provide a place for hybridization reaction in a liquid phase, and a region in which an electric field is formed in the liquid phase by generating a potential difference between the electrodes.

In addition, in this reaction region, in addition to the interaction between the single stranded nucleotides, that is, hybridization, a desired nucleotide chain is formed with a detection nucleotide chain, and the paired nucleotide and the peptide (or protein) interact with each other, and the enzyme response. Other intermolecular interactions can be made. For example, in the case of using the double-stranded nucleotide, binding of a receptor molecule such as a hormone receptor, which is a transcription factor, and a response array DNA portion, or the like can be analyzed.

Next, in the bioassay substrate which concerns on this invention, the said "detection part" is made into the cell detection part which has a cell structure in which the detection surface was formed in one wall surface, and the form which arrange | positioned a plurality of this cell detection part on the disk-shaped board | substrate is employ | adopted. can do. In addition, a "cell detection part" is defined as a site | part which has a burn-up reaction area partitioned with the surrounding substrate area | region.

The "cell detection unit" can be disposed at an appropriate position on the substrate. If the cells are arranged side by side so as to show a radial view from above, the space on the substrate can be effectively used, so that the density of information can be increased. That is, it is possible to provide a (disc) DNA chip with a large amount of record information accumulation.

In addition, since the cell detectors are partitioned so as not to be confused with each other, the other detector nucleotide chains are fixed to a cell detector unit or a plurality of grouped cell detector units, and a separate independent condition is set for each detection nucleotide chain. The redidation reaction can be advanced.

For example, a marker gene of disease occurrence can be fixed by grouping on a substrate. This makes it possible to check the expression of a plurality of diseases at the same time using one substrate.

Moreover, based on the difference of Tm or GC content rate, it becomes possible to group the nucleotide chain for detection to immobilize. As a result, it is possible to meticulously select the reaction conditions such as the buffer composition, the concentration, and the like, the washing conditions, and the dropping sample solution concentration, which can obtain an active hybridization reaction, according to the nature of the detection nucleotide chain. The work can significantly reduce the risk of false positives or false negatives.

Next, in the bioassay substrate according to the present invention, the reaction region of the detection portion is formed or arranged in a stem groove radially extended on the disk-shaped substrate, and the detection surface is placed on the inner wall surface of the stem groove. The arrangement of arranging can also be adopted.

In addition, the "stem groove" formed in a board | substrate means the elongate microchannel structure elongated in stripe form, and one field to which the bioassay board | substrate which has this stem groove belongs is a disk type microchannel array. Can be. Hereinafter, the detection part of the structure in which the reaction area is provided in the stem groove is called a "stem groove detection part" for convenience along with said "cell detection part."

When this "stem groove detection part" is adopted, a liquid transfer method using a capillary environment or a liquid transfer method utilizing the centrifugal force generated by rotating a disk-shaped substrate by a predetermined method can be used. For example, it is possible to smoothly and reliably transfer a liquid such as a sample solution or a cleaning liquid for removing excess target material not actively bound after the reaction from the substrate center region to the stem groove (that is, the reaction region). .

In addition, even in the case of the stem groove detection unit, another detection nucleotide chain may be fixed to the stem groove unit or the plurality of grouped stem groove units.

In the above-described bioassay substrate, when a means for providing the positional information and the rotational synchronous information on the detection surface portion is provided, the detection nucleotide-containing solution and the target nucleotide-containing solution are based on the positional information and the rotational synchronous information. Can be dripped accurately following a predetermined reaction region.

The means may be based on wobbling or address pits provided on the substrate. In addition, "wobble" means that a group (guide groove) for recording data by a user meanders slightly to the center of the track in order to record the information of the physical address (address) on the disk in advance on the disk. will be. Usually, for a frequency higher than the tracking servo band, FM modulation with a slight frequency deviation is performed, and the sinusoidal modulation signal amplitude is inscribed on the substrate as a group radial displacement.

In the reaction region provided on the substrate for bioassay described above, it is also possible to fill a substance (referred to as "phase change substance") in which reversible phase change of gel and sol can occur between room temperature and a temperature suitable for the reaction. . An example of a phase change substance is agarose gel.

