JP4337432B2 - Biochemical reaction substrate, biochemical reaction apparatus, and hybridization method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biochemical reaction substrate such as a DNA chip, a biochemical reaction apparatus, and a hybridization method for hybridizing nucleotide chains.
[0002]
[Prior art]
Currently, so-called DNA chips or DNA microarrays (hereinafter collectively referred to as DNA chips) in which predetermined DNA (full length or part) is finely arranged by microarray technology are used for gene mutations, SNPs (single nucleotides). Type) analysis, gene expression frequency analysis, etc., and has begun to be widely used in drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and other fields.
[0003]
A DNA analysis method using a DNA chip is a method for generating sample DNA by PCR amplification of mRNA (messenger RNA) extracted from cells, tissues, etc. while incorporating a fluorescent probe dNTP by reverse transcription PCR (Polymerase Chain Reaction) reaction, etc. The sample DNA is dropped onto the probe DNA immobilized (immobilized) on the DNA chip, and the probe DNA and the sample DNA are hybridized. Then, a fluorescent labeling agent is inserted into the double helix of DNA, and the fluorescence is measured with a predetermined detector. Thus, it is determined whether or not the sample DNA and the probe DNA have the same base sequence.
[0004]
Here, utilizing the fact that the DNA is negatively charged, a technique for moving the suspended sample DNA in the direction of the probe DNA using a direct current electric field in the direction toward the probe DNA, thereby speeding up the hybridization. Is known (see Patent Document 1).
[0005]
[Patent Document 1]
Japanese Patent Laid-Open No. 2001-238664 [0006]
[Problems to be solved by the invention]
However, since single-stranded DNA does not become linear in solution but forms a random coil, there is a large steric hindrance to bind probe DNA and sample DNA, and hybridization can be performed at high speed. Have difficulty. Even if the floating sample DNA is moved in the direction of the probe DNA by an electric field, the degree of steric hindrance does not change.
[0007]
Accordingly, an object of the present invention is to provide a biochemical reaction substrate and a biochemical reaction apparatus that can perform hybridization and the like at high speed with a simple configuration in order to solve such a conventional problem.
[0008]
Another object of the present invention is to provide a hybridization method capable of performing hybridization at high speed with a simple configuration.
[0009]
[Means for Solving the Problems]
A biochemical reaction substrate according to the present invention is a biochemical reaction substrate that is a substrate used for a biochemical reaction, and a reaction region that performs the biochemical reaction, and an electric field applied to the reaction region. An electrode portion; a hole portion into which a rotation shaft for holding and rotating the substrate is inserted; and a second contact portion in contact with the first contact portion formed on the rotation shaft . The contact portion is electrically connected to the electrode portion.
[0010]
A biochemical reaction apparatus according to the present invention is a biochemical reaction apparatus that performs a biochemical reaction using a substrate having a hole, and is a rotating shaft that is inserted into the hole of the substrate to hold and rotate the substrate. A first contact portion that is in contact with a second contact portion that is electrically connected to an electrode that is formed on the rotating shaft and formed in the reaction region of the substrate; and power that is applied to the first contact portion. Power supply means for supplying power.
[0011]
The hybridization method according to the present invention is a field of interaction between a probe substance and a sample substance, and a plurality of wells in which the inside of the well is treated so that one end of the probe substance can be fixed, and an electric field is formed in the well. an electrode portion for applying a center hole chucking mechanism for holding and rotating the substrate is inserted, the first in which the chucking mechanism is formed on the chucking mechanism when inserted and a second contact portion for contacting a contact portion with respect to said first contact portion and the hybridization substrate on which the electrode portions and are electrically connected, one end of the wells of the nucleotide strand for the probe attached to the bottom surface, with respect to the interior of the well to which one end of the nucleotide chain for the probe is coupled to the bottom surface, the nucleotide chain for the sample Was added dropwise comprising, between the chucking King mechanism holding the hybridization substrate by inserting into said central hole is placed an external electrode on the well, the external electrode and the chucking mechanism AC power is applied to the probe to hybridize the probe nucleotide chain and the sample nucleotide chain.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, as a specific embodiment to which the present invention is applied, a bioassay substrate for DNA analysis to which the present invention is applied and a DNA analysis method using the bioassay substrate will be described.
[0013]
(Bioassay substrate)
FIG. 1 schematically shows an upper surface of the bioassay substrate 1 according to the embodiment of the present invention, and FIG. 2 schematically shows a cross section of the bioassay substrate 1 according to the embodiment of the present invention. The figure is shown.
[0014]
The overall shape of the bioassay substrate 1 is a flat plate whose main surface is circular, like an optical disk such as a CD (Compact Disk) and a DVD (Digital Versatile Disk). A central hole 2 is formed at the center of the main surface of the bioassay substrate 1. A chucking mechanism for holding and rotating the bioassay substrate 1 is inserted into the center hole 2 when the bioassay substrate 1 is mounted on the DNA analyzer.
[0015]
As shown in FIG. 2, the bioassay substrate 1 is formed of a substrate layer 3, a transparent electrode layer 4, a solid phase layer 5, and a well forming layer 6 from the lower layer side. The surface on the well forming layer 6 side of the bioassay substrate 1 is referred to as the upper surface 1a, and the surface on the substrate layer 3 side is referred to as the lower surface 1b.
