JP2004045376A - Substrate for bioassay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substrate for bioassay having high accuracy of a hybridization reaction. <P>SOLUTION: This substrate 1(4) for bioassay is equipped with at least a detection surface 21(51) to which surface processing is applied, capable of immobilizing a terminal portion of a nucleotide chain D for detection on a disc-shaped substrate capable of optically reading record information, positive and negative electrodes 22(52) for forming an electric field for elongating the nucleotide chain D for detection in the immobilized state on the detection surface 21(51), and a reaction area 23(53) which is a field for the hybridization reaction between the nucleotide chain D for detection and a target nucleotide chain T. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a disc-shaped information recording medium which is a particularly useful bioassay tool in the field of bioinformatics (bioinformatics). More specifically, the present invention relates to a bioassay substrate that is devised so that a hybridization reaction is performed with high accuracy by dropping a solution containing a target nucleotide chain onto a substrate surface portion on which a nucleotide chain for detection is immobilized. .
[0002]
[Prior art]
[0003]
The main prior art of the present invention will be described below. At present, an integrated substrate for a bioassay called a DNA chip or a DNA microarray (hereinafter, collectively referred to as “DNA chip”) in which predetermined DNAs are finely arranged by microarray technology is used for gene mutation analysis, SNPs (single nucleotide). (Polymorphism) analysis, gene expression frequency analysis, etc., and have begun to be widely used in drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and other fields.
[0004]
This DNA chip has a large number of DNA oligo chains and cDNA (complementary DNA) integrated on a glass substrate or a silicon substrate, so that comprehensive analysis of intermolecular reactions such as hybridization can be performed. It is characterized by points.
[0005]
Briefly describing 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, and the like to a fluorescent probe dNTP. This is a technique in which PCR amplification is carried out while the DNA is incorporated, hybridization is performed on the substrate, and fluorescence measurement is performed using a predetermined detector.
[0006]
Here, DNA chips can be classified into two types. The first type synthesizes oligonucleotides directly on a predetermined substrate by using a photolithography technique to which a semiconductor exposure technique is applied, and is typically obtained by Affymetrix (Affymetrix). (For example, see Japanese Patent Publication No. 4-5055763). This type of chip has a high degree of integration, but has a limit in DNA synthesis on a substrate, and is about several tens of bases long.
[0007]
The second type is also referred to as a “Stanford method”, and is produced by dispensing and solidifying DNA prepared in advance on a substrate using a split pin ( For example, see Japanese Patent No. 3272365). This type of chip has a lower degree of integration than the former, but has the advantage that a DNA fragment of about 1 kb can be immobilized.
[0008]
[Problems to be solved by the invention]
However, in the above-described conventional DNA chip technology, the number of integration and the integration density of the DNA chip itself were low, so that the amount of analysis that can be achieved in one assay is not sufficient, and the type and number of the substance for detection, and It is difficult for the user to freely set the arrangement (grouping) on the substrate.
[0009]
Further, in a conventional DNA chip, a nucleotide chain fixed in advance on a substrate surface and used for detection is not necessarily kept in a linear form, and therefore, hybridization with a target nucleotide is caused by steric hindrance or the like. The accuracy of the reaction varied, and the reaction required a long time.
[0010]
Further, in a conventional DNA chip having a structure in which a detection substance is arranged on a substrate surface having a two-dimensional planar spread of DNA probes having Tm (melting Temperature) or GC contents not uniform, the same is used. There is a problem that there is a high risk of showing false positives or false negatives when exposed to hybridization conditions and washing conditions.
[0011]
Therefore, the present invention provides a bioassay substrate in which the amount of a detection substance to be immobilized is large, the substance can be freely grouped, and the cost is low. The main purpose is to provide.
[0012]
[Means for Solving the Problems]
In order to solve the above technical problems, the present application first provides the following “substrate for bioassay”. In the present application, “bioassay” means a biochemical analysis based on hybridization or other interaction between substances.
[0013]
The “substrate for bioassay” according to the present invention is surface-treated on a disc-shaped substrate from which recorded information can be read optically so that at least (1) the terminal portion of the nucleotide chain for detection can be fixed. (2) Positive and negative electrodes forming an electric field for extending the outgoing nucleotide chain immobilized on the detection surface, (3) Hybridization reaction between the detection nucleotide chain and the target nucleotide chain A “detection unit” including a reaction region serving as a field for the detection is provided.
