JP2005091251A - Bioassay method and bioassay apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enhance the efficiency of the interaction (hydridization or the like) of a detecting substance and a target substance in a substrate for bioassay such as a DNA chip, a protein chip or the like. <P>SOLUTION: The reaction region 12 of the substrate 1 for bioassay (DNA chip or the like), which is provided with at least a reaction region 12 becoming the interaction field between the detecting substance and the target substance, is irradiated with microwaves to regulate the temperature of the reaction region 12. Further, the bioassay apparatus 2 equipped with a microwave irradiation means 21 is provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a technique related to microarray technology such as a DNA chip and a protein chip. More specifically, the present invention relates to a method of adjusting the temperature of a reaction region provided on a bioassay substrate such as a DNA chip by performing microwave irradiation.

  The main prior art of the present invention will be described below. Currently, integrated substrates for bioassays called DNA chips or DNA microarrays (hereinafter collectively referred to as “DNA chips”), in which predetermined DNA is finely arranged by microarray technology, are used for gene mutation analysis, SNPs (single nucleotides). Polymorphism) analysis, gene expression frequency analysis, etc., and has begun to be widely used in drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and other fields.

  Since this DNA chip has a large number of DNA oligo chains and cDNA (complementary DNA) integrated on a glass substrate or silicon substrate, comprehensive analysis of intermolecular interactions such as hybridization becomes possible. It is characterized by dots.

  To briefly explain an analysis method using a DNA chip, a fluorescent probe dNTP is obtained by reverse transcription PCR reaction or the like by extracting mRNA extracted from cells, tissues, etc. with respect to a DNA probe solid-phased on a glass substrate or a silicon substrate. In this method, PCR amplification is carried out while incorporating, hybridization is performed on the substrate, and fluorescence is measured with a predetermined detector.

  Here, DNA chips can be classified into two types. The first type is one in which oligonucleotides are directly synthesized on a predetermined substrate using a photolithographic technique applying a semiconductor exposure technique, and a typical one is by Affymetrix (Affymetrix). (For example, refer to Patent Document 1). Although this type of chip has a high degree of integration, there is a limit to DNA synthesis on the substrate, and the length is about several tens of bases.

The second type is also referred to as “Stanford method”, and is prepared by dispensing and solidifying a prepared DNA on a substrate using a tip-breaking pin ( For example, see Patent Document 2). This type of chip is less integrated than the former, but has the advantage that a DNA fragment of about 1 kb can be immobilized.

  In addition, a solution that contains a target substance for this detection substance is solidified on a minute detection surface portion provided in a thin flat plate shape, which is currently called a biosensor chip including a protein chip. Biosensor technology has been developed to observe and analyze the interaction between both substances using the principle of surface plasmon resonance, quartz oscillator, etc., such as antigen-antibody reaction and hormone response reaction. It has become a useful technique in analyzing the interaction between substances.

And in order to raise reaction efficiency of these series of DNA chips, protein chips, etc., temperature is adjusted using a heater etc. and reaction efficiency is raised.
Special table hei 4-505633 report Japanese National Patent Publication No. 10-503841

  However, the conventional techniques have the following problems to be solved.

  In the above-described conventional DNA chip technology and biosensor technology, in a narrow reaction part of a two-dimensional substrate, a detection nucleic acid such as a DNA probe or protein is immobilized (immobilized), and a hybridization reaction or antigen is detected. Because it is a technology that promotes interactions between substances such as antibody reactions, the degree of freedom of reaction products is limited spatially, and interactions such as hybridization are performed exclusively based on Brownian motion of the target nucleotide chain, etc. I was letting. For this reason, the conventional DNA chip technology or biosensor technology has a technical problem that the interaction efficiency is low and the reaction time is long.

  In addition, when the temperature of the reaction region is increased using a heater or the like, although there is a tendency for some reactions such as hybridization to be promoted, there is a problem that not only temperature management is difficult, but also the apparatus becomes large. there were. Further, there is a problem that temperature unevenness occurs in the reaction region and a desired reaction efficiency cannot be obtained.

  Furthermore, when the medium in the reaction region is composed of a gel, no convection of the liquid layer occurs in the reaction region. Therefore, temperature propagation is performed exclusively by radiation, which further deteriorates the hybridization efficiency.

  Therefore, the present invention makes it possible to increase the efficiency of the interaction between the detection substance and the target substance (hybridization, etc.) by adjusting the temperature of the reaction region where the interaction between substances such as hybridization occurs. The main object is to provide a bioassay method and a bioassay device.

  In order to solve the above technical problem, the present invention provides the following means.

  In the present invention, first, a method for adjusting the temperature of a reaction region by irradiating microwaves to the reaction region serving as an interaction field between substances is provided. By irradiating the reaction region with microwaves, the temperature of the medium (buffer solution, gel, etc.) in the reaction region can be raised. Therefore, the target interaction can be promoted by adjusting the temperature of the reaction region by microwave irradiation so that the reaction region has a temperature most suitable for the interaction between substances.

  This method can be used, for example, for interaction (hybridization) between single-stranded nucleic acids. Hybridization refers to the formation of a double-stranded nucleic acid by hydrogen bonding between base pairs in which single-stranded nucleic acids are complementary.

  The double-stranded nucleic acid is unwound from the double-stranded structure by applying heat to become single-stranded. The temperature at which a double-stranded nucleic acid melts into a single strand is referred to as a melting temperature Tm (Melting Temperature), and the melting temperature Tm of each nucleic acid is different based on the GC content of the base sequence of the nucleic acid forming the double strand. In general, it is said that hybridization is most accelerated when the temperature is about 5 ° C. lower than the melting temperature Tm. Therefore, target hybridization can be promoted by adjusting the temperature in the reaction region so that the temperature is most suitable for hybridization.

  Further, by setting the temperature of the reaction region to be higher than the melting temperature Tm by microwave irradiation before the target hybridization reaction, the single-stranded nucleic acid used for the reaction is mishybridized before the reaction. This can be prevented.