[0016]
The substrate layer 3 is a material that transmits excitation light that excites a fluorescent labeling agent, which will be described in detail later, and fluorescence of the fluorescent labeling agent. For example, the substrate layer 3 is formed of a material such as quartz glass, silicon, polycarbonate, or polystyrene.
[0017]
The transparent electrode layer 4 is a layer formed on the substrate layer 3. The transparent electrode layer 4 is formed of a light-transmitting and conductive material such as ITO (indium-tin-oxide) or aluminum. The transparent electrode layer 4 is formed to a thickness of about 250 nm on the substrate layer 3 by, for example, sputtering or electron beam evaporation.
[0018]
The solid phase layer 5 is a layer formed on the transparent electrode layer 4. The solid phase layer 5 is formed of a material for solidifying one end of the probe DNA. In this example, the solid phase layer 5 is a layer in which SiO ₂ whose surface can be modified with silane is formed to a thickness of about 50 nm by, for example, sputtering or electron beam evaporation.
[0019]
The well formation layer 6 is a layer formed on the solid phase layer 5. The well forming layer 6 is a layer in which a plurality of hollow wells 8 opened on the upper surface 1a side of the bioassay substrate 1 are formed.
[0020]
The internal space of the well 8 serves as a field for interaction between the probe DNA (detection nucleotide chain) and the sample DNA (target nucleotide chain), specifically, a hybridization reaction field. The well 8 has such a depth and size that the liquid can be retained when a liquid such as a solution containing the sample DNA is dropped. For example, the well 8 has an opening having a size of 100 μm square, a depth of about 5 μm, and the solid phase layer 5 is exposed on the bottom surface 11.
[0021]
As shown in FIG. 3, the well forming layer 6 is coated with the photosensitive polymer 13 on the solid phase layer 5 by spin coating or the like to a thickness of about 5 μm (step S1). Subsequently, a photomask 14 having a predetermined pattern is formed on the coated photosensitive polymer 13 , and the photosensitive polymer 13 is exposed and developed using the photomask 14 (step S2). As a result, a plurality of wells 8 are formed in the well formation layer 6 (step S3).
[0022]
Furthermore, the bottom surface 11 of the well 8 (the portion where the solid-phased layer 5 is exposed) is surface-modified with a corresponding functional group so that a probe DNA whose one end is modified with the functional group is bound. For example, as shown in FIG. 4, the well 8 has the bottom surface 11 (the solid phase layer 5 formed of SiO ₂ ) modified with a silane molecule 16 having an SH group 15. For this reason, for example, probe DNA whose one end is modified with an SH group can be bound to the bottom surface 11 of the well 8. Thus, in the bioassay substrate 1, one end of the probe DNA can be bound to the bottom surface 11 of the well 8. Therefore, as shown in FIG. 5, the probe DNA has a chain extending vertically from the bottom surface 11. (P) can be combined.
[0023]
Further, in the bioassay substrate 1, as shown in FIG. 1, a plurality of wells 8 are arranged at equal intervals, for example, at intervals of about 400 μm on a plurality of rows radially extending from the center of the main surface toward the outer periphery. Has been placed.
[0024]
In the bioassay substrate 1, the transparent electrode layer 4 is exposed on the upper surface 1 a side in the peripheral portion of the center hole 2. That is, the solid phase layer 5 and the well forming layer 6 are not formed in the peripheral portion of the center hole 2 of the bioassay substrate 1. Therefore, in the bioassay substrate 1, the transparent electrode layer 4 serving as an intermediate layer can be contacted from the upper surface 1a side. A portion exposed to the upper surface 1a side of the transparent electrode layer 4 is hereinafter referred to as a second contact portion 9.
[0025]
The bioassay substrate 1 is formed with address pits that can be read by irradiating laser light from the lower surface 1b side of the bioassay substrate 1. The address pit is information for specifying the position of each well 8 on the plane of the bioassay substrate 1. By optically reading information from the address pits, it is possible to identify which one of the plurality of wells 8 is currently irradiated with laser light. By providing such address pits, it is possible to control the dropping position of the solution by a dropping apparatus, which will be described later, and specify the fluorescence detection position by the objective lens.
[0026]
Since the bioassay substrate 1 as described above is formed in a disc shape, a focusing servo control for controlling the focusing position of the laser beam by using a reproduction system similar to the optical disc system, radial direction Positioning servo control for controlling the irradiation position of the laser beam on the laser beam and the dropping position by the dropping device, and information detection processing of the address pits can be performed. In other words, the information content recorded in the address pit is associated with the well 8 in the vicinity of the address pit so that the address pit information is read out, so that only one specific well 8 is read. By irradiating the laser beam, the position of the well 8 where the fluorescence is emitted is specified, or the relative position between the specific one well 8 and the dropping device is controlled, and the specific one well 8 is controlled. The solution can be added dropwise.
[0027]
Furthermore, in the bioassay substrate 1 as described above, a parallel electric field can be formed between the electrode and the transparent electrode layer 4 by bringing the electrode close to the well 8 from the upper side. Therefore, for example, when DNA hybridization is performed, by applying an alternating electric field to the well 8, DNA floating in the well can be extended to promote the progress of hybridization.