[0014]
In the bioassay substrate having the “detection portion”, the electric field is applied to the detection nucleotide immobilized on the detection surface to elongate the nucleotide chain in a linear manner, and then a sample solution containing the target nucleotide chain is applied. It is possible to allow the hybridization reaction between the nucleotide chain for detection and the target nucleotide chain to proceed by accurately dropping the droplet at the reaction region of the detection section. As a result, an advantageous effect that the hybridization reaction can be efficiently performed in a short time is obtained.
[0015]
The effect is as follows: a nucleotide chain consisting of a large number of polarization vectors composed of a negative charge of a nucleotide chain having a phosphate ion and the like and a positive charge of an ionized hydrogen atom is elongated by application of an electric field, and bases are overlaid. As a result, steric hindrance during the reaction is eliminated and the hybridization reaction between the nearby target nucleotide and the detection nucleotide is smoothly performed.
[0016]
Here, the principle of the extension or movement of the nucleotide chain is explained in detail. Ion cloud is formed by the phosphate ion (negative charge) forming the skeleton of the nucleotide chain and the hydrogen atom (positive charge) formed by the water around it. It is considered that the polarization vector (dipole) generated by these negative and positive charges is directed in one direction as a whole by applying 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 lines of force are partially concentrated is applied, the nucleotide chain moves toward the site where the lines of electric force are concentrated (Seiichi Suzuki, Takeshi Yamanishi, Shin-ichi Tagawa, Osamuro Kurosawa). and Masao Washizu: "Quantitative analysis on electrostatic orientation of DNA instationary AC electic field using on fluoresce ancestry, Ecosystem, USA.
[0017]
Here, in the present application, the term “nucleotide chain” means a polymer of a phosphate ester of a nucleoside in which a purine or pyrimidine base and a sugar are glycoside-linked, and an oligonucleotide including a DNA probe, a polynucleotide, a purine nucleotide and a pyrimidine nucleotide. Widely includes DNA (full length or a fragment thereof), cDNA (cDNA probe) obtained by reverse transcription, RNA, and the like.
[0018]
“Detection surface” refers to a surface site that has been subjected to a suitable surface treatment that is capable of immobilizing the end of a nucleotide chain by a coupling reaction or other chemical bond, and is not to be construed narrowly. For example, a detection surface that has been surface-treated with streptavidin is suitable for immobilizing a biotinylated nucleotide chain.
[0019]
Here, when an electric field is applied to the detection surface so that the lines of electric force are concentrated, as a result, the nucleotide chain extended (described above) moves toward the detection surface due to the electric field, and the terminal site is detected. By colliding with the surface, the nucleotide chain for detection can be reliably fixed to the detection surface.
[0020]
The “reaction region” is a partitioned region or space that can provide a field for a hybridization reaction in a liquid phase, and a region where an electric field is formed in the liquid phase by generating a potential difference between the positive and negative electrodes. It is.
[0021]
In this reaction region, in addition to the interaction between single-stranded nucleotides, that is, hybridization, a desired double-stranded nucleotide is formed from the nucleotide chain for detection, and the interaction between the double-stranded nucleotide and the peptide (or protein) is performed. Reactions, enzyme response reactions and other intermolecular interactions can also be performed. For example, when the double-stranded nucleotide is used, binding between a receptor molecule such as a hormone receptor as a transcription factor and a response element DNA portion can be analyzed.
[0022]
Next, in the bioassay substrate according to the present invention, the aforementioned “detection unit” is a cell detection unit having a cell structure in which a detection surface is formed on one wall surface, and a plurality of the cell detection units are provided on a disc-shaped substrate. It is possible to adopt a form in which they are arranged. Note that the “cell detection unit” is defined as a region having a small chamber-like reaction region partitioned from the surrounding substrate region.
[0023]
This “cell detector” can be disposed at an appropriate position on the substrate. However, if the “cell detector” is disposed side by side so as to exhibit a radial view from above, the space on the substrate can be effectively used. Can be increased. That is, it is possible to provide a (disk-shaped) DNA chip having a large amount of accumulated recording information.