  In addition, according to the melting temperature Tm of each nucleic acid, by appropriately adjusting the temperature of the reaction region by microwave irradiation, only the mishybridized nucleic acid generated in the target hybridization reaction is melted into a single strand, It can also be removed.

  Microwaves can adjust the intensity of irradiation energy. Therefore, by irradiating the reaction region with an appropriate amount of microwaves, it is possible to affect not only the medium in the reaction region but also the nucleic acid itself. Microwaves are mainly used for nucleic acids: (1) the action of allowing nucleic acids to move actively within the reaction region; (2) the activation of the Brownian movement of nucleic acids and the opportunity for association of single-stranded nucleic acids. (3) Dissociates mishybridized nucleic acid bonds and promotes correct hybridization.

  As described above, microwave irradiation is performed at an appropriate intensity, at an appropriate time, at an appropriate interval, and at an appropriate timing, and by appropriately combining such irradiations, the desired interaction such as hybridization can be achieved. Can be promoted efficiently.

  In the present invention, nucleic acid means a polymer of a phosphate ester of a nucleotide in which a purine or pyrimidine base and a sugar are glycosidically bonded, and an oligonucleotide, a polynucleotide containing a DNA probe, a purine nucleotide and a pyrimidine nucleotide are polymerized. Widely includes DNA (full length or a fragment thereof), cDNA obtained by reverse transcription (cDNA probe), RNA and the like.

  Next, the present invention provides a method for adjusting the temperature of a reaction region in which microwave irradiation is performed on the reaction region of a bioassay substrate provided with at least a reaction region serving as an interaction field between substances. Bioassay substrates include DNA chips and protein chips.

  For example, in the case of a DNA chip, one single-stranded nucleic acid is previously present in the reaction region as a substance for detection, and the other single-stranded nucleic acid is dropped as a target substance into the reaction region. As a result, when both single-stranded nucleic acids are complementary, interaction (hybridization) between the single-stranded nucleic acids occurs.

  In the case of a protein chip, one substance (protein, etc.) is preliminarily present in the reaction area as a detection substance, and the other substance (protein, nucleic acid, etc.) is added as a target sample to the reaction area. To do. As a result, when there is an interaction between the two, interactions such as an antigen-antibody reaction, protein binding, an action between an enzyme (protein) and its substrate (protein, nucleic acid, etc.), etc. occur. .

  Many of the interactions that occur in the reaction region of a bioassay substrate such as a DNA chip or protein chip have the most suitable temperature for the reaction. For example, as described above, it is said that hybridization between nucleic acids is most likely to occur at a temperature about 5 ° C. lower than the melting temperature Tm. In the case of an enzyme, since there is an optimum temperature at which the reaction rate is maximized, it is necessary to adjust the temperature of the medium in the reaction region. Therefore, in the present invention, the temperature in the reaction region is adjusted by performing microwave irradiation on the reaction region of the bioassay substrate. As a result, the temperature of the reaction region of the bioassay substrate can be adjusted to a temperature at which the target interaction is most likely to occur.

  Similarly to the above, since the intensity of the irradiation energy of the microwave can be adjusted, by irradiating the reaction region with an appropriate amount of microwave, not only the medium in the reaction region but also the reaction. It is also possible to act on the substance itself in the region.

  Therefore, microwave irradiation is performed at an appropriate intensity, an appropriate time, an appropriate interval, and an appropriate timing, and by appropriately combining such irradiations, the target mutual in the reaction region of the bioassay substrate can be obtained. The action can be promoted efficiently.

  In addition, in the case of a DNA chip, it is possible to add not only single-stranded nucleic acids but also double-stranded nucleic acids as target substances to the reaction region. Since the temperature of the reaction region can be adjusted by microwave irradiation according to the present invention, if the target material is dropped onto the reaction region, the temperature of the medium in the reaction region is increased to Tm (melting temperature) or more of the target material. The target substance melts into a single strand. After that, if the temperature of the medium in the reaction region is lowered, most of the target substance returns to double-stranded nucleic acid, but part of the target substance interacts with the detection substance (single-stranded nucleic acid) (hybridization). Wake up. Therefore, according to the present invention, it is possible to simplify the procedure for using the DNA chip.

  The substrate for bioassay is not limited to a DNA chip or a protein chip, but is a reaction that acts as a field for interaction between substances such as a reaction between polymers, a reaction between molecules, a reaction between a polymer and a molecule. It is intended to broadly encompass a bioassay substrate provided with at least a region.

  Subsequently, an apparatus for detecting an interaction between substances using a bioassay substrate provided with a reaction region in which a detection substance exists, in which a sample solution containing a target substance is dropped onto the reaction area. There is provided a bioassay device comprising at least means for performing microwave irradiation on the reaction region, and means for controlling the microwave irradiation.

  For example, when the bioassay substrate is a DNA chip, a single-stranded nucleic acid is present in advance as a detection substance in the reaction region of the bioassay substrate. When a sample solution containing a nucleic acid serving as another target substance is dropped onto the reaction region of the bioassay substrate using the bioassay device according to the present invention, if the target substance is the target substance, an interaction (hybridization) ) Occurs.

  Since the bioassay device according to the present invention includes means for irradiating the reaction region with microwaves, the temperature of the medium in the reaction region is adjusted to a temperature most suitable for interaction (hybridization). can do. As described above, it is generally said that hybridization is most promoted at a temperature about 5 ° C. lower than the melting temperature Tm of nucleic acid. Therefore, the target interaction is promoted by adjusting the temperature of the reaction region to a predetermined temperature by microwave irradiation.