[0028]
(DNA analysis method)
Next, a DNA analysis method using the bioassay substrate 1 as described above will be described.
[0029]
First, the bioassay substrate 1 is loaded into a DNA analyzer, and the bioassay substrate 1 is held horizontally such that the upper surface 1a side is in an upward direction, and the bioassay substrate 1 is rotated.
[0030]
The bioassay substrate 1 is held and rotated using a chucking mechanism 20 similar to the optical disk drive provided in the DNA analyzer.
[0031]
As shown in FIG. 6, the chucking mechanism 20 is connected to a rotational drive shaft and rotates a bioassay substrate 1. The chucking mechanism 20 is formed in a plate shape perpendicular to the central shaft 21. And a chucking claw 23 that is inserted into the center hole 2 from the lower surface 1b side and supports the bioassay substrate 1 from the upper surface 1a side. The turn plate 22 and the chucking claw 23 are respectively attached to the central shaft 21 and rotate together with the central shaft 21.
[0032]
In such a chucking mechanism 20, the chucking claw 23 is inserted from the lower surface 1b side into the center hole 2 of the bioassay substrate 1, and a part of the chucking claw 23 comes out to the upper surface 1a side by a spring or the like. The bioassay substrate 1 is biased downward from the upper surface 1a side. Thus, in the chucking mechanism 20, the bioassay substrate 1 can be sandwiched and held between the turn plate 22 and the chucking claw 23, and the bioassay substrate 1 can be horizontally placed with the upper surface 1a facing up. And the bioassay substrate 1 can be rotated. In the chucking mechanism 20, a first contact portion 24 is formed in a portion that contacts the second contact portion 9 (a portion where the transparent electrode layer 4 is exposed). The first contact part 24 is electrically connected to the AC power generation part inside the DNA analyzer via the central axis 21 of the chucking mechanism 20.
[0033]
Subsequently, in the state where the bioassay substrate 1 is held by the chucking mechanism 20 as described above, the solution S containing the probe DNA (P) whose one end is modified with an SH group, as shown in FIG. For example, it is dropped into a predetermined well 8 using the dropping nozzle 25. At this time, one bioassay substrate 1 is rotationally driven to drop the probe DNA while controlling the relative position between the specific well 8 and the dropping nozzle 25. Further, a plurality of types of probe DNAs are dropped into each well 8 of the bioassay substrate 1. However, one type of probe DNA is allowed to enter one well 8. Note that to determine which type of probe DNA is to be dropped into each well 8, an arrangement map showing the correspondence between the wells and the probe DNA is generated in advance, and dropping is controlled based on the arrangement map.
[0034]
Further, the dropping control of the solution S is performed by driving and controlling the bioassay substrate 1 in the same manner as the optical disk driving system. That is, as shown in FIG. 6, the bioassay substrate 1 is rotationally driven while being held parallel with the upper surface 1a facing upward, and the laser beam V is irradiated from the lower side (lower surface 1b side) of the bioassay substrate 1. Then, the position of the well 8 is specified by detecting the address pit, and the dropping position may be controlled.
[0035]
Subsequently, as shown in FIG. 8, a disk-shaped plate electrode plate 30 having substantially the same shape as the bioassay substrate 1 is covered on the well forming layer 6 from the outside on the upper surface 1 a side of the bioassay substrate 1. To be placed. Subsequently, an AC voltage is applied between the flat electrode plate 30 and the first contact portion 24 by the AC power generating portion 31. At this time, since the first contact portion 24 and the second contact portion 9 are in contact with each other, power is applied to the transparent electrode layer 4. Therefore, an alternating electric field is applied to the bioassay substrate 1 between the flat electrode plate 30 and the transparent electrode layer 4. Therefore, an alternating electric field perpendicular to the main surface of the bioassay substrate 1 is applied to each well 8. For example, an AC electric field of about 1 MV / m or 1 MHz is applied in the well 8.
[0036]
When the alternating electric field is applied to the predetermined well 8 in this way, the probe DNA (P) suspended in the solution in the well 8 is perpendicular to the main surface of the bioassay substrate 1 as shown in FIG. While extending in the direction, the probe DNA moves in a direction perpendicular to the bioassay substrate 1. For this reason, the modified end of the probe DNA is bonded to the bottom surface 11 that has been surface-modified in advance, and the probe DNA can be immobilized (immobilized) on the bottom surface 11 of the well 8.
[0037]
The reason why the single-stranded DNA (nucleotide chain) extends and moves when an alternating electric field is applied is as follows. That is, in the nucleotide chain, it is considered that an ion cloud is formed by the phosphate ion (negative charge) forming the skeleton of the nucleotide chain and the hydrogen ion (positive charge) obtained by ionizing water in the vicinity. This is because the polarization vector generated by the negative charge and the positive charge is directed in one direction as a whole by applying a high frequency high voltage, and as a result, the nucleotide chain is elongated. Furthermore, when an inhomogeneous electric field where electric lines of force are concentrated in part is applied, the nucleotide chain also moves toward the site where electric lines of force are concentrated (Seiichi Suzuki, Takeshi Yamanashi, Shin-ichi Tazawa, Osamu Kurosawa and Masao Washizu: "Quantitative analysis on electrostatic orientation of DNA in stationary AC electric field using fluorescence anisotropy", IEEE Transaction on Industrial Application, Vol. 34, No. 1, p. 75-83 (1998)).
