JP2005345181A - Intersubstance interaction detecting part, bioassay substrate provided with detecting part, and intersubstance interaction detecting method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To impart light transmission and an insulating property, and to effectively prevent nonspecific adsorption of a substance on a detection face. <P>SOLUTION: This intersubstance interaction detecting part/the like is provided with a light transmitting substrate (base material layer 11), a light-transmissive and insulating carbon compound layer 12 layered on the substrate, a reaction area 2 formed in an upper face side of the carbon compound layer 12 to provide an intersubstance interaction field, and counter electrodes E<SB>1</SB>, E<SB>2</SB>capable of impressing an electric field to a medium M stored or held in the reaction area 2, and the carbon compound layer 12 is formed of diamond or/and diamond-like carbon modified with a functional group charged with a negative charge. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a technique for detecting an interaction between substances. More specifically, the present invention relates to a technique for detecting an interaction between substances that effectively uses the properties of light-transmitting and insulating diamond and / or diamond-like carbon modified with a negatively charged functional group.

  A technique for forming a diamond thin film or a diamond-like carbon thin film (hereinafter abbreviated as “DLC thin film”) on a substrate surface is known.

  "Diamond thin film" can be formed using a gas phase synthesis method such as plasma CVD, and has high thermal conductivity, high insulation resistance, heat resistance, corrosion resistance, radiation resistance, X-ray absorption characteristics, inertness to chemical substances, It has characteristics such as negative electron affinity.

  The “DLC thin film” is literally a thin film made of a carbon-based material having the above-mentioned characteristics of diamond. For example, a hydrocarbon gas is decomposed by arc discharge plasma in a high vacuum, and ions and excited molecules in the plasma are used as a substrate. It can be formed by a method such as a method of making it collide with ion or an ionized vapor deposition method. This DLC thin film has been used mainly for cutting tools, wear parts, and the like because it has a high surface smoothness due to a dense amorphous structure and exhibits excellent trabology (friction wear).

  At present, these thin films are used for semiconductors, flat panel displays, radiation and X-ray monitoring devices, sensor devices that detect strain, pressure, temperature, magnetic field, and electrode materials. I'm starting.

  In recent years, the interaction between biological materials on minute and narrow surfaces using various measurement principles, with the main background of advances in molecular biology, microfabrication technology represented by nanotechnology, bioinformatics, etc. Has been developed and put into practical use (hereinafter referred to as “sensor technology”).

  In the field of this sensor technology, a technology using the above-described diamond thin film or DLC thin film has been proposed. For example, Patent Document 1 discloses that a carbon-based material layer formed by diamond, DLC, graphite, or a mixture thereof is formed on a substrate surface having a surface roughness Ra of 1 to 500 nm subjected to a surface roughening treatment. A roughened slide glass is disclosed in which biological materials such as oligonucleotide fragments, genes, proteins, and peptides are supported on a coating of a system material. Further, Patent Document 1 describes that the DLC film is preferably a transparent film in view of fluorescence intensity measurement.

Patent Document 2 discloses a semiconductor sensing device such as a DNA sensor in which an organic monomolecular film is formed as a direct detection unit on a substrate formed from diamond. In this case, diamond is a material of the substrate itself.
JP 2002-211954 A (refer to claim 1, claim 8, claim 9, paragraph 0011, paragraph 0015, etc.). Japanese Patent Application Laid-Open No. 2004-4007 (refer to claims 6 and 11).

  In bioassay substrates such as various DNA chips that are currently proposed, a reaction region that provides a field of interaction between substances is set on the substrate in advance, and probe DNA or the like that is dropped into the reaction region, etc. The specific interaction (for example, hybridization) between the detection substance in the sample and the target substance in the sample solution is allowed to proceed, and this is detected by the optical pickup means (fluorescence reading means).

  However, there is a problem that non-specific adsorption of the substance to the detection surface in the reaction region occurs and hinders the progress of the interaction. Further, the non-specific adsorption causes a problem that the background noise value at the time of reading the fluorescence intensity is increased. Specifically, assuming that hybridization is detected using a fluorescently labeled intercalator, the intercalator emits fluorescence when adsorbed nonspecifically on the surface of the reaction region. Therefore, normal fluorescence based on hybridization is used. It becomes an obstacle to intensity measurement, and in turn causes a decrease in detection accuracy.