  In addition, since this apparatus also includes means for controlling the microwave irradiation, the microwave irradiation is performed at an appropriate intensity, at an appropriate time, at an appropriate interval, and at an appropriate timing. Can be combined as appropriate. Therefore, the temperature of the reaction region of the bioassay substrate can be appropriately adjusted according to the purpose. In addition, since the irradiation energy of the microwave can be adjusted, as described above, not only the medium in the reaction region but also the nucleic acid itself is (1) an action that enables the nucleic acid to move actively in the reaction region. , (2) the action of activating the Brownian movement of nucleic acids and increasing the chance of association of single-stranded nucleic acids, (3) the action of dissociating mishybridized nucleic acids and promoting correct hybridization, etc. Can be exerted. Therefore, it is possible to efficiently promote the interaction such as the target hybridization.

  In addition, since the temperature of the reaction region of the bioassay substrate can be adjusted by this apparatus, the nucleic acid serving as the target substance is not limited to a single-stranded nucleic acid as described above. Can be used.

  Note that this apparatus is not limited to the case of using a DNA chip, but a field of interaction between substances such as a protein chip, a polymer, a low molecule, a reaction between a polymer and a low molecule. The case where a substrate for bioassay provided with at least a reaction region is used is widely included. For example, in the case of a protein chip, the protein often has an optimum temperature at which the reaction rate is maximized. Therefore, the present apparatus that can adjust the temperature of the reaction region of the bioassay substrate is effective.

  In addition to the above-described means, the apparatus can include means for irradiating the reaction region with excitation light for detecting interaction. By providing a means for irradiating the reaction region with excitation light, if the detection substance and the target substance interact in the reaction region of the bioassay substrate, the light excited from the substance in the reaction region Are effective as means for detecting and measuring whether or not an interaction occurs between the detection substance and the target substance.

  Examples of the method for emitting a signal such as light when irradiated with excitation light include, for example, a method of fluorescently labeling a target substance in advance, a method of mixing a fluorescent substance when dropping the target substance, and dropping the reaction substance onto the reaction region, a target substance And a method in which the fluorescent substance is dripped approximately simultaneously or in time series and separately. By using these methods, it is possible to detect and measure the interaction between substances accurately and with high sensitivity. In addition, when detecting an interaction (hybridization) between nucleic acids, there is a method using an intercalator. The “intercalator” is incorporated into the hybridized double-stranded nucleic acid so as to be inserted into the hydrogen bond between the bases of the detection nucleic acid substance and the target nucleic acid substance. This shifts the fluorescence wavelength to the longer wavelength side compared to the fluorescence wavelength from a double-stranded nucleic acid not containing an intercalator, and between the fluorescence intensity and the amount of intercalator incorporated into the double-stranded nucleotide chain. Since there is a correlation, quantitative detection is possible based on this correlation.

  This apparatus can be provided with an optical means for collecting light emitted from the substance in the reaction region and detecting the light intensity by irradiating the excitation light. As a result, it is possible to collect the light excited when the interaction between the substances occurs in the reaction region and detect the light intensity, thereby improving the detection performance of the interaction between the substances. .

  In addition, the bioassay device according to the present invention further includes a recording / reproducing part for recording / reproducing signals recorded on the bioassay substrate, a part having an Internet connection function for connecting to the Internet, a recording / reproducing part, and an Internet connection. A control part that controls functions, processes and generates predetermined data, records information on a bioassay substrate, reads information, and the like can be employed. As a result, a series of procedures can be automated, and the apparatus can be controlled by simple input means or the like. In addition, information such as pre-recorded samples and data such as experimental results can be easily processed and processed, and the information can be stored on a bioassay substrate. It becomes possible.

  The effects produced by the present invention are as follows.

  The temperature in the reaction region can be adjusted by irradiating the reaction region serving as an interaction field between substances with microwaves. Since the temperature of the reaction region can be adjusted to a temperature optimal for the interaction between substances, the interaction can be promoted.

  Microwave irradiation acts not only on the medium in the reaction region, but also on the interacting substance itself such as nucleic acid and protein. Therefore, the target interaction (hybridization or the like) can easily occur by adjusting the microwave irradiation energy.

  Microwave irradiation is performed at an appropriate intensity, at an appropriate time, at an appropriate interval, at an appropriate timing, and by appropriately combining such irradiations, the desired interaction such as hybridization is efficiently promoted. Can be made.

  By irradiating the reaction region of a bioassay substrate such as a DNA chip or protein chip with microwaves, the same effects as described above can be obtained.

  Since the bioassay device according to the present invention comprises a microwave irradiation means for the reaction region and a means for controlling the microwave irradiation, the reaction region of the bioassay substrate such as a DNA chip or a protein chip, Microwave irradiation can be performed at an appropriate size, an appropriate time, an appropriate interval, and an appropriate timing. Therefore, the interaction between substances in the reaction region of the bioassay substrate can be regulated and promoted.

  The bioassay device according to the present invention can detect and measure a target interaction with high sensitivity by using a fluorescent substance, an intercalator, or the like.

(Microwave irradiation pattern)
In the microwave irradiation according to the present invention, irradiation intensity (irradiation energy), irradiation time, irradiation interval, and irradiation timing can be appropriately combined depending on the purpose.

  When a bioassay substrate is used as a DNA chip, it is said that the interaction (hybridization) between single-stranded nucleic acids occurs most efficiently at a temperature about 5 ° C. lower than the melting temperature Tm. In the present invention, as shown in FIG. 1, by irradiation with microwaves, the temperature of the reaction region can be raised to a temperature about 5 ° C. lower than the melting temperature Tm, and the microwaves are intermittently irradiated. By doing so, the temperature of the reaction region can be kept constant to some extent.

  As shown in FIG. 2, when intermittently irradiating microwaves, the temperature of the reaction region can be kept constant by shortening the irradiation interval and gradually decreasing the irradiation intensity. It becomes. In addition, by raising the temperature of the reaction region once to the target temperature by microwave irradiation, and then irradiating microwaves with low irradiation intensity without interruption, the temperature of the reaction region can be reduced to the target temperature. Can be kept constant.