[0038]
In this way, when an alternating electric field is applied, the probe DNA is stretched in a direction parallel to the electric field, resulting in a state with little steric hindrance, and the probe DNA and the bottom surface 11 are easily bonded. The probe DNA can be immobilized (immobilized) on the bottom surface 11 of the well 8 by binding the probe DNA to the bottom surface 11.
[0039]
Subsequently, the planar electrode plate 30 is removed, and the solution containing the sample DNA extracted from the living body is dropped into each well 8 on the bioassay substrate 1 using the dropping nozzle 25.
[0040]
Subsequently, after the sample DNA is dropped, the plate electrode plate 30 is placed again so as to cover the well forming layer 6 from the outside on the upper surface 1a side of the bioassay substrate 1, and the plate electrode plate 30 and the first electrode plate 30 are covered . An AC voltage is applied between the contact portion 24 and the AC power generator 31. Along with the application of the AC voltage, the ambient temperature is maintained at about 60 ° C. That is, an AC electric field in the vertical direction is applied to the main surface of the bioassay substrate 1 while heating. For example, an AC electric field of about 1 MV / m or 1 MHz is applied in the well 8.
[0041]
When such processing is performed, the sample DNA and the probe DNA are stretched in the vertical direction so that the steric hindrance is reduced and the sample DNA moves in the vertical direction with respect to the bioassay substrate 1. As a result, when sample DNA and probe DNA having complementary base sequences are in the same well 8, they cause hybridization.
[0042]
Subsequently, after causing hybridization, the flat electrode plate 30 is removed, and a fluorescently labeled intercalator or the like (for example, POPO-1 or TOTO-1) is dropped into the well 8 of the bioassay substrate 1. Such a fluorescent labeling agent is inserted and bonded between the double helix of the probe DNA and the sample DNA that have undergone hybridization.
[0043]
Subsequently, the surface 1a of the bioassay substrate 1 is washed with pure water or the like to remove the sample DNA and the fluorescent labeling agent in the well 8 where hybridization has not occurred. As a result, the fluorescent labeling agent remains only in the well 8 where hybridization has occurred.
[0044]
Subsequently, the fluorescence from the well 8 is detected by driving and controlling the bioassay substrate 1 in the same manner as the optical disk drive system. That is, while rotating the bioassay substrate 1 while holding it, the position of the well 8 is specified by detecting the address pit by irradiating the laser beam V from the lower side (lower surface 1b side) of the bioassay substrate 1. To do. At the same time, the excitation light is irradiated from the lower side (lower surface 1b side) of the bioassay substrate 1, and the fluorescence generated according to the excitation light is also detected from the lower surface 1b side. Then, it is detected from which well 8 fluorescence is generated.
[0045]
Subsequently, a map indicating the position of the well 8 where fluorescence on the bioassay substrate 1 was generated is created. Then, the base sequence of the sample DNA is analyzed based on the created map and an arrangement map showing what kind of base sequence of the probe DNA has been dropped on each well 8.
[0046]
(DNA analyzer)
Next, a DNA analysis apparatus 51 that performs DNA analysis using the bioassay substrate 1 according to the embodiment of the present invention will be described with reference to FIG.
[0047]
As shown in FIG. 10, the DNA analyzer 51 holds the bioassay substrate 1 and rotates the disk loading unit 52, stores various solutions for hybridization, and wells 8 of the bioassay substrate 1. Are provided with a dropping unit 53 for dropping the solution, an excitation light detecting unit 54 for detecting excitation light from the bioassay substrate 1, and a control / servo unit 55 for managing and controlling each of the above parts. .
[0048]
The disc loading unit 52 is inserted into the center hole 2 of the bioassay substrate 1 to hold the bioassay substrate 1, and the chucking mechanism 20 drives the bioassay substrate 1 by driving the chucking mechanism 20. and a spindle motor 62 for rotating. The chucking mechanism 20 is the same as the mechanism shown in FIG. The disc loading unit 52 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. In the disc loading section 52, the problem that the solution dropped on the well 8 hangs down can be avoided by holding the bioassay substrate 1 horizontally.
[0049]
The dropping unit 53 includes a storage unit 63 that stores the sample solution S and the fluorescent labeling agent S ′, and a dropping head 64 that drops the sample solution S and the fluorescent labeling agent S ′ in the storage unit 63 onto the bioassay substrate 1. Have. The dropping head 64 is disposed above the upper surface 1a of the bioassay substrate 1 loaded horizontally. Furthermore, the dropping head 64 controls the relative position with respect to the bioassay substrate 1 in the radial direction based on the position information read from the address pits of the bioassay substrate 1 and the rotation synchronization information, and the sample DNA (target nucleotide chain T The sample solution S containing) is dropped accurately following the predetermined well 8. In the storage unit 63, the combination of the storage unit 63 and the dropping head 64 is the number of sample solutions used for hybridization.