[0024]
In addition, since the cell detection units are partitioned so as not to contaminate each other, different detection nucleotide chains are fixed for each cell detection unit or for each of a plurality of grouped cell detection units, and for each detection nucleotide chain. Separate and independent conditions can be set to allow the hybridization reaction to proceed.
[0025]
For example, a marker gene for disease onset can be grouped and fixed on a substrate. Thus, it is possible to simultaneously confirm the occurrence of a plurality of diseases using one substrate.
[0026]
In addition, it is possible to group detection nucleotide chains to be immobilized based on the difference in Tm or GC content. This makes it possible to precisely select reaction conditions such as buffer composition, concentration, etc., washing conditions, the concentration of the sample solution to be dropped, and the like, in which an active hybridization reaction is obtained, according to the properties of the nucleotide chain for detection. The risk of showing false positives or false negatives in the analysis work can be significantly reduced.
[0027]
Next, in the bioassay substrate according to the present invention, the reaction region of the detection unit is formed or arranged in a groove extending radially on the disc-shaped substrate, and on the inner wall surface of the groove, A configuration in which the detection surface is provided can also be adopted.
[0028]
The term "groove" formed on the substrate means a strip-shaped elongated microchannel structure. One field to which a bioassay substrate provided with the groove belongs is a disc-shaped microchannel. It can be called a channel array. Hereinafter, the detection unit having a configuration in which the reaction region is provided in the groove will be referred to as a “line detection unit” for convenience, following the “cell detection unit”.
[0029]
When this "groove detecting unit" is adopted, it is possible to use a liquid sending method utilizing a capillary phenomenon or a liquid sending method utilizing a centrifugal force generated by rotating a disk-shaped substrate by a predetermined method. . For example, it is possible to smoothly and reliably transfer a sample solution or a cleaning solution for removing excess target substances that did not bind actively after the reaction from the substrate central region into the groove (that is, the reaction region). It becomes.
[0030]
Note that, even in the case of the groove detecting unit, different nucleotide chains for detection can be fixed to the groove unit or a plurality of grouped groove units.
[0031]
In the bioassay substrate described above, when a means for providing the position information and the rotation synchronization information of the detection surface portion is provided, based on the position information and the rotation synchronization information, the detection nucleotide-containing solution and the target nucleotide-containing solution are used. Can be dropped while accurately following a predetermined reaction region.
[0032]
The means may be by wobbling or address pits provided on the substrate. Note that "wobbling" means that a groove (guide groove) for recording data by a user is slightly shifted from the center of the track in order to record information of a physical address (address) on the disk in advance on the disk. Meandering from side to side. Normally, FM modulation having a slight frequency deviation is performed on a frequency higher than the tracking servo band, and the amplitude of the sine wave modulation signal is carved on the substrate as a groove radial displacement.
[0033]
For the above-described reaction region provided on the bioassay substrate described above, a substance capable of causing a reversible phase change of a gel or sol between room temperature and an optimal reaction temperature (referred to as a “phase change substance”) .) Is also possible. An example of a phase change material is agarose gel.
[0034]
In this means, the phase change material is converted into a sol under a high temperature, the voltage is applied in this state to arrange nucleotide chains such as DNA, and then the temperature is lowered to gelate. Sometimes, a procedure of sol formation can be carried out under the optimal temperature condition of the reaction, and if the phase change substance is kept in a gel state during hybridization, the hybridization can be performed with the nucleotide chain of DNA or the like extended. It is preferable because it can be performed. When the phase change substance is in a gel state, hybridization may not proceed well when the sample solution containing the target nucleotide chain is dropped onto the detection unit. A vertical groove may be formed at a point in advance, and the sample solution may be dropped into this groove.
[0035]
In the present invention, either a means for labeling the target nucleotide chain with a fluorescent dye or a means using an intercalator can be employed. The “intercalator” is incorporated into the hybridized double-stranded nucleotide chain such that it is inserted into a hydrogen bond between the bases of the detection nucleotide chain and the target nucleotide chain. As a result, the fluorescence wavelength shifts to the longer wavelength side, and since there is a correlation between the fluorescence intensity and the amount of the intercalator incorporated into the double-stranded nucleotide chain, quantification is performed based on this correlation. Detection becomes possible.
[0036]
As described above, the present invention provides a novel bioassay substrate that can be used for gene mutation analysis, SNPs (single nucleotide polymorphism) analysis, gene expression frequency analysis, and the like, for drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine, and the like. Has the technical significance of providing it to related industries.