  As described above, the temperature of the reaction region can be adjusted by appropriately adjusting the microwave irradiation intensity (irradiation energy), irradiation time, irradiation interval, irradiation timing, and the like.

(Bioassay substrate)
FIG. 3 is a diagram schematically showing an upper surface and a cross section of the bioassay substrate 1. This bioassay substrate 1 has a flat plate shape with a circular main surface, like an optical disc such as a CD (Compact Disc) and a DVD (Digital Versatile Disc), and is composed of quartz glass, silicon, polycarbonate, polystyrene, It is formed of a synthetic resin that can be molded into a disc shape, preferably a synthetic resin that can be injection molded. By using an inexpensive synthetic resin substrate, a low running cost can be realized as compared with a conventional glass chip. The bioassay substrate 1 is used for a DNA chip, protein chip, etc., and is not limited to the shape and material of the bioassay substrate 1 in FIG. Widely encompass.

  A central hole 11 is formed at the center of the bioassay substrate 1. A chucking mechanism or the like for holding and rotating the bioassay substrate 1 is inserted into the center hole 11 when the bioassay substrate 1 is mounted on the bioassay device.

  The circular main surface of the bioassay substrate 1 is divided into two parts, a recording part 3 and a reaction part 4 formed concentrically in the radial direction. In this example, the recording site 3 is located on the inner circumference side, and the reaction site 4 is located on the outer circumference side. The recording part 3 is a part where information is optically recorded and reproduced by irradiating a laser beam in the same manner as the optical disc information recording medium. The reaction site 4 is a site serving as a field for the interaction (hybridization or the like) between the detection substance and the target substance.

  The layer structure of the bioassay substrate 1 is formed of an information layer 5 and a reaction layer 6 as shown in FIG. Here, it is assumed that the information layer 5 is located in the lower layer and the reaction layer 6 is located in the upper layer. Further, the surface on the reaction layer 6 side of the bioassay substrate 1 is referred to as an upper surface 1a, and the surface on the information recording layer 5 side is referred to as a lower surface 1b.

  The information layer 5 is formed with a signal recording film 7 for reproducing or recording / reproducing data by irradiating a portion corresponding to the recording portion 3 with a laser beam such as a pit or a phase change material. . Such a signal recording film 7 can be formed by a disc production method similar to that for optical discs such as CD and DVD.

  The signal recording film 7 is subjected to signal reproduction or recording reproduction by irradiating laser light from the lower surface 1 b side of the bioassay substrate 1. The information layer 5 is formed of a material that transmits the excitation light and control light irradiated during analysis and the wavelength of fluorescence emitted from the fluorescent labeling agent L during analysis. For example, the information layer 5 is made of a material such as quartz glass, silicon, polycarbonate, or polystyrene. Details of excitation light, control light, and fluorescence will be described later.

  On the upper surface (main surface) of the bioassay substrate 1, reaction regions 12 that are finely recessed in the back surface direction are accumulated. This reaction region 12 is a region serving as an interaction field between the detection substance and the target substance. For example, in the case of a DNA chip, a probe DNA (detection substance, detection nucleotide chain) and sample DNA ( This is a region for interaction with a target substance, a target nucleotide chain), specifically, a region for hybridization reaction.

  FIG. 4 is a schematic cross-sectional view schematically showing one of the reaction regions 12 on the bioassay substrate 1. In the reaction region 12 having a shape recessed from the main surface of the bioassay substrate 1, a medium such as a buffer solution or a gel is used in order to maintain the stability of the interacting substance and to cause the interaction efficiently. Can be accommodated.

For example, when the bioassay substrate 1 is used as a DNA chip or the like, a material for immobilizing one end of a nucleic acid used as a detection substance on the bottom surface 13 of the reaction region 12 can be used. For example, as shown in FIG. 4, SiO ₂ whose surface can be modified with silane can be formed into a layer on the bottom surface 13 by forming a film with a thickness of about 50 nm by sputtering or electron beam evaporation. Thereby, the nucleic acid having one end modified with the NCO group can be bound to the bottom surface 13 of the reaction region 12. In addition, when the bottom surface 13 is treated with streptavidin, it is suitable for immobilizing a biotinylated nucleotide chain. In addition, if a magnetic bead is attached to the end of the nucleic acid and the nucleotide chain is temporarily fixed with a magnet or the like from the back surface, there is no need for special surface treatment.

  For example, the nucleic acid N used as the detection substance can be immobilized on the bottom surface 13 of the reaction region 12 as shown in FIG. When a sample solution or the like containing the target substance N ′ is dropped in the reaction region 12, the detection substance N and the target substance N ′ cause an interaction (hybridization), and the target interaction can be detected. .

  For example, when a gel is used as the medium, the detection substance only needs to be confined in the gel, and therefore it is not always necessary to immobilize the detection substance on the bottom surface 13. Therefore, the present invention is not limited to the above-described case where the detection substance is limited to the bottom surface 13. Moreover, although the above-mentioned example uses a DNA chip, the present invention is not limited to the DNA chip, and widely includes substrates used for bioassays including protein chips. In particular, since the bioassay substrate 1 according to the present invention can use a gel or the like as a medium, proteins and other macromolecules, low molecular substances, and the like that are difficult to be immobilized on the bottom surface 13 can also be used as a gel or the like. By confining, there is an advantage that the reaction region 12 can be fixed.

(Bioassay device)
Next, a bioassay device 2 that analyzes an interaction between substances using the above-described bioassay substrate 1 will be described with reference to FIG.