By this means, the phase change material is solvated at a high temperature, voltage is applied in this state to arrange nucleotide chains such as DNA, followed by gelation by lowering the temperature, and subsequent hybridization. The step of solvating at a temperature suitable for the reaction can be carried out, and if the phase change material is kept in a gel state at the time of hybridization, the hybridization can be performed in the state where the nucleotide chain such as DNA is elongated. Do. In addition, when the phase change material is in a gelled state, when the sample solution containing the target nucleotide chain is dropped into the detection unit, the hybridization may not proceed well. The longitudinal groove may be formed in advance, and the sample solution may be dropped into the groove.

In the present invention, the target nucleotide chain can be either a means for labeling with fluorescent dye or a means for using an intercalator. The "integrator" is incorporated into the hybridization double-stranded nucleotide chain so as to be inserted during the hydrogen bond between the base of the detection nucleotide chain and the target nucleotide chain. As a result, the fluorescence wavelength shifts on the long wavelength side, and there is a correlation between the fluorescence intensity and the amount of the intercalator incorporated into the double-stranded nucleotide chain, so that quantitative detection is possible based on this correlation. As described above, the present invention has a technical meaning of providing a novel technology related to a DNA chip or a biosensor chip.

In addition, the present invention provides novel bioassay substrates that can be used for gene mutation analysis, SNPs (monopolymorphic) analysis, gene expression analysis, etc. to related industries such as new drug development, clinical diagnosis, pharmacological genomics, and forensic science. Has a technical significance.

1 is a flow diagram illustrating very suitable steps of a bioassay method according to the invention and a very suitable embodiment of the bioassay method according to the invention.

2 is a waveform diagram illustrating an embodiment of an on / off step of voltage application of the bioassay method or apparatus.

3 is an external perspective view of the bioassay substrate according to the present invention as viewed from above.

4 is an external perspective view showing an enlarged view of one of the cell detection units provided on the substrate.

Fig. 5 is an enlarged view showing the vicinity of the detection surface of the reaction region of the cell detection unit.

Fig. 6 is a view showing a substrate for bioassay, which is another preferred embodiment according to the present invention, (A) is a plan view when viewed from the top of the substrate, and (B) is an enlarged plan view of part X in (A). .

EMBODIMENT OF THE INVENTION Hereinafter, based on an accompanying drawing, the preferred embodiment of this invention is described.

1 (A) to (F) are flow diagrams for explaining very suitable steps of a bioassay method according to the present invention and a very suitable embodiment of the bioassay device according to the present invention, and FIG. It is a waveform diagram for explaining an embodiment of the on / off step of applying voltage applied in the apparatus.

In addition, in the following, the present invention will be described as a representative example where the detection substance and the target substance are both nucleotide chains and the interaction is hybridization. On the basis of this explanation, the detection substance and the target substance are not limited to nucleotide chains, and the interaction reactions of the present invention are not limited to hybridization.

First, the bioassay method or apparatus according to the present invention includes a detection surface S on which surface treatment has been performed such that the detection substance indicated by the symbol D in FIG. 1 can be fixed, and the detection surface S. A reaction zone (R) providing a place for interaction between the fixed substance (D) and the added target substance (T) in a fixed state, and creating a potential difference in the reaction zone (R), It is assumed that the detection unit 1 having at least an electrode E1 and an electrode E2 serving as an electric field forming means E for forming an electric field in R) is used. Reference numeral 2 denotes a part of the substrate including the detection unit 1, and reference numeral V denotes a power source for applying electric power to the electrode E1 and the electrode E2.

FIG. 1A succinctly shows a state in which a terminal portion of one strand of a nucleotide chain for detection (for example, a DNA probe) D1 is fixed to the detection surface S. FIG. In this state, the higher-order structure of the detection nucleotide chain (D1) is not linear, for example, in many cases, the chains are entangled with each other as shown in Fig. 1A. For this reason, since all the nucleotide sequences are not exposed to the reaction region (R), efficient hybridization with the target nucleotide chain (T) added later to the reaction region (R) is considered to be difficult to obtain. .