[0037]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. First, FIG. 1 is an external perspective view of a first embodiment of a bioassay substrate according to the present invention.
[0038]
In FIG. 1, a bioassay substrate (hereinafter abbreviated as “substrate”) denoted by reference numeral 1 is a substrate used for a disc-shaped substrate (disk) used for an optical information recording medium such as a CD, DVD, or MD. It is formed from a material. That is, the substrate 1 can optically read recorded information.
[0039]
The base material is formed in a disc shape using quartz glass, silicon, polycarbonate, polystyrene, or another disc-shaped synthetic resin, preferably a synthetic resin that can be injection-molded. By using an inexpensive synthetic resin substrate, a lower running cost can be realized as compared with a conventional glass chip.
[0040]
On one surface of the substrate 1, an aluminum deposition layer having a thickness of about 40 nm as a reflection film is formed, and this layer functions as a reflection film. This reflection film has a surface reflection of 4% or more from a substrate having a refractive index of 1.5 or more. A light transmitting layer made of transparent glass, transparent resin, or the like is formed on the reflective film.
[0041]
In the case where the base material is a material having a high reflectance, the surface of the base material itself functions as a reflection surface, so that the reflection film need not be formed. If a high reflectance film such as a metal film is formed, the fluorescence intensity of the fluorescent-labeled target substance can be detected with high sensitivity. The material and layer structure of the substrate 1 described above are the same in the substrate 4 (see FIG. 4) described later.
[0042]
Here, a large number of cell detectors 2 are arranged in the light transmitting layer so as to extend radially upward from a peripheral region at the center of the substrate 1. FIG. 2 is an external perspective view showing one of the cell detectors 2 in an enlarged manner.
[0043]
The cell detector 2 includes a detection surface 21 that has been subjected to a surface treatment so that the terminal portion of the nucleotide chain for detection indicated by the symbol D can be immobilized, and a nucleotide chain D for detection that has been immobilized on the detection surface 21 in advance. It has a positive electrode 22a and a negative electrode 22b that form an electric field (electric field) to be extended, and a reaction region 23 that is a field for a hybridization reaction between the detection nucleotide chain D and the target nucleotide chain T. The reaction region 23 is formed as a rectangular cell that opens upward, as viewed from above, for example, a cell having a depth of 1 μm, a length of 100 μm, and a width of 50 μ, but is not limited to the illustrated shape and size.
[0044]
The detection surface 21 is formed on an inner wall portion of the negative electrode 22b facing the reaction region 23 side. The detection surface 21 is surface-treated so that the terminal of the detection nucleotide chain D is fixed by a chemical bond such as a coupling reaction.
[0045]
That is, the detection surface 21 is not limited to a narrow one, as long as it has been subjected to a surface treatment suitable for immobilizing a previously processed terminal portion of the nucleotide chain D for detection such as a DNA probe. For example, in the case of the detection surface 21 that has been surface-treated with streptavidin, the end of the biotinylated nucleotide chain can be immobilized.
[0046]
Specifically, when an electric field is applied to the detection surface 21 so that the lines of electric force are concentrated, the nucleotide chain that has been subjected to elongation by the application of a high-frequency high voltage moves toward the detection surface 21, and the terminal site is detected. By colliding with the surface 21, the detection nucleotide chain D can be reliably immobilized on the detection surface 21. For this reason, the negative electrode 22b has a comb shape (see FIG. 2).
[0047]
Reference numeral 3 in FIG. 2 indicates a tip portion of a nozzle into which the sample solution S (S ′) is dropped. The nozzle 3 accurately transfers the sample solution S containing the detection nucleotide chain D and the sample solution S ′ containing the target nucleotide chain T to the reaction region 23 based on the position information and the rotation synchronization information provided from the substrate 1. And is configured to be dropped.
[0048]
Many of the detection nucleotide chains D dropped into the reaction region 23 are fixed to the detection surface 21 in a state where bases are superimposed, and therefore, during the hybridization reaction with the target nucleotide chain T dropped later. Causes the problem of steric hindrance.