  As shown in FIG. 6, the bioassay device 2 includes a microwave irradiation unit 21 that irradiates microwaves to the reaction region 12 of the bioassay substrate 1, and a disk that holds and rotates the bioassay substrate 1. From the loading unit 22, a dropping unit 23 that stores various solutions for interaction such as hybridization, and drops the solution to the reaction region 12 of the bioassay substrate 1, and the reaction region 12 of the bioassay substrate 1 An excitation light detection unit 24 for detecting excitation light, a control / servo unit 25 for managing and controlling each of the above components, and a recording for recording and reproducing signals on the signal recording film 7 of the bioassay substrate 1 Playback unit 26, Internet connection function 27 for connecting to the Internet, microwave irradiation unit 21, recording / playback unit 26, Internet connection By controlling the capacity 27 and the like, integrated device M, and a control unit 28 that performs predetermined data processing.

  The microwave irradiation unit 21 is disposed on the upper side of the horizontally loaded bioassay substrate 1, that is, on the upper surface 1a side. The microwave irradiation unit 21 irradiates the microwave toward the reaction region 12 provided on the upper surface 1 a of the bioassay substrate 1.

  The disk loading unit 22 is inserted into the center hole 11 of the bioassay substrate 1 to hold the bioassay substrate 1 and rotates the bioassay substrate 1 by driving the chucking mechanism 31. And a spindle motor 32 to be driven. The disc loading unit 22 rotationally drives the bioassay substrate 1 in a state where the bioassay substrate 1 is held horizontally so that the upper surface 1a side is upward.

    The dropping unit 23 includes a storage unit 33 that stores the sample solution, and a dropping head 34 that drops the sample solution in the storage unit 33 onto the bioassay substrate 1. The dropping head 34 is disposed above the upper surface 1a of the bioassay substrate 1 loaded horizontally. Further, the dropping head 34 controls the relative position with respect to the bioassay substrate 1 in the radial direction based on the position information and rotation synchronization information read from the address pits 9 of the bioassay substrate 1 shown in FIG. (N), a sample solution containing the sample DNA (N ′) or the fluorescent labeling agent L is configured to drop following the predetermined reaction region 12 accurately.

  The excitation light detector 24 includes (1) an optical head 40, (2) an excitation light source 44 that emits the excitation light P, a collimator lens 45 that uses the excitation light P emitted from the excitation light source 44 as a parallel light beam, and a collimator lens. A portion composed of a first dichroic mirror 46 that refracts the excitation light P, which has been made into a parallel light beam by 45, on the optical path X and irradiates the light guide mirror 43; And a second dichroic mirror 49 that refracts the fluorescence F emitted onto the optical path X from the optical head 40 and irradiates the avalanche photodiode 47 with the condenser lens 48 that collects the fluorescence F. (4) The control light source 50 that emits the control light V, the collimator lens 51 that uses the control light V emitted from the control light source 50 as a parallel light beam, and the reflected light R of the control light V are detected. A photodetection circuit 52, a cylindrical lens 53 that collects the reflected light R on the photodetection circuit 52 by generating astigmatism, and an optical separator 54 that separates the control light V and the reflected light R. Part.

  The optical head 40 includes an objective lens 41, a biaxial actuator 42 that supports the objective lens 41 so as to be movable, and a light guide mirror 43. The objective lens 41 is supported by the biaxial actuator 42 so that the central axis thereof is substantially perpendicular to the surface of the bioassay substrate 1. Therefore, the objective lens 41 can collect the light beam incident from the lower side of the bioassay substrate 1 on the bioassay substrate 1. The biaxial actuator 42 supports the objective lens 41 so as to be movable in two directions perpendicular to the surface of the bioassay substrate 1 and in the radial direction of the bioassay substrate 1. By driving the biaxial actuator 42, the focal point of the light collected by the objective lens 41 can be moved in the direction perpendicular to the surface of the bioassay substrate 1 and in the radial direction. Therefore, the optical head 40 can perform the same control as the just focus control and positioning control in the optical disc system.

  The light guide mirror 43 is disposed at an angle of 45 ° with respect to the optical path X. The optical path X is an optical path through which the excitation light P, fluorescence F, control light V, and reflected light R enter and exit the optical head 40. Excitation light P and control light V are incident on the light guide mirror 43 from the optical path X. The light guide mirror 43 reflects the excitation light P and the control light V to be refracted by 90 ° and enters the objective lens 41. The excitation light P and the control light V incident on the objective lens 41 are collected by the objective lens 41 and applied to the bioassay substrate 1. Further, the fluorescent light F and the reflected light R of the control light V are incident on the light guide mirror 43 through the objective lens 41 from the bioassay substrate 1. The light guide mirror 43 reflects the fluorescent light F and the reflected light R, refracts them 90 °, and emits them onto the optical path X. A drive signal for moving the optical head 40 by a sled and a drive signal for driving the biaxial actuator 42 are supplied from the control / servo unit 25.

  The excitation light source 44 is a light emitting means having a laser light source having a wavelength capable of exciting the fluorescent labeling agent. Here, the excitation light P emitted from the excitation light source 44 is laser light having a wavelength of 405 nm. The wavelength of the excitation light P may be any wavelength as long as it can excite the fluorescent labeling agent. The collimator lens 45 turns the excitation light P emitted from the excitation light source 44 into a parallel light beam. The first dichroic mirror 46 is a reflecting mirror having wavelength selectivity, reflects only the light having the wavelength of the excitation light P, and transmits the light having the wavelength of the fluorescence F and the control light V (its reflected light R). To do. The first dichroic mirror 46 is inserted on the optical path X with an angle of 45 °, reflects the excitation light P emitted from the collimator lens 45 to be refracted by 90 °, and excites the light guide mirror 43. Light P is irradiated.

  The avalanche photodiode 47 is a very sensitive photodetector, and can detect the fluorescent light F with a weak light amount. The wavelength of the fluorescence F detected by the avalanche photodiode 47 is about 470 nm here. Further, the wavelength of the fluorescence F varies depending on the type of the fluorescent labeling agent. The condensing lens 48 is a lens for condensing the fluorescence F on the avalanche photodiode 47. The second dichroic mirror 49 is inserted at an angle of 45 ° on the optical path X, and is disposed downstream of the first dichroic mirror 46 when viewed from the light guide mirror 43 side. Therefore, the fluorescence F, the control light V, and the reflected light R are incident on the second dichroic mirror 49, and the excitation light P is not incident. The second dichroic mirror 49 is a reflecting mirror having wavelength selectivity, reflects only light having the wavelength of the fluorescence F, and transmits light having the wavelength of the control light (reflected light R). The second dichroic mirror 49 reflects and refracts the fluorescence F emitted from the light guide mirror 43 of the optical head 40 and irradiates the avalanche photodiode 47 with the fluorescence F via the condenser lens 48. .