  In addition, when an electric action or effect is applied to a substance existing in a medium stored or held in the reaction region and used for detecting an interaction, an ionic medium that can be stored or held in the reaction region In order to prevent the electrochemical reaction due to the above, it is considered that the surface forming the reaction region is required to have insulating properties.

  Therefore, the present invention provides an inter-substance detection unit capable of effectively preventing nonspecific adsorption of a substance on a detection surface, having a light transmission property and an insulating property, a bioassay substrate including the detection unit, and The main object is to provide a method for detecting an interaction between substances.

  In the present invention, first, a light-transmitting substrate (base material layer), a light-transmitting and insulating carbon compound layer laminated on the substrate, and an upper surface side of the carbon compound layer are formed. A reaction region providing an interaction field such as hybridization, and a counter electrode capable of applying an electric field to a medium stored or held in the reaction region, wherein the carbon compound layer is negatively charged. Provided is a bioassay substrate comprising an interaction detection part between substances formed of diamond or / and diamond-like carbon that is modified with a functional group bearing a carbon atom, a DNA chip on which the interaction detection part is disposed, and the like.

  Diamond or diamond-like carbon, or a mixture of these carbon compound layers, has optical transparency and insulation properties. Therefore, an optical pickup for interaction detection and an assay using electric field application in the reaction region It is suitable for.

  By modifying the carbon compound layer with a functional group such as a negatively charged carboxyl group, non-specific adsorption of the negatively charged substance existing in the reaction region to the surface of the reaction region is electrically repelled. Can be effectively prevented.

  For example, in a configuration using a fluorescent intercalator that is inserted and bonded to a complementary strand (double strand) to detect hybridization, non-specific adsorption of the fluorescent intercalator to the reaction region surface can be prevented. As a result, the background noise value of the fluorescence intensity measurement can be greatly reduced, which greatly contributes to improvement in detection accuracy.

  By measuring the fluorescence intensity obtained by irradiating excitation light of a predetermined wavelength from the back surface side of the substrate toward the reaction region by making both the substrate and the carbon compound layer laminated on the substrate light-transmissive. It becomes possible to detect the interaction between substances. In the case of a substrate configuration in which an electrode is interposed in the traveling path of light directed to the reaction region and fluorescence emitted from the reaction region, this electrode is also a light-transmitting conductor such as ITO (indium-tin-oxide). Form.

  As can be understood from the above description, in the present invention, a light-transmitting substrate, a carbon compound layer formed of light-transmitting and insulating diamond and / or diamond-like carbon laminated on the substrate, and the carbon A reaction region formed on the upper surface side of the compound layer and providing a field of interaction between the materials, and a counter electrode capable of applying an electric field to a medium stored or held in the reaction region. Using an interaction detection unit, the fluorescence intensity obtained by irradiating excitation light of a predetermined wavelength from the back surface side of the substrate toward the reaction region is measured, whereby the interaction between substances that detect the interaction is measured. An action detection method can be provided.

  In this method, the optical pickup means from the back side of the substrate can be adopted, so that a group of devices for supplying a predetermined medium by dropping or injecting the reaction medium into the reaction region can be concentrated in the upper region of the substrate, A group of optical systems used for the optical pickup can be integrated.

  In this method, when the interaction to be detected is hybridization, a method of measuring the fluorescence intensity of a fluorescence intercalator that is inserted and bonded to a complementary strand obtained by the hybridization can be employed. By adopting a carbon compound layer formed of diamond or / and diamond-like carbon modified with a negatively charged functional group, nonspecific adsorption of the fluorescent intercalator to the carbon compound layer can be reliably prevented. is there. In this method, in addition to the detection of hybridization using a fluorescent intercalator, it is also possible to detect hybridization using a fluorescently labeled target nucleic acid.

  Here, definition of main technical terms used in the present invention is performed.