Therefore, in the present invention, a high frequency high voltage is applied to the liquid phase (salt solution, etc.) of the reaction region R between the electrode E1 and the electrode E2, thereby generating a potential difference in the reaction region R, thereby causing an electric field (electric field). To form. This electric field exerts the effect | action which extends the detection nucleotide chain D1 linearly (linearly) according to an electric field. As a result, the detection nucleotide chain D1 is elongated (shown by reference numeral D2), and the base sequence of the detection nucleotide chain D1 is exposed to the liquid phase (see FIG. 1 (B)).

Next, the sample including the target substance T (target nucleotide chain T1) in the reaction region R with the nozzle 3 of a predetermined structure is turned off or the application of the high frequency high voltage is turned off. Add solution. The target nucleotide chain (T1) is not necessarily linear as shown in Fig. 1 (C) at the beginning of addition, but in the liquid phase to which a high frequency voltage is applied, as shown in Fig. 1 (D), It becomes the state extended in chain form (it shows with the code | symbol T2), and it becomes a state in which it is easy to form a complementary chain with the nucleotide chain for detection (D2) which became linear in the same way.

Then, by repeatedly turning on / off the application of the rectangular wave voltage, both nucleotide chains D2 and T2 are approached in stages, or the target nucleotide chain is moved back and forth, or the timing of the reaction is adjusted. do.

The reason for turning off the square wave voltage is that the complementary chain formation reaction between the linear target nucleotide chain (T2) and the linear detection nucleotide chain (D2), that is, hybridization, is left to Brownian motion only. To do that. 1 (E) briefly shows a reaction product (D2 + T2) in which the complementary base sequence portions of both chains (D2, T2) are hydrogen-bonded and paired.                 

In addition, in the present invention, in addition to the purpose of detecting the target nucleotide chain (T1) (T2) having a base sequence complementary to the detection nucleotide chain (D1) (D2), the target double-stranded nucleotide chain using the above steps (DNA), a method of detecting the interaction between a specific protein such as a transcription factor and a specific reaction sequence portion of the double-stranded nucleotide chain (DNA), and also searching for and detecting endocrine disruptors using the method. You may develop.

FIG. 1 (F) shows a step of detecting and reading a target nucleotide chain T2 labeled (e.g., fluorescently labeled) based on the optical means 4, and in the present invention, the labeling method is narrow. It is not limited. For example, you may use a fluorescent interpolator. The fluorescence intercalator is incorporated into the hybridized double-stranded nucleotide chain so as to be inserted during hydrogen bonding between the detection nucleotide chain (D) and the base of the target nucleotide chain (T). As a result, the fluorescence wavelength is shifted to the long wavelength side, and quantitative detection is possible based on the correlation between the fluorescence intensity and the amount of the fluorescence intercalator incorporated into the double-stranded DNA. As a fluorescent dye used for the fluorescent interpolator, POPO-1, TOTO-3, etc. can be considered.

In the detecting step, by applying the high frequency high voltage, by adjusting the higher order structure of the reaction product (double-chain nucleotide chain) formed in the reaction region R to a structure without a middle layer, it is accurately detected by the optical means 4. can do.

Subsequently, an embodiment of the application on / off step will be described based on FIG. 2. This embodiment also corresponds to the steps after Fig. 1C.                 

<Step a>

By placing a nucleotide chain under an electric field formed by application of a high frequency high voltage, the induced polarization of the nucleotide chain is achieved, thereby extending the nucleotide chain. In this step a, by applying a high frequency high voltage, the target nucleotide chain T1 is extended, and similarly, the detection nucleotide chain D1 is also extended (the state shown in Figs. 1 (C) and 1 (D)). See).

In addition, it is thought that the high frequency high voltage is about 1 × 10 ⁶ V / m, and about 1 MHz is optimal (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)).

<Step b>

At the timing at which both nucleotide chains D2 and T2 are considered to have moved relative to each other, the application of the voltage is turned off, and the hybridization proceeds solely based on the Brownian motion (the state shown in Fig. 1E). Reference).

<Step c>

The application of the rectangular wave voltage in the + direction is turned on and the target nucleotide chain T2 still in the inaccessible state is moved to the Coulomb force, thereby making it a position suitable for hybridization with the detection nucleotide chain D2 (Fig. See the process of transitioning from the state of 1 (D) to the state of FIG. 1 (E)).                 