[0049]
Therefore, in the present invention, an electric field is generated between the positive and negative electrodes 22 (22a, 22b) provided in the cell detection unit 2 so that an electric field is generated in the liquid phase (salt solution) stored and held in the reaction region 23. (Electric field), and the action of the detection nucleotide chain D extending linearly along the direction of the electric field is obtained. The condition of the electric field is considered to be 1 × 106 V / m and about 1 MHz as a preferable example of the condition (Masao Washizu and Osamu Kurosawa: “Electrostatic Manipulation of DNA in Microelectronics Structured Electronic Equipment. , No. 26, P. 1165-1172 (1900)).
[0050]
When the cell detectors 2 are arranged in a row on the substrate 1, either the configuration in which the positive and negative electrodes 22a and 22b are provided for each cell detector 2 or the configuration in which the positive and negative electrodes 22a and 22b are used as common electrodes are adopted. May be.
[0051]
The hydrogen bond (complementary bond) between the base of the linearly extended detection nucleotide chain D and the target nucleotide chain T labeled with a fluorescent dye or the like progresses efficiently because steric hindrance is reduced. . That is, the result is obtained that 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 favorable result that the reaction time of the hybridization is shortened and the probability of showing a false positive or false negative is also reduced.
[0052]
Here, FIG. 3 is an enlarged schematic view showing the vicinity of the detection surface 21 of the reaction region 23 of the cell detection unit 2. In FIG. 3, a detection nucleotide chain D whose terminal portion is fixed to the detection surface 21 and a target nucleotide chain T having a base sequence complementary to the base sequence of the detection nucleotide chain D are hybridized. The state of forming a heavy chain is schematically shown.
[0053]
For the reaction region 23 (similarly for a reaction region 53 described later), a phase change substance such as agarose gel or the like, which can cause a reversible phase change of a gel or sol between room temperature and an optimum reaction temperature (shown in FIG. ) Can also be filled. In this embodiment, the phase-change material is converted into a sol under a high temperature, the voltage is applied in this state to arrange nucleotide chains such as DNA, and then the temperature is lowered to gel, and further, At the time of hybridization, there is an advantage that a sol-forming procedure can be performed under the optimal reaction temperature condition. In addition, it is preferable that the phase change substance is kept in a gel state during hybridization, so that hybridization can be performed in a state where nucleotide chains such as DNA are extended.
[0054]
When the phase change substance is in a gelled state, when the sample solution S ′ containing the target nucleotide chain T is dropped onto the detection unit 2, the hybridization may not proceed properly. Similarly, a vertical groove may be formed in advance at the drop point, and the sample solution may be dropped into this groove.
[0055]
Here, the target nucleotide chain T may be labeled with a fluorescent dye, or an intercalator may be used. The intercalator is incorporated into the hybridized double-stranded nucleotide chain so as to be inserted into a hydrogen bond between the bases of the detection nucleotide chain D and the target nucleotide chain T. 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 intercalator incorporated into the double-stranded DNA. As the fluorescent dye used as the intercalator, POPO-1 and TOTO-3 can be considered.
[0056]
This is, for example, a target nucleotide complementary to a detection nucleotide chain D containing a marker gene known to be expressed when a specific disease occurs in a sample solution S ′ extracted from a predetermined cell or the like. The fact that a chain is present is recognizable, so that the cell is expected to have the disease.
[0057]
Next, a second embodiment of the substrate according to the present invention will be described with reference to FIG. 4A is a plan view of the substrate according to the second embodiment as viewed from above, and FIG. 4B is an enlarged plan view of a portion X in FIG. 4A.
[0058]
The substrate indicated by reference numeral 4 in FIG. The groove detecting section 5 includes a groove in which the reaction region 53 is radially extended on a disk-shaped substrate, and the inner surface I on one side in the longitudinal direction forming the groove has the same shape as the detection surface 21 described above. Are arranged at predetermined intervals. In addition, positive and negative electrodes 52a and 52b are provided opposite to each other where the detection surface 51 is formed so as to sandwich the reaction region 53 (see FIG. 4B). Note that the positive electrodes 52a, 52a,... (See FIG. 4B) disposed so as to be separated from each other can be used as a common electrode, and similarly, the positive electrodes 52a, 52a are disposed so as to be separated from each other. The negative electrodes 52b, 52b... (See FIG. 4B) can also be made common electrodes. That is, the positive common electrode and the negative common electrode can be arranged side by side on the substrate so as to face each other with the reaction region 53 interposed therebetween. According to this configuration, the needle-shaped probe can be pressed against the positive and negative common electrodes from above to supply electricity.