  The control light source 50 is a light emitting unit having a laser light source that emits laser light having a wavelength of, for example, 780 nm. The wavelength of the control light V is set to a wavelength at which the address pit 9 can be detected and information can be recorded and reproduced with respect to the signal recording film 7. Further, the wavelength of the control light V is set to a wavelength different from the wavelengths of the excitation light P and the fluorescence F. As long as such a wavelength is used, the wavelength of the control light V is not limited to 780 nm and may be any wavelength. The collimator lens 51 turns the control light V emitted from the control light source 50 into a parallel light beam. The control light V made into a parallel light beam is incident on the optical separator 54.

  The photodetector circuit 52 generates a detector for detecting the reflected light R, a focus error signal, a positioning error signal, a reproduction signal for the address pit 9, and a reproduction signal for the signal recording film 7 from the detected reflected light R. And a signal generation circuit. Since the reflected light R is light generated by reflecting the control light V with the bioassay substrate 1, the wavelength thereof is 780 nm, which is the same as that of the control light.

  Note that when the optical head 40 irradiates laser light to the reaction site 4 (outer periphery side, see FIG. 3) of the bioassay substrate 1, the focus error signal is the light collected by the objective lens 41. An error signal indicating the amount of displacement between the in-focus position and the reaction layer 6 of the bioassay substrate 1 is obtained. The positioning error signal indicates the amount of displacement between the position of the predetermined reaction region 12 and the focal position in the disc radial direction. Signal. When the optical head 40 irradiates the recording site 3 (inner peripheral side, see FIG. 3) of the bioassay substrate 1 with a laser beam, the focus error signal is the sum of the light collected by the objective lens 41. The error signal indicates the amount of positional deviation between the focal position and the signal recording film 7, and the positioning error signal is a signal indicating the amount of positional deviation between the track position and the focal position of the signal recording film 7 in the disc radial direction.

  The reproduction signal of the address pit 9 is detected only when the reaction site 4 (outer side) is irradiated with laser light, and is described in the address pit 9 recorded on the bioassay substrate 1. It is a signal indicating the contents. By reading out this information content, it is possible to identify the reaction region 12 that is currently irradiated with the control light V.

  The reproduction signal of the signal recording film 7 is detected only when the recording part 3 (inner peripheral part) is irradiated with laser light, and is a signal indicating the information content recorded on the track of the signal recording film 7. is there.

  The photodetect circuit 52 supplies the control / servo unit 25 with a focus error signal, a positioning error signal, and a reproduction signal for the address pit 9 generated based on the reflected light R.

  The cylindrical lens 53 is a lens for condensing the reflected light R on the photodetection circuit 52 and causing astigmatism. By generating astigmatism in this way, the focus error signal can be generated by the photodetect circuit 52.

  The optical separator 54 includes a light separation surface 54a made of a deflecting beam splitter and a quarter wavelength plate 54b. In the optical separator 54, light incident from the opposite side of the quarter wavelength plate 54b is transmitted through the light separation surface 54a, and when the reflected light of the transmitted light is incident from the quarter wavelength plate 54b side, the light is separated. The separation surface 54a has a function of reflecting. The light separator 54 has a light separation surface 54 a inserted at an angle of 45 ° on the optical path X, and is disposed downstream of the second dichroic mirror 49 when viewed from the light guide mirror 43 side. Therefore, the optical separator 54 transmits the control light V emitted from the collimator lens 51 and makes the control light V incident on the light guide mirror 43 in the optical head 40 and guides the optical head 40. The reflected light R emitted from the mirror 43 is reflected to be refracted by 90 °, and the reflected light R is irradiated to the photodetection circuit 52 through the cylindrical lens 53.

  The control / servo unit 25 performs various servo controls based on the focus error signal, the positioning error signal, and the address pit 9 reproduction signal detected by the excitation light detection unit 24.

  When the reaction site 4 (outer side) is irradiated with laser light, the control / servo unit 25 drives the biaxial actuator 42 in the optical head 40 based on the focus error signal to The distance between the reaction area 12 is controlled, and based on the positioning error signal, the biaxial actuator 42 in the optical head 40 is driven to control the positional relationship in the radial direction between the objective lens 41 and the reaction area 12, and the address pit The sled movement control of the optical head 40 is performed based on the reproduction signal 9 to move the optical head 40 to a predetermined radial position.

  When the recording part 3 (inner peripheral part) is irradiated with the laser beam, the control / servo unit 25 drives the biaxial actuator 42 in the optical head 40 based on the focus error signal, and the objective lens 41. And the signal recording film 7 is controlled, and the biaxial actuator 42 in the optical head 40 is driven based on the positioning error signal, whereby the positional relationship in the radial direction between the objective lens 41 and the recording track of the signal recording film 7 is controlled. To control.

    The recording / reproducing unit 26 demodulates and decodes the reproduction signal of the data recorded on the information recording layer 7 and outputs the data, and also encodes and modulates the recording data to be recorded on the information recording layer 7. At the time of reproduction, the recording / reproducing unit 26 receives the reproduction signal output from the excitation light detecting unit 24 and outputs the demodulated and decoded data to the outside. In recording, the recording / reproducing unit 26 inputs recording data from the outside, supplies the encoded and modulated data to the excitation light detecting unit 24, and drives the control light source 50 that emits the control light V. .