<Step d>

The application of the voltage is again turned off, and the hybridization proceeds solely based on the Brownian motion (see the state of FIG. 1E).

<Step e>

Next, the application of the rectangular wave voltage in the − direction is turned on, and the target nucleotide chain T2 which is still in an inaccessible state is moved (the state transitions from the state of FIG. 1D to the state of FIG. 1E). See course).

<Step f>

Again, the application of the voltage is turned off, and hybridization proceeds solely based on the Brownian motion (see the state shown in Fig. 1E).

<Step g>

The application of the high square wave voltage in the + direction is again turned on, and the movement of the target nucleotide chain T2 still in the inaccessible state is performed (the process of transitioning from the state of FIG. 1 (D) to the state of FIG. 1 (E)). See).

<Step h>

Again, the application of the voltage is turned off, and hybridization proceeds solely based on the Brownian motion (see the state shown in Fig. 1E).

<Step i>

The application of the rectangular wave voltage in the-direction is again turned on, and the target nucleotide chain T2 which is still in an inaccessible state is moved (the process of transitioning from the state of FIG. 1 (D) to the state of FIG. 1 (E)). Reference).

<Step j>

The application of the voltage is turned off again, and the hybridization proceeds solely based on the Brownian motion (see the state shown in Fig. 1E).

<Step k>

The double-stranded nucleotide chain (D2 + T2), which is a reaction product by hybridization, is stretched under high frequency high voltage and subjected to fluorescence reading (see FIG. 1 (F)).

Incidentally, the steps of applying on / off of the rectangular wave voltage and the like described above can select and determine a suitable step and timing according to the kind of reaction product to be handled, and are not limited to the above steps and timing. Moreover, according to the objective, the magnitude | size of a frequency and a voltage can also be determined suitably.

Subsequently, the bioassay board | substrate which can be employ | adopted by this invention and the bioassay apparatus which concerns on this board | substrate are demonstrated.

Fig. 3 is an external perspective view of a bioassay substrate in which a large number of detection portions having a predetermined configuration are arranged, and Fig. 4 is an enlarged external view of a preferred embodiment of the “detection portion” provided on the substrate. In addition, the substrate which can be employed in the present invention is not limited to the illustrated form.

First, the bioassay substrate 2 (hereinafter, abbreviated as &quot; substrate 2 &quot;) shown in FIG. 3 is employed in a disc substrate (disc) used for optical information recording media such as CD, DVD, MD, and the like. It is formed of a base material. That is, the board | substrate 2 is able to read record information optically.                 

The said base material is formed in disk shape by synthetic resin which can be shape | molded in disk shape, such as quartz glass, silicone, polycarbonate, polystyrene, Preferably it is synthetic resin which can be injection-molded. In addition, by using an inexpensive synthetic resin substrate, it is possible to realize a low running cost as compared with the glass chips that have been conventionally used. In the center of the board | substrate 2, the spindle fixing hole (not shown) used when rotating a board | substrate 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 reflective film, is formed, and the layer functions as a reflective film. This reflecting film is made into 4% or more of surface reflections from the base | substrate single body of refractive index 1.5 or more. On the upper layer of the reflective film, a light transmitting layer made of transparent glass, transparent resin, or the like is formed.

In the case where the substrate is a material having a high reflectance, the surface of the substrate itself functions as a reflective surface, so the reflective film may not be formed. Formation of a high reflectivity film such as a metal film can detect the fluorescence intensity of the fluorescently labeled target material with excellent sensitivity. The material and layer structure of the board | substrate 2 demonstrated above are the same also in the board | substrate 20 (refer FIG. 6) mentioned later.

In the light transmitting layer, a plurality of detectors 1 are arranged so that the peripheral region at the center of the substrate 2 extends radially from above. 4 is an external perspective view showing an enlarged view of one of these cell detection units 1. In addition, although the description by the detection part 1 below makes a case where both a detection substance and a target substance are single-stranded nucleotide chains, it does not limit the target reaction substance of the detection part 1 to this.                 