[0059]
The reaction area 53 may be formed in pits (not shown) arranged in each groove. By dropping a small droplet on the reaction area of the pit, almost the same spot size is realized and reproduced. Good fluorescence intensity detection can be realized.
[0060]
When the groove detecting section 5 having the above configuration is employed, a liquid sending means utilizing a capillary phenomenon or a liquid sending means utilizing a centrifugal force generated by rotating a disk-shaped substrate by a predetermined method is also used. be able to.
[0061]
More specifically, a liquid reservoir 6 is provided at the center of the substrate 4, and the liquid reservoir 6 is used to remove the sample solution S (S ′) and excess target substances that have not been actively bonded after the reaction. By injecting a cleaning liquid or the like and rotating the substrate 4, the liquid can be smoothly and reliably fed from the central region of the substrate into the groove (ie, the reaction region 53).
[0062]
In either case of the cell detection unit 2 or the groove detection unit 5, different detection nucleotide chains can be fixed to the detection unit or the plurality of grouped detection units.
[0063]
Hereinafter, the position information and the rotation synchronization information of the substrate 1 (4) will be briefly described. In the rotation direction of the substrate 1 (4), a number of address pits formed in advance by an optical disk mastering process are formed. When the substrate 1 (4) is considered as an optical disk, the reaction area 23 (53), which is a drop detection position, is considered as a user data area, and other areas are arranged with synchronous pits by a sample servo method or the like, and are used as tracking servos. Also, the position information is given by inserting an address part (geographical address on the disk) immediately afterwards.
[0064]
The address portion starts from a sector mark which is a head pattern, and includes a VFO (Variable Frequency Oscillator) for giving a rotation phase of a disk which is actually rotating, an address mark for giving a start position of address data, and a track and sector number. An ID (Identifier) and the like are combined.
[0065]
In order to perform a bioassay using the substrate 1 (4) having the above configuration, an apparatus having at least the following means and mechanism is used. That is, a substrate rotating means for rotatably holding the substrate 1 (4), and a solution S 'containing a nucleotide chain for detection and a solution S' containing a target nucleotide chain while rotating the substrate 1 (4) by the substrate rotating means. And a focus servo for maintaining a constant distance between (the nozzle of) the dropping means and the substrate 1 (4) in a predetermined order and timing to the reaction region 23 (53). A tracking servo mechanism for causing the drops of the solutions S and S ′ to follow the reaction region 23 (53) of the substrate 1 (4) based on the position information and the rotation synchronization information provided from the substrate 1 (4). It is a device (not shown) provided at least.
[0066]
Instead of using the above address pits, wobbling is formed on the track, the wobbling meander is adjusted so as to have clock information corresponding to the position, and addressing is performed by acquiring position information on the disk. May be. At the same time, tracking servo can be performed by using the wobbling frequency component. Further, by forming address pits and wobbling together, more accurate addressing and tracking servo become possible.
[0067]
In addition, it is preferable to use an inkjet printing method for the dropping unit. The reason is that the minute droplet can be accurately dropped by accurately following the predetermined reaction region 23 (53).
[0068]
The “ink-jet printing method” is a method in which a nozzle used in an ink-jet printer is applied, and is a method in which a substance for detection is ejected from a printer head to a substrate using electricity to fix the same as in an ink-jet printer. This method includes a piezoelectric ink jet method, a bubble jet (registered trademark) (registered trademark) (registered trademark) (registered trademark) method, and an ultrasonic jet method.
[0069]
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. The bubble jet (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 serving as a heater is buried in the nozzle, and a uniform bubble is created by controlling the temperature at about 300 ° C./s to eject a droplet. However, care must be taken when using it for biological material samples, since the liquid will be exposed to high temperatures. The ultrasonic jet method is a method in which an ultrasonic beam is applied to a free surface of a liquid to apply a locally high pressure to discharge droplets from the portion. A droplet having a diameter of about 1 μm can be formed at a high speed without the need for a nozzle.
[0070]
In the present invention, a "piezoelectric ink-jetting method" can be suitably adopted as the "ink-jet printing method". By changing the shape of the pulse to be applied, the size of a droplet (microdroplet) can be controlled, which is suitable for improving analysis accuracy. When the radius of curvature of the surface of the droplet 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. Further, 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.