  The Internet connection function 27 is a function for accessing information using URL information recorded in advance in any area of the bioassay substrate 1 or a URL specified by an input means (not shown).

  As shown in FIG. 6, the control unit 28 includes an input unit and a display unit, and controls the microwave irradiation unit 21, the recording / reproducing unit 26, and the Internet connection function unit 27.

  The irradiation intensity (irradiation energy), irradiation time, irradiation interval, irradiation timing, and the like of the microwave irradiated from the microwave irradiation unit 21 are designed so as to be adjusted by an instruction from the control unit 28.

  The image image based on the hybridization result read by the recording / reproducing unit 26 is compressed into a predetermined format (for example, JPG) and recorded on the bioassay substrate 1 via the recording / reproducing unit 26. Further, the Internet connection function 27 is controlled to collect related information, and the data is similarly recorded.

  In addition, when the bioassay substrate 1 is mounted on the bioassay device 2, it is determined from the written information whether the substrate 1 has already been used for the inspection, and when it is determined that the substrate has already been used for the inspection. Controls to display the written image and collected data, or to display a warning that has already been inspected when the board is used for inspection again, and to limit the measurement operation. The part 28 is designed.

  In order to determine whether or not the disk has been used for inspection, a 3.5-inch floppy disk, a switch indicating that MD has been written, a claw for preventing erasure in a video tape, and the like may be provided.

  In the bioassay device 2 configured as described above, for example, when a nucleic acid bioassay is performed as a DNA chip, the following operation is performed.

  While rotating the bioassay substrate 1, the bioassay apparatus 2 drops a solution containing the sample DNA (N ′) into the reaction region 12 as shown in FIG. 7, and the probe DNA (N) and the sample DNA (N ′) ) Are allowed to interact with each other (hybridization).

  A buffer solution containing a fluorescent labeling agent L is dropped on the bioassay substrate 1 after the hybridization treatment, and as shown in FIG. 7, the probe DNA (N) and sample DNA (N ′) are in a double helix. Fluorescent labeling agent L is inserted into In addition, the bioassay device 2 rotates the bioassay substrate 1 after the fluorescent labeling agent L is dropped, and causes the excitation light P to enter from the lower surface 1b side of the bioassay substrate 1 so that the fluorescence in the reaction region 12 is emitted. The labeling agent L is irradiated, and the fluorescence F generated from the fluorescent labeling agent L according to the excitation light P is detected from below the bioassay substrate 1.

  Here, in the bioassay device 2, the excitation light P and the control light V are irradiated onto the bioassay substrate 1 through the same objective lens 41. Therefore, the bioassay device 2 can specify the irradiation position of the excitation light P, that is, the emission position of the fluorescence F by performing focus control, positioning control, and address control using the control light V. The probe DNA (N) bound to the sample DNA (N ′) can be identified from the fluorescence emission position.

  In addition, the bioassay device 2 stops the emission of the excitation light P, emits only the control light V, performs servo control while rotating the bioassay substrate 1, and controls the track on the signal recording film 7. Data recording or reproduction can be performed.

(DNA analysis method)
Next, as an example of a method for analyzing an interaction between substances, a case where the present invention is used in a DNA analysis method will be described as an example.

  First, the bioassay substrate 1 is horizontally loaded into the disk loading unit 22 of the bioassay device 2. Next, the bioassay substrate 2 was rotated by the bioassay device 2 while performing position control based on the address pits 9, and the dripping head 34 contained the probe DNA (N) whose one end was modified with an NCO group or the like. The solution is dropped into the predetermined reaction area 12. At this time, a plurality of types of probe DNAs (N) are dropped on one bioassay substrate 1. However, one kind of probe DNA (N) is allowed to enter one reaction region 12. In addition, which kind of probe DNA (N) is dropped on each reaction region 12 is read out in advance from the signal recording film 7 as an arrangement map showing the correspondence between the reaction region 12 and the probe DNA (N). Alternatively, information is collected using the Internet connection function 27, and control is performed based on the arrangement map.

  Subsequently, an electrode is inserted into the solution in the reaction region 12 from the upper surface 1 a side of the bioassay substrate 1, and an alternating electric field of about 1 MV / m and 1 MHz is applied to each reaction region 12. When the alternating electric field is applied in this manner, the probe DNA (N) extends in the vertical direction with respect to the bioassay substrate 1, and the probe DNA (N) is moved in the vertical direction to the bioassay substrate 1 in advance. The modified end of the probe DNA (N) is bound to the bottom surface 13 subjected to the surface modification treatment, and the probe DNA (N) can be immobilized (immobilized) in the reaction region 12 (Masao Washizu and Osamu Kurosawa: “Electrostatic Manipulation of DNA in Microfabricated Structures”, IEEE Transaction on Industrial Application Vol. 26, No 26, P, 1165-1172 (1900)).

  Subsequently, the solution containing the sample DNA (N ′) is dropped from the dropping head 34 to the reaction regions 12 on the bioassay substrate 1 together with the solution containing the buffer salt by the bioassay device 2.

  Subsequently, after the sample DNA (N ′) is dropped, the reaction region 12 of the bioassay substrate 1 is irradiated with microwaves using the microwave irradiation unit 21 to heat the inside of the reaction region 12 to several tens of degrees Celsius. Then, an AC electric field of about 1 MV / m and 1 MHz is applied in the heated state. When such a treatment is performed, the sample DNA (N ′) and the probe DNA (N) extend in the vertical direction to be in a state where there is little steric hindrance, and the sample DNA (N ′) is applied to the bioassay substrate 1. To move vertically. As a result, when the sample DNA (N ′) and the probe DNA (N) corresponding to each other in the base sequence are in the same reaction region 12, they cause hybridization.

  Subsequently, after causing hybridization, a fluorescent labeling agent L such as an intercalator is dropped from the dropping head 34 into the reaction region 12 of the bioassay substrate 1 by the bioassay device 2. Such a fluorescent labeling agent L is inserted and bonded between the double helix of the probe DNA (N) and the sample DNA (N ′) in which hybridization has occurred.