Here, the detection part 1 is a detection surface S which has been surface-treated so that the terminal part of the detection nucleotide chain shown by the code | symbol D can be fixed, and said fixed surface previously fixed to the detection surface S The site of the hybridization reaction between the electrode (E1) and the electrode (E2) for forming an electric field extending the detection nucleotide chain (D) and the detection nucleotide chain (D) and the target nucleotide chain (T) It has a reaction zone (R). Here, the reaction region R is formed as a rectangular cell, for example, a cell having a depth of 1 μm, a length of 100 μm, and a width of 50 μ when viewed from above, which opens upward. It is not limited.

The detection surface S is formed in the inner wall surface part facing the reaction area | region R side of the electrode E2 side. This detection surface S is surface-treated so that the terminal of the detection nucleotide chain D may be fixed by chemical bonds, such as a coupling reaction.

In other words, the detection surface S may be a surface treatment that is very bonded to fix the preprocessed terminal portion of the detection nucleotide chain D such as a DNA probe, and is not limited thereto. For example, in the case of the detection surface S surface-treated with streptavidin, it is suitable for immobilization of biotinylated nucleotide chain ends.

Here, a suitable method for fixing the detection nucleotide chain to the detection surface (S) will be described. A non-uniform electric field is applied to the reaction zone (R), thereby obtaining the effect of the electric field (free to the reaction zone (R). The nucleotide chain for detection (in the present state) moves toward the portion of the detection surface S where the electric force lines are concentrated, and the terminal portion collides with the detection surface S. This makes it possible to reliably fix the nucleotide chain to the detection surface (S). In order to perform this method suitably, the electrode E2 is comb-shaped as shown in FIG. In addition, the code | symbol V of FIG. 4 is a power supply which supplies electric power to the electrode E1 and the electrode E2.

Here, the code | symbol 3 in FIG. 4 has shown the front-end | tip part of the nozzle which dripped the sample solution 5. As shown in FIG. The nozzle 3 contains a sample solution 5 containing the detection nucleotide chain D and a sample solution containing the target nucleotide chain T based on the positional information and rotation synchronization information provided from the substrate 2. It is set as the structure which 5 'is followed correctly and dripped at the reaction area | region R position.

As a dripping means, the inkjet printing method can be employ | adopted suitably. The reason for this is that it can follow the predetermined reaction region R precisely and drop the microdroplets accurately (the same applies to the substrate 20 described later).

"Inkjet printing method" is a method of applying a nozzle used in an inkjet printer, and is a method of spraying and fixing a detection substance onto a substrate from a print head like an inkjet printer using electricity. This method includes a piezoelectric inkjet method, a bubblejet (registered trademark) method, and an ultrasonic jet method.

The piezoelectric inkjet method is a method of blowing droplets by the pressure of the displacement caused by each time a pulse is applied to the piezoelectric body. The bubble jet (registered trademark) method is a thermal method, in which a droplet is blown by the pressure of bubbles generated by heating a heater in a nozzle. The silicon substrate used as a heater is embedded in the nozzle, and controlled at about 300 ° C / s to create the same bubble to push out the droplets. However, care must be taken when using them in biological samples because the liquid will be exposed to high temperatures. The ultrasonic jet method is a method of releasing small droplets from a location by applying a local pressure on a free surface of an ultrasonic beam and a liquid. It is possible to form small droplets of about 1 탆 in diameter at high speed without the need for a nozzle.

In the present invention, the "piezoelectric ink jetting method" can be suitably employed as the "inkjet printing method". By changing the shape of the pulse to be applied, the size of the droplets (micro droplets) can be controlled, which is very suitable for improving the analysis accuracy. When the radius of curvature of the droplet surface is small, the droplet can be made small. 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 rapidly changing the pulse in the negative direction.

Here, since most of the detection nucleotide chains D dropped in the reaction region R are fixed to the detection surface S in a state in which the bases are laminated (see FIG. 1 (A)), they are dropped in the back. In hybridization with the target nucleotide chain (T), problems of steric hindrance occur and reaction efficiency is not good.

Thus, based on the bioassay method according to the present invention, by applying electric power to the electrodes E1 and E2 disposed in the detection unit 1 to generate a potential difference in the reaction region R, the reaction region R is stored in the reaction region R. An electric field is formed in a liquid phase (salt solution), and the nucleotide chain for detection (D) and the target nucleotide chain are extended in a straight line (straight line) according to the direction of the electric field of the electric field.