[0050]
In addition, in the dropping unit 53, for example, a dropping method based on a so-called “inkjet printing method” is employed in order to accurately drop the sample solution S at a predetermined position on the bioassay substrate 1. The “ink jet printing method” is a method in which an ink jetting mechanism used in a so-called ink jet printer is applied to the dropping head 64, and the sample solution S is jetted onto the bioassay substrate 1 from a nozzle head such as an ink jet printer. .
[0051]
The excitation light detection unit 54 has an optical head 70. The optical head 70 is disposed on the lower side of the bioassay substrate 1 loaded horizontally, that is, on the lower surface 1b side. The optical head 70 is movable in the radial direction of the bioassay substrate 1 by, for example, a thread mechanism (not shown).
[0052]
The optical head 70 includes an objective lens 71, a biaxial actuator 72 that supports the objective lens 71 so as to be movable, and a light guide mirror 73. The objective lens 71 is supported by the biaxial actuator 72 so that the central axis thereof is substantially perpendicular to the surface of the bioassay substrate 1. Therefore, the objective lens 71 can collect the light beam incident from the lower side of the bioassay substrate 1 on the bioassay substrate 1. The biaxial actuator 72 supports the objective lens 71 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 72, the focal point of the light collected by the objective lens 71 can be moved in the direction perpendicular to the surface of the bioassay substrate 1 and in the radial direction. Therefore, the optical head 70 can perform the same control as the just focus control and the positioning control in the optical disc system.
[0053]
The light guide mirror 73 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, servo light V, and reflected light R enter and exit the optical head 70. Excitation light P and servo light V are incident on the light guide mirror 73 from the optical path X. The light guide mirror 73 reflects the excitation light P and the servo light V to be refracted by 90 ° and enters the objective lens 71. The excitation light P and the servo light V incident on the objective lens 71 are collected by the objective lens 71 and irradiated onto the bioassay substrate 1. Further, the reflected light R of the fluorescence F and the servo light V enters the light guide mirror 73 through the objective lens 71 from the bioassay substrate 1. The light guide mirror 73 reflects the fluorescence F and the reflected light R, refracts 90 °, and emits the light onto the optical path X.
[0054]
A drive signal for moving the optical head 70 by a sled and a drive signal for driving the biaxial actuator 72 are supplied from the control / servo unit 55.
[0055]
The excitation light detection unit 54 includes an excitation light source 74 that emits the excitation light P, a collimator lens 75 that converts the excitation light P emitted from the excitation light source 74 into a parallel light beam, and an excitation light that has been converted into a parallel light beam by the collimator lens 75. A first dichroic mirror 76 that refracts the light P on the optical path X and irradiates the light guide mirror 73.
[0056]
The excitation light source 74 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 74 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 75 turns the excitation light P emitted from the excitation light source 74 into a parallel light beam. The first dichroic mirror 76 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 servo light V (its reflected light R). To do. The first dichroic mirror 76 is inserted on the optical path X at an angle of 45 °, reflects the excitation light P emitted from the collimator lens 75 to be refracted by 90 °, and excites the light guide mirror 73. Light P is irradiated.
[0057]
The excitation light detection unit 54 refracts the fluorescence F emitted from the optical head 70 onto the optical path X by refracting the avalanche photodiode 77 that detects the fluorescence F, the condensing lens 78 that collects the fluorescence F, and the like. And a second dichroic mirror 79 for irradiating the avalanche photodiode 77.
[0058]
The avalanche photodiode 77 is a very sensitive photodetector and can detect the fluorescent light F with a weak light amount. Here, the wavelength of the fluorescence F detected by the avalanche photodiode 77 is about 470 nm. Further, the wavelength of the fluorescence F varies depending on the type of the fluorescent labeling agent. The condensing lens 78 is a lens for condensing the fluorescence F on the avalanche photodiode 77. The second dichroic mirror 79 is inserted at an angle of 45 ° on the optical path X, and is disposed downstream of the first dichroic mirror 76 when viewed from the light guide mirror 73 side. Therefore, the fluorescence F, the servo light V, and the reflected light R are incident on the second dichroic mirror 79, and the excitation light P is not incident. The second dichroic mirror 79 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 servo light (reflected light R). The second dichroic mirror 79 reflects and refracts the fluorescence F emitted from the light guide mirror 73 of the optical head 70 and irradiates the avalanche photodiode 77 with the fluorescence F via the condenser lens 78. .
[0059]
The avalanche photodiode 77 generates an electrical signal corresponding to the light amount of the fluorescence F thus detected, and supplies the electrical signal to the control / servo unit 55.
[0060]
The excitation light detection unit 54 detects the servo light source 80 that emits the servo light V, the collimator lens 81 that uses the servo light V emitted from the servo light source 80 as a parallel light beam, and the reflected light R of the servo light V. A photodetector circuit 82; a cylindrical lens 83 that collects the reflected light R on the photodetector circuit 82 by generating astigmatism; and an optical separator 84 that separates the servo light V and the reflected light R. ing.