[0071]
Next, the substrate information is read by irradiating the substrate 1 (4) with laser light (for example, blue laser light) to excite each reaction region 23 (53), and detecting the magnitude of the fluorescence intensity with a detector (not shown). ) To determine the binding reaction status between the detection nucleotide chain D and the labeled target nucleotide chain. Finally, the fluorescence intensity for each reaction region 23 (53) is visualized by A / D conversion and the distribution of the binding reaction ratio is displayed on the screen of the computer C.
[0072]
【The invention's effect】
(1) Since the bioassay substrate according to the present invention can form a large number and a large number of detection parts, the amount of accumulated information is large.
[0073]
(2) By applying an electric field to the nucleotide for detection fixed on the detection surface of the detection section to extend the nucleotide chain in a linear manner, the sample solution containing the target nucleotide chain is subjected to the reaction in the detection section. Since the configuration is such that the droplet is accurately dropped onto the region and the hybridization reaction between the detection nucleotide chain and the target nucleotide chain proceeds, the hybridization reaction can be performed efficiently in a short time.
[0074]
(3) Since the assay can be performed by selecting an optimal reaction condition or the like in units of detection units or grouped detection units, the incidence of false positive and false negative results can be significantly reduced. it can. Therefore, according to the bioassay substrate, comprehensive and efficient analysis can be performed with high accuracy, and the cost per recorded information is low.
[0075]
(4) The bioassay substrate according to the present invention is particularly useful as a DNA chip or a biosensor chip. Further, a disk-shaped microchannel array having a novel structure can be provided. When this substrate is used as a DNA chip, it can be used for gene mutation analysis, SNPs (single nucleotide polymorphism) analysis, gene expression frequency analysis, etc., and is widely used in drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and other fields. Can be used.
[Brief description of the drawings]
FIG. 1 is an external perspective view of a bioassay substrate (1) according to a preferred embodiment of the present invention when viewed upward.
FIG. 2 is an external perspective view showing, on an enlarged scale, one of cell detectors (2) provided on a substrate (1).
FIG. 3 is an enlarged view showing the vicinity of a detection surface (21) of a reaction area (23) of the cell detector (2).
FIG. 4A is a plan view of a bioassay substrate (4) according to a second embodiment as viewed from above.
(B) An enlarged plan view of a portion X in the previous figure (A).
[Explanation of symbols]
1,4 Bioassay substrate
21, 51 Detection surface
23,53 Reaction zone
D nucleotide chain for detection
T (labeled) target nucleotide chain

Claims (10)

On a disc-shaped substrate that allows optically reading recorded information,
A bioassay substrate provided with a detection unit including at least the following (1) to (3).
(1) A detection surface that has been subjected to a surface treatment so that the terminal site of the nucleotide chain for detection can be fixed.
(2) Positive and negative electrodes that form an electric field for extending the detection nucleotide chain fixed to the detection surface.
(3) A reaction region where a hybridization reaction between the detection nucleotide chain and the target nucleotide chain takes place. The bioassay according to claim 1, wherein the detection unit is a cell detection unit having the detection surface formed on one wall surface, and a plurality of the cell detection units are arranged on the disc-shaped substrate. Substrate. The bioassay substrate according to claim 2, wherein the cell detection unit is disposed on the disc-shaped substrate so as to exhibit a radial view from above. 4. The bioassay substrate according to claim 3, wherein different detection nucleotide chains are fixed to the cell detection unit or the plurality of grouped cell detection units. The reaction region of the detection unit is provided in a groove extending radially on a disk-shaped substrate, and the detection surface is provided on an inner wall surface of the groove. 2. The substrate for bioassay according to 1. The bioassay substrate according to claim 5, wherein different detection nucleotide chains are immobilized on the groove units or on a plurality of grouped groove units. 2. The bioassay substrate according to claim 1, further comprising means for providing position information and rotation synchronization information of the detection surface portion. The bioassay substrate according to claim 7, wherein the means is based on wobbling or address pits provided on the substrate. The positive substrate for bioassay according to claim 1, wherein the reaction region is filled with a substance capable of causing a reversible phase change of a gel or a sol between room temperature and an optimum reaction temperature. The bioassay substrate according to claim 1, wherein the prehybridization reaction is detected using an intercalator.

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