Hydrogen bonds (complementary bonds) between the linear nucleotide chains for detection (D) and the target nucleotide chains (T) labeled with fluorescent dyes, for example, have less steric hindrance and the like. The arrays are easily accessed and progress efficiently.

In other words, the hybridization reaction between the detection nucleotide chain (D) and the target nucleotide chain (T) proceeds efficiently. As a result, it is possible to obtain a desirable result that the reaction time of hybridization is shortened and the probability of showing false positive or false voice at the time of reading is also reduced.

5 is a schematic diagram which shows the detection surface S vicinity of the reaction area | region R of the detection part 1 on the enlarged scale. In FIG. 5, a target nucleotide chain (T) having a base sequence complementary to the base sequence of the detection nucleotide chain (D) and the detection nucleotide chain (D) whose immobilization is fixed to the detection surface (S). ) Is hybridized to form a double chain.

Regarding the reaction zone R (the same applies to the reaction zone R 'described later), a phase change substance such as agarose gel, in which reversible phase change of the gel and the sol may occur between room temperature and a temperature suitable for the reaction ( (Not shown). In this embodiment, the phase change material is solvated at a high temperature, and in this state, a voltage is applied to arrange nucleotide chains such as DNA, followed by gelation by lowering the temperature, followed by hybridization. In this case, there is an advantage that the step of solving at a temperature condition suitable for the reaction can be performed. In addition, it is preferable to keep the phase change material in a gel state during hybridization, since hybridization such as DNA and nucleotide chains can be extended.

In addition, when the phase change material is in a gelled state, when the sample solution 5 'containing the target nucleotide chain T is dropped into the detection unit 1, the progress of hybridization may not be improved. As in the case of electrophoresis, a longitudinal groove may be formed in advance at the dropping point, and the sample solution may be dropped into the groove.

Here, the target nucleotide chain (T) may be labeled with fluorescent dyes, or an interlocker may be used. The intercalator is incorporated into the hybridized double-stranded nucleotide chain so that it is inserted during hydrogen bonding between the detection nucleotide chain (D) and the base of the target nucleotide chain (T). As a result, the fluorescence wavelength is shifted to the long wavelength side, and quantitative detection is possible on the basis of the correlation between the fluorescence intensity and the amount of the intercalator incorporated into the double-stranded DNA. As a fluorescent dye used as an interlocker, POPO-1, TOTO-3, etc. can be considered.

For example, in the sample solution 5 'extracted from predetermined cells or the like, a target nucleotide chain having complementarity with a detection nucleotide chain (D) containing a marker gene that is known to be expressed when a specific disease occurs. The fact that it exists exists, and as a result, it is predicted that the said disease has arisen in the said cell.

In addition, the reading of the substrate information is performed by irradiating a laser beam (for example, blue laser beam) on the substrate 2 to excite each reaction region R to detect the magnitude of the fluorescence intensity by a detector (not shown). The binding reaction state between the detection nucleotide chain (D) and the labeled target nucleotide chain is determined. Finally, the fluorescence intensity of each reaction region R is visualized by A / D conversion and the binding reaction ratio is distributedly displayed on the screen of the computer C (the same applies to the substrate 20 described later).

Next, with reference to FIG. 6, the 2nd Example of the board | substrate which can be used by this invention is demonstrated. Fig. 6A is a plan view when viewed from above the substrate 20 of the second embodiment, and Fig. 6B is an enlarged plan view of an X portion in Fig. 6A.

The substrate shown by reference numeral 20 in FIG. 6 includes a stem groove detector 10. The detection unit 10 is formed in a stem groove shape in which the reaction region R 'extends radially on the disc-shaped substrate, and the detection unit 1 is formed on one inner wall surface V in the longitudinal direction forming the stem groove. The detection surface S 'which has the same structure as the detection surface S of () is arrange | positioned at predetermined intervals. Moreover, the electrodes E3 and E4 are provided in each site | part in which the detection surface S 'was formed so that the reaction area | region R' might be interposed (refer FIG. 5 (B)). In addition, the stem groove detection unit 10 may be said to have a configuration in which the cell-shaped detection unit 1 is arranged in the stem groove.