[0061]
The servo light source 80 is a light emitting unit having a laser light source that emits laser light having a wavelength of 780 nm, for example. Note that the wavelength of the servo light V is such that the address pits can be detected, and may be any wavelength as long as it is different from the wavelengths of the excitation light P and the fluorescence F. The collimator lens 81 turns the servo light V emitted from the servo light source 80 into a parallel light beam. The servo light V converted into a parallel light beam enters the optical separator 84.
[0062]
The photodetection circuit 82 includes a detector that detects the reflected light R, and a signal generation circuit that generates a focus error signal, a positioning error signal, and an address pit reproduction signal from the detected reflected light R. Since the reflected light R is light generated by reflecting the servo light V with the bioassay substrate 1, the wavelength thereof is 780 nm, which is the same as that of the servo light.
[0063]
The focus error signal is an error signal indicating the amount of positional deviation between the in-focus position of the light collected by the objective lens 71 and the substrate layer 3 of the bioassay substrate 1. When the focus error signal becomes 0, the distance between the objective lens 71 and the bioassay substrate 1 is optimized. The positioning error signal is a signal indicating a positional deviation amount with respect to the disk radial direction between the position of the predetermined well 8 and the focal position. When the positioning error signal becomes 0, the irradiation position of the servo light V in the disk radial direction coincides with the arbitrary well 8. The address pit reproduction signal is a signal indicating the information content described in the address pit recorded on the bioassay substrate 1. By reading this information content, it is possible to identify the well 8 that is currently irradiating the servo light V.
[0064]
The photodetect circuit 82 supplies the control / servo unit 55 with a focus error signal, a positioning error signal, and an address pit reproduction signal generated based on the reflected light R.
[0065]
The cylindrical lens 83 is a lens for collecting the reflected light R on the photodetection circuit 82 and causing astigmatism. By generating astigmatism in this way, the focus error signal can be generated by the photodetect circuit 82.
[0066]
The optical separator 84 includes a light separation surface 84a made up of a deflecting beam splitter and a quarter wavelength plate 84b. In the optical separator 84, light incident from the opposite side of the quarter-wave plate 84b is transmitted through the light separation surface 84a, and when the reflected light of the transmitted light is incident from the quarter-wave plate 84 side, light is transmitted. The separation surface 84a has a function of reflecting. The optical separator 84 has a light separation surface 84a inserted at an angle of 45 ° on the optical path X, and is disposed at the rear stage of the second dichroic mirror 79 when viewed from the light guide mirror 73 side. Accordingly, the optical separator 84 transmits the servo light V emitted from the collimator lens 81 and causes the servo light V to enter the light guide mirror 73 in the optical head 70 and guide the optical head 70. The reflected light R emitted from the mirror 73 is reflected to be refracted by 90 °, and the reflected light R is irradiated to the photodetector circuit 82 through the cylindrical lens 83.
[0067]
The control / servo unit 55 performs various types of servo control based on the focus error signal, the positioning error signal, and the address pit reproduction signal detected by the excitation light detection unit 54.
[0068]
That is, the control / servo unit 55 drives the biaxial actuator 72 in the optical head 70 based on the focus error signal to control the interval between the objective lens 71 and the bioassay substrate 1, and the focus error signal is 0. Servo control is performed so that The control / servo unit 55 controls the movement of the objective lens 71 in the radial direction of the bioassay substrate 1 by driving the biaxial actuator 72 in the optical head 70 based on the positioning error signal, and the focus error signal is 0. Servo control is performed so that Further, the control / servo unit 55 performs sled movement control of the optical head 70 based on the address pit reproduction signal, moves the optical head 70 to a predetermined radial position, and moves the objective lens 71 to the target well position.
[0069]
Further, the control / servo unit 55 controls the AC power generation unit 31 during the hybridization and also controls the supply of power via the chucking mechanism 20.
[0070]
The DNA analyzer 51 having the above configuration performs the following operation.
[0071]
The DNA analyzer 51 drops the solution containing the sample DNA on the well 8 while rotating the bioassay substrate 1, and causes the probe DNA and the sample DNA to interact (hybridize). The above-described electric field control is also performed during hybridization. Further, a buffer solution containing a fluorescent labeling agent is dropped onto the bioassay substrate 1 that has been subjected to the hybridization treatment.
[0072]
In addition, the DNA analyzer 51 rotates the bioassay substrate 1 after the fluorescent labeling agent is dropped, and causes the excitation light P to enter from the lower surface 1b side of the bioassay substrate 1 so that the fluorescent label in the well 8 is present. The agent is irradiated, and the fluorescence F generated from the fluorescent labeling agent according to the excitation light P is detected from below the bioassay substrate 1.
[0073]
Here, in the DNA analyzer 51, the excitation light P and the servo light V are applied to the bioassay substrate 1 through the same objective lens 71. Therefore, the DNA analyzer 51 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 servo light V. The probe DNA bound to the sample DNA can be identified from the fluorescence emission position.
[0074]
In the bioassay substrate 1 as described above, one end of the probe DNA suspended in the solution is extended and moved in the vertical direction by applying an AC electric field in the vertical direction to the surface of the substrate 1, and one end of the bottom surface 11 of the well 8. Connect to. Therefore, since the electric field is applied perpendicularly to the bioassay substrate 1, for example, it is not necessary to generate the electrode by patterning the electrode on the substrate. Can be fixed.