  Subsequently, the surface 1a of the bioassay substrate 1 is washed with pure water or the like to remove the sample DNA (N ′) and the fluorescent labeling agent L in the reaction region 12 where hybridization has not occurred. As a result, the fluorescent labeling agent L remains only in the reaction region 12 where hybridization has occurred.

  Subsequently, the bioassay apparatus 2 rotates the bioassay substrate 1 while performing focus servo control, positioning servo control, and address control using the control light, and irradiates the predetermined reaction region 12 with the excitation light P. Along with the irradiation of the excitation light P, whether or not the fluorescence F is generated is detected while detecting the address information.

  Subsequently, the bioassay device 2 calculates a measurement value corresponding to the position of each reaction region 12 on the bioassay substrate 1 and the intensity of the emission of the fluorescence F, and the result and the captured image are calculated on the bioassay substrate 1. Recording and storing on the signal recording film 7. Subsequently, the presence / absence of the target substance is determined from the measured value, and a determination map on the disk image is created. Then, the base sequence of the sample DNA (N ′) is analyzed based on the created map and an arrangement map indicating what kind of base sequence of the probe DNA has been dropped on each reaction region 12. With reference to the result of this analysis, for example, the relationship with the disease may be analyzed based on the information on the relationship between the probe DNA (N) recorded on the signal recording film 7 and the disease. Furthermore, an expression gene may be made to correspond to the gene network recorded on the bioassay substrate 1, and the function thereof may be illustrated.

  The bioassay device 2 records and stores the analysis result, the detected arrangement map, and the like on the signal recording film 7 of the bioassay substrate 1.

  In the present invention, a disk-shaped DNA disk using a magneto-optical disk has been described. However, the shape may be appropriately set (for example, a square, a rectangle, or an ellipse). In addition to the DNA disk, the same method can be used as appropriate when analyzing the interaction of proteins and other substances. In addition, although an example using a magneto-optical disk as a data recording layer was shown, magnetic recording tape with a predetermined recording function, semiconductor memory (EEPROM, FRAM, MRAM, etc.), printing technology, etc. are used as long as there is no problem. It shall be possible. Note that the microwave irradiation can be appropriately performed according to the purpose, and the timing, interval, irradiation time, irradiation energy, and the like of the microwave irradiation can be changed according to circumstances.

  The present invention is a method of adjusting the temperature of a reaction region by irradiating microwaves to the reaction region of a bioassay substrate such as a DNA chip or a protein chip. When detection / measurement or the like is performed using a bioassay substrate, temperature adjustment is often required during the procedure in order to efficiently perform the reaction. Therefore, according to the present invention, the temperature of the reaction region can be adjusted with a simple detection / measurement instrument, which is a useful technique for using a bioassay substrate.

  In addition, microwave irradiation itself has the effect of promoting interaction between substances in the reaction region depending on the irradiation intensity. Therefore, detection and measurement using a substrate for bioassay is highly sensitive and efficient. It is also a useful technique for making things easy.

  Furthermore, the bioassay apparatus according to the present invention is industrially useful because it has a microwave irradiation means and a mechanism for detecting and measuring reactions such as interactions in the reaction region with high sensitivity. Since this apparatus has means for controlling microwave irradiation, means for managing and controlling a series of detection / measurement procedures, means for processing and recording detected / measured data, Internet connection means, etc. This is industrially useful in that a series of detection and measurement procedures are simplified, automated, and easy to operate.

The figure which showed the relationship between the microwave irradiation pattern at the time of intermittently irradiating a microwave, and reaction region temperature. The figure which showed the relationship between the microwave irradiation pattern and reaction region temperature when the irradiation intensity was gradually weakened while intermittently irradiating the microwave. The top view and side sectional view showing typically the disc-shaped substrate for bioassay. The typical side surface sectional view of a reaction field. The typical external appearance perspective view of the reaction area. The cross-sectional schematic diagram which showed the structure and mechanism of the bioassay apparatus 2 which concern on this invention. FIG. 3 is an external perspective view schematically showing a state where a target substance is dropped into a reaction region 12. The schematic diagram which showed a mode that the fluorescent substance was couple | bonded with the nucleic acid fix | immobilized by the bottom face 13 of the reaction area | region 12. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Bioassay board | substrate 12 Reaction area | region 2 Bioassay apparatus 21 Microwave irradiation part 23 Dripping part 24 Excitation light detection part 28 Control part 44 Excitation light source 47 Photodetector 52 Photodetect circuit L Fluorescent labeling agent N Probe DNA (Detection substance )
N 'sample DNA (target substance)

Claims (9)

  A method for adjusting a temperature of a reaction region, characterized in that microwave irradiation is performed on a reaction region that is a field of interaction between substances.   The temperature control method according to claim 1, wherein the interaction is hybridization.   A method for adjusting a temperature of a reaction region, characterized in that microwave irradiation is performed on the reaction region of a bioassay substrate provided with at least a reaction region serving as an interaction field between substances.   The temperature control method according to claim 3, wherein the bioassay substrate is a DNA chip. An apparatus for detecting an interaction between substances using a bioassay substrate provided with a reaction region in which a substance for detection exists,
Means for dropping a sample solution containing a target substance to the reaction region;
Microwave irradiation means for the reaction region;
A bioassay device comprising at least means for controlling the microwave irradiation.   The bioassay according to claim 5, comprising: means for irradiating the reaction region with excitation light for detecting an interaction; and optical means for collecting light emitted from a substance in the reaction region to detect light intensity. apparatus.   7. The bioassay device according to claim 6, wherein the optical means is means for detecting fluorescence intensity labeled on the substance.   The bioassay device according to claim 6, wherein the optical means is means for detecting light derived from an intercalator that binds to a substance in the reaction region.   The bioassay device according to claim 5, wherein the interaction is hybridization.

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