The electrodes E3, E3 ... can be formed into a common electrode, and similarly, the electrodes E4, E4 ... can also be formed into a common electrode. That is, the electrode E3 and the electrode E4, which are common electrodes, may be disposed to face each other with the reaction region R 'interposed therebetween. In this structure, electric power can be applied by pressing the needle-shaped probe upward on the electrode E3 and the electrode E4.

The reaction region R 'may be formed in pits (not shown) arranged in each stem groove. By dropping microdroplets into the reaction region in the pit, it is possible to realize almost the same spot size, and to realize good fluorescence intensity detection with good reproducibility.

In addition, when the stem groove detection unit 10 having the above-described configuration is employed, liquid transfer means using capillary action or liquid transfer means utilizing the centrifugal force generated by rotating the disc-shaped substrate by a predetermined method can also be used.

Specifically, the liquid flow part 21 is provided in the center part of the board | substrate 20, and sample liquid 5 (5 ') (refer FIG. 4) to this liquid flow part 21, and was not actively coupled after reaction. By injecting a cleaning solution or the like for removing excess target material and rotating the substrate 20, it is possible to smoothly and reliably transfer liquid from the center region of the substrate to the stem groove (that is, the reaction region R ').

In any case of the stem groove detector 10 provided in the detector 1 or the substrate 20 of the substrate 2, the other detection substance D can be fixed to the detector unit or a plurality of grouped detector units. have.

Here, the positional information and the rotational synchronous information of the substrates 2 and 20 will be described briefly. In the rotational direction of the substrates 2 and 20, a plurality of address pits formed in advance by the optical disk mastering process are formed. When the substrate 2 (20) is regarded as an optical disk, the reaction region R (R '), which is a drop detection position, is regarded as a user data region, and the other regions are aligned and tracked at the same time by the sample servo method. It is also used as a servo, and position information is given immediately by inserting an address section (geographical address on disk).                 

The address section starts with a sector mark, which is the first pattern, and a VFO (Variable Frequency Oscillater) giving the rotational phase of the actually rotating disk, an address mark giving the starting position of the address data, an ID containing the track and sector numbers, and the like. This is done in combination.

In order to perform bioassay using the board | substrate 2 (20) of the said structure, it is very preferable to use the apparatus provided with the following means and mechanism at least. That is, the substrate rotating means for rotatably holding the substrates 2 and 20, the solution containing the detection nucleotide chain 5 and the target nucleotide chain while rotating the substrate 2 and 20 by the substrate rotating means. A dropping means for dropping the containing solution 5 'at a predetermined stage and timing with respect to the reaction region R (R'), and the distance between the dropping means (the nozzle) and the substrate 2, 20 is fixed. The dropping of the solution (5, 5 ') on the reaction region of the substrate (2) (26) based on the focus servo mechanism for maintaining it, and the positional information and rotational synchronization information provided from the substrate (2) (20) (R) An apparatus (not shown) provided with at least the tracking servo mechanism that follows (R ').

Instead of using the address pits, wobbling may be formed on the track, and the meandering of the wobbling may be adjusted so that the clock information according to the position may be obtained so that the position information on the disc may be acquired and addressed. At the same time, tracking servo can be performed by using the wobbling frequency component. In addition, by forming the address pit and wobbling together, more accurate addressing and tracking servos are possible.

By employing the substrates 2 and 20 described above and the bioassay apparatus using these, the bioassay method according to the present invention described above can be suitably implemented.

In particular, according to the above-described bio assay substrate, the following unique effects can be obtained.

In other words, in the conventional DNA chip technology, since the DNA chip itself has a small number of accumulations and density, it cannot be said that the amount of analysis that can be achieved in one assay is sufficient. It was difficult for the user to freely set the arrangement (grouping) on the substrate.

In the conventional DNA chip, since the nucleotide chains fixed in advance on the substrate surface and used for detection are not necessarily held linearly, variations in the accuracy of the hybridization reaction with the target nucleotide due to steric hindrance or the like are caused. The reaction required a long time.