[0075]
Further, in the bioassay substrate 1 as described above, the sample DNA suspended in the solution and the probe DNA having one end fixed to the bottom surface of the well 8 are applied with a vertical AC electric field to extend and extend in the vertical direction. Move. Therefore, since both the sample DNA and the probe DNA extend and move in the same direction, hybridization is promoted. Furthermore, since it is not necessary to form an electrode for applying such an electric field on the substrate by patterning the electrode, the probe DNA can be immobilized using an electrode having a very simple layer structure.
[0076]
Further, in the bioassay substrate 1 as described above, since electric power is supplied via the chucking mechanism 20 that holds the bioassay substrate 1, the bioassay substrate 1 and the DNA analyzer (or the hybridization device) are supplied. It is possible to make an electrical connection with the device very easily.
[0077]
In the embodiment of the present invention, the plate electrode plate 30 is placed so as to be covered from the outside on the upper surface 1a side of the bioassay substrate 1, and an AC voltage is applied for hybridization to perform hybridization. When the process is completed, the plate electrode plate 30 is removed, but the plate electrode 30 may be formed integrally with the bioassay substrate 1.
[0078]
However, in this case, since the probe DNA and the sample DNA cannot be dropped into each well as they are, the well forming layer 6 has a plurality of wells 8 arranged radially, as shown in FIG. A plurality of grooves 41 communicating with a row of wells 8 extending from the outer periphery to the outer periphery are formed. Further, as shown in FIG. 12, a plate electrode 30 having an opening 42 formed at a portion corresponding to one end of the groove 41 is bonded onto the well forming layer 6. By forming the bioassay substrate 1 in this way, the probe DNA and the sample DNA can be injected into the well 8 from the opening 42.
[0079]
Further, in the case of forming integrally Thus the flat electrode plate 30 to the bioassay substrate 1 can be energized through the flat electrode plate 30 to the chucking mechanism 20 also.
[0080]
As shown in FIG. 13, the chucking mechanism 20 in this case may be provided with the third contact portion 43 together with the central shaft 21, the turn plate 22, the chucking claw 23, and the first contact portion 24. The third contact portion 43 contacts the fourth contact portion 44 formed on the flat plate electrode plate 30 when the chucking mechanism 20 is inserted into the center hole 2.
[0081]
【The invention's effect】
A biochemical reaction substrate according to the present invention is a biochemical reaction substrate that is a substrate used for a biochemical reaction, and a reaction region that performs the biochemical reaction, and an electric field applied to the reaction region. An electrode portion; a hole portion into which a rotation shaft for holding and rotating the substrate is inserted; and a second contact portion in contact with the first contact portion formed on the rotation shaft . The contact portion is electrically connected to the electrode portion.
[0082]
As a result, the biochemical reaction substrate according to the present invention can perform hybridization and the like at high speed with a simple configuration.
[0083]
A biochemical reaction apparatus according to the present invention is a biochemical reaction apparatus that performs a biochemical reaction using a substrate having a hole, and is a rotating shaft that is inserted into the hole of the substrate to hold and rotate the substrate. A first contact portion that is in contact with a second contact portion that is electrically connected to an electrode that is formed on the rotating shaft and formed in the reaction region of the substrate; and power that is applied to the first contact portion. Power supply means for supplying power.
[0084]
Thereby, in the biochemical reaction apparatus according to the present invention, hybridization and the like can be performed at high speed with a simple configuration.
[0085]
In the hybridization method according to the present invention, the following substrate is used.
[0086]
The substrate serves as a field of interaction between the probe substance and the sample substance, and a plurality of wells in which the inside of the well is treated so that one end of the probe substance can be fixed, and an electric field is applied to the inside of the well. The electrode portion, a central hole into which a chucking mechanism for holding and rotating the substrate is inserted, and a first contact portion formed in the chucking mechanism when the chucking mechanism is inserted. A second contact portion, and the first contact portion and the electrode portion are electrically connected .
[0087]
The hybridization method according to the present invention applies an electric field to a well via a chucking mechanism using the substrate as described above.
[0088]
Thus, in the hybridization method according to the present invention, hybridization can be performed at high speed with a simple configuration.
[Brief description of the drawings]
FIG. 1 is a plan view of a bioassay substrate according to an embodiment of the present invention.
FIG. 2 is a cross-sectional view of a bioassay substrate according to an embodiment of the present invention.
FIG. 3 is a diagram showing a step of forming a well of a bioassay substrate.
FIG. 4 is a diagram showing a silane molecule having an OH group modified on the bottom surface of a well.
FIG. 5 shows probe DNA bound to the bottom of the well.
FIG. 6 is a diagram for explaining solution dropping control on a bioassay substrate.
FIG. 7 is a diagram for explaining a state in which a solution is dropped on a predetermined well using a dropping nozzle.
FIG. 8 is a diagram for explaining a method of applying an alternating electric field to a bioassay substrate.
FIG. 9 is a diagram for explaining an AC electric field applied to a well and a state of DNA at this time.