Furthermore, in the case of a DNA probe having a Tm (melting point) or GC content inconsistent, the detection material is arranged on the surface of the substrate having two-dimensional planar expansion. In the conventional DNA chip, the same hybridization conditions and cleaning are performed. There was a problem that there is a high risk of exposing false positives or false negatives due to exposure to conditions.

In comparison with this, according to the bioassay substrate which concerns on this invention, the following outstanding effect can be acquired.

(1) Since the bioassay substrate which concerns on this invention can form a large quantity and a large number of detection parts, the amount of information accumulation is large.                 

(2) By extending the nucleotide chain in a straight chain by applying an electric field to a detection nucleotide fixed to the detection surface of the detector, the sample solution containing the target nucleotide chain is aimed at the reaction region of the detector. By accurately dropping the mixture, the hybridization reaction between the detection nucleotide chain and the target nucleotide chain can be carried out. Thus, the hybridization reaction can be efficiently performed in a short time.

(3) As the detection unit or the grouped detection unit, the assay can be performed by selecting the optimal reaction conditions and the like, and thus the incidence rate of false positives and false negatives can be significantly reduced. Therefore, according to the above-described bioassay substrate, it is possible to perform comprehensive and efficient analysis with high accuracy, and the cost per recording information is also low.

(4) The bioassay substrate according to the present invention is particularly useful as a DNA chip or a biosensor chip. It is also possible to provide a disk-shaped micro channel array having a novel structure. When the substrate is used as a DNA chip, it can be used for gene mutation analysis, SNPs (monopolymorphic polymorphism) analysis, gene expression analysis, etc., and can be widely used in new drug development, clinical diagnosis, pharmacological genomics, forensic medicine, and other fields. .

The present invention has the following unique effects.

(1) In the bioassay method or bioassay apparatus according to the present invention, the reaction of the interaction between the detection substance and the target substance in the reaction zone is controlled by controlling the timing of the electric field formation in the reaction zone provided in the detection unit. The efficiency can be improved and the timing of the reaction can be controlled.

(2) Particularly useful for bioassay methods based on DNA chips or biosensor chips, which can be used for gene mutation analysis, SNPs (monopolymorphic) analysis, gene expression analysis, new drug development, clinical diagnosis, and pharmacological genomics. The present invention can be widely used in forensic medicine and other fields, and furthermore, it can be used for the examination of antigen-antibody reactions, the assay of endocrine disrupting substances, and the like.

Claims (14)

A detection surface that is surface-treated so that a detection material can be fixed, A reaction zone providing a place for interaction between the detection substance and the target substance in a fixed state on the detection surface; A bioassay method comprising detecting an interaction between materials by a detecting portion including at least electric field forming means for generating an electric field in the reaction region by generating a potential difference in the reaction region. The detection material and the target material are nucleotide chains, and the interaction is hybridization, The method includes at least turning on / off the electric field formation by the electric field forming means at a predetermined timing, The method further comprises Forming an electric field to extend the detection nucleotide chain having a terminal moiety fixed to the detection surface; Adding a target nucleotide chain to said reaction zone after said step, Forming an electric field after the step to relatively move the detection nucleotide chain and the target nucleotide chain in the reaction zone; And performing hybridization with the electric field removed after said step. delete delete delete The method of claim 1, A bioassay method comprising detecting material-to-material interactions by a plurality of detectors on an optically readable disc substrate. The method of claim 5, And the detection unit is a cell detection unit in which the detection surface is formed on one wall surface, and a plurality of cell detection units are disposed on the disc-shaped substrate. The method of claim 6, And said cell detector is arranged on said disc-shaped substrate so as to be radial from above. The method of claim 7, wherein A bioassay method in which a different detection nucleotide chain is fixed to each cell detector or each group of cell detectors. The method of claim 5, The reaction region of the detection unit is provided in each stem groove radially extended on the disc-shaped substrate, and the detection surface is disposed on the inner wall surface of the stem groove. 10. The method of claim 9, A bioassay method in which a different detection nucleotide chain is fixed to each stem groove or each stem groove group. The method of claim 5, And the substrate further comprises means for providing positional information and rotational synchronization information of the detection surface portion. 12. The method of claim 11, And the means comprises a wobbled groove or address pit mounted on the substrate. delete The method of claim 5, A bioassay method for detecting the hybridization reaction using an interlocker.

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