FIG. 10 is a configuration diagram of a DNA analysis apparatus that performs DNA analysis using the bioassay substrate according to the embodiment of the present invention.
FIG. 11 is a view showing a bioassay substrate on which a plate electrode is integrally formed.
FIG. 12 is a diagram for explaining a plurality of grooves communicating with a row of wells extending from the inner periphery toward the outer periphery together with a plurality of wells arranged radially, and openings formed in the plate electrode.
FIG. 13 is a diagram for explaining a method of applying an alternating electric field to a bioassay substrate on which a plate electrode is formed.
[Explanation of symbols]
1 Bioassay substrate, 2 center hole, 3 substrate layer, 4 transparent electrode layer, 5 solid-phased layer, 6 well forming layer, 8 well

Claims (15)

In the substrate for biochemical reaction, which is a substrate used for biochemical reaction,
A reaction region for performing the biochemical reaction;
An electrode portion for applying an electric field in the reaction region;
A hole into which a rotating shaft for holding and rotating the substrate is inserted;
A second contact portion in contact with the first contact portion formed on the rotating shaft,
The first contact part is a biochemical reaction substrate electrically connected to the electrode part. The first contact portion is formed in a chucking mechanism disposed on the rotating shaft,
2. The biochemical reaction substrate according to claim 1, wherein the second contact portion is disposed so as to come into contact with the first contact portion when the chucking mechanism is inserted into the hole portion. The raw chemical reaction is a hybridization reaction of the nucleotide chain,
The biochemical reaction substrate according to claim 1, wherein the reaction region is subjected to an internal treatment capable of fixing the nucleotide chain.   The biochemical reaction substrate according to claim 1, wherein the electrode part is a first flat electrode layer formed in a lower layer of the reaction region.   The biochemical reaction substrate according to claim 4, wherein the first flat plate electrode layer is a transparent electrode film. A second plate electrode layer formed on an upper layer of the reaction region,
To the chucking mechanism when said chucking mechanism is inserted into the hole portion, a third contact portion for contacting with the second plate electrode layer is formed,
The first flat electrode layer formed in the lower layer of the reaction region is electrically connected to the first contact portion, and the second flat electrode layer formed in the upper layer of the reaction region is the third contact. The biochemical reaction substrate according to claim 4, which is electrically connected to the portion. A biochemical reaction apparatus for performing a biochemical reaction using a substrate having a hole,
A rotating shaft that is inserted into the hole of the substrate to hold and rotate the substrate;
A first contact portion that contacts the second contact portion that is electrically connected to the electrode formed on the rotating shaft and formed in the reaction region of the substrate;
A biochemical reaction device comprising: power supply means for supplying power to the first contact portion.   The biochemical reaction device according to claim 7, wherein the power supply means supplies AC power. Further comprising chucking means disposed on the rotating shaft,
The biochemical reaction device according to claim 7, wherein the first contact portion is formed in the chucking means so as to come into contact with the second contact of the substrate. An external electrode disposed to face the electrode of the substrate;
The biochemical reaction device according to claim 7, wherein the power supply means supplies power between the electrode in the reaction region and the external electrode electrically connected to the first contact portion. The electrode of the substrate is a first plate electrode layer formed on the lower layer of the reaction region of the substrate,
Second flat plate electrode layer is formed on the upper layer of the reaction region of the substrate, further comprising a third contact portion that contacts the fourth contact portion that is connected to the second flat plate electrode layer,
It said power supply means, a biochemical reaction apparatus according to claim 7 that to supply power to the first contact portion and the third contact portion. A plurality of wells in which the inside of the well is treated so that one end of the probe substance can be fixed, and an electrode part for applying an electric field to the inside of the well; a second contact for contacting the center hole chucking mechanism for holding and rotating the substrate is inserted, the first contact portion in which the chucking mechanism is formed on the chucking mechanism when inserted A hybridization substrate in which the first contact portion and the electrode portion are electrically connected, and one end of a probe nucleotide chain is bonded to the bottom surface of the well,
Against the interior of the well to which one end of the nucleotide chain for the probe is coupled to the bottom surface, was added dropwise a solution containing a nucleotide chain of samples,
The chucking mechanism holding the hybridization substrate by inserting into said central hole is placed an external electrode on the wells, the AC power is applied between the external electrode and the chucking mechanism A hybridization method of hybridizing the probe nucleotide chain and the sample nucleotide chain.   The hybridization method according to claim 12, wherein the electrode portion is a first flat electrode layer formed in a lower layer of a portion where the plurality of wells are formed.   The hybridization method according to claim 13, wherein the first flat plate electrode layer is a transparent electrode film. The hybridization substrate includes a second flat plate electrode layer formed above the portion where the plurality of wells are formed, and a fourth contact portion that is connected with the second plate electrode layer,
Said plurality of first plate electrode layer formed below the well, the is the first contact portion and electrically connected to said plurality of said second flat electrode layer formed above the well, Formed in the chucking mechanism when the chucking mechanism is inserted into the center hole, and electrically connected to a third contact portion that contacts the fourth contact portion ;
Said second contact portion, the hybridization method of claim 12 wherein applying an AC power between said fourth contact portion in contact with the third contact portion.

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