  “Light transmittance” means that it has the property of transmitting at least the excitation light of a predetermined wavelength and the fluorescence of the predetermined wavelength used for detecting the interaction, and the light transmittance is as long as the fluorescence intensity can be measured. Good.

  The “reaction region” is a region or space that can provide a reaction field for hybridization or other interaction, and examples thereof include a reaction field having a well shape that can store a liquid phase, a gel, and the like. The interaction performed in this reaction region is not narrowly limited as long as the object and effect of the present invention are met. Further, the shape and size of the reaction region are not particularly limited, but the length, width, and depth are about several μm to several hundred μm, respectively. It can be determined based on the minimum possible dripping amount of the solution (detection substance-containing medium, target substance-containing medium).

  “Interaction” broadly means non-covalent bonds, covalent bonds, chemical bonds or dissociations including hydrogen bonds between substances, for example, hybridization that is a complementary bond between nucleic acids (nucleotide strands), polymer-high Widely includes molecules, macromolecule-small molecules, small molecule-small molecule specific bonds or associations.

  “Hybridization” means a complementary strand (double strand) formation reaction between complementary base sequence structures. “Mishybridization” means the complementary strand formation reaction that is not normal, and is often abbreviated as “mishybridization” in the present invention.

  A “fluorescence intercalator” is a fluorescently labeled substance that can be inserted and bound to a double-stranded nucleic acid, and is a substance used for hybridization detection. For example, POPO-1, TOTO-1, SYBERGreen I, PicoGreen, etc. can be used.

  A “detection substance” is a substance that exists in a medium stored or held in a reaction region and functions as a detector for detecting a substance that specifically interacts with the substance. It exists fixed or free on the surface. A typical example is a nucleic acid for detection such as a DNA probe, but widely includes other biological materials such as proteins.

  The “target substance” is a substance that shows a specific interaction with the detection substance.

  “Nucleic acid” means a polymer (nucleotide chain) of a nucleoside phosphate ester in which a purine or pyrimidine base and a sugar are glycosidically bonded, and an oligonucleotide, polynucleotide, purine nucleotide and pyrimidine nucleotide containing a probe DNA are polymerized. DNA (full length or a fragment thereof), cDNA obtained by reverse transcription (c probe DNA), RNA, polyamide nucleotide derivative (PNA) and the like are widely included.

  “Bioassay substrate” means a substrate for detecting the interaction by causing an interaction between substances to proceed in a predetermined reaction region on the substrate, and widely includes regardless of the type of the substance, The detection principle of the interaction is not limited.

  “DNA chip” is one of the bioassay substrates, and means a hybridization detection substrate in which nucleic acids for detection such as DNA probes are in a free state or in a state of being immobilized and finely arranged, It also includes the concept of DNA microarray.

  According to the present invention, nonspecific adsorption of a substance on a detection surface can be effectively prevented. Moreover, an optical pickup means from the back surface of the substrate can be employed, and furthermore, an electric field assay can be performed in the reaction region.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the accompanying drawings. Each embodiment shown in the attached drawings shows an example of a typical embodiment of the thing and method concerning the present invention, and, thereby, the scope of the present invention is not interpreted narrowly.

  First, FIG. 1 is a diagram schematically showing a basic layer structure of a substrate that can be used in a substance interaction detection unit according to the present invention.

  A substrate 1a in FIG. 1 is formed of a light-transmitting base material layer 11, a light-transmitting carbon compound layer 12 laminated on the base material layer 11, and a polyimide laminated on the carbon compound layer 12. The synthetic resin layer 13 is formed with a well-shaped minute reaction region 2 formed in the synthetic resin layer 13. The bottom surface of the reaction region 2 is constituted by the carbon compound layer 12. In FIG. 1, a medium stored or held in the reaction region 2 is indicated by a symbol M.

  The reaction region 2 may be a fine region or space that can store or hold the medium M and can be an interaction field between substances. In order to form the reaction region 2 having such a fine structure, a photolithography method, an etching method, or the like known in the field of normal semiconductor or mechanical microfabrication technology can be used.

  The base material layer 11 can be formed from a base material similar to an optical information recording medium such as a CD (Compact Disc), a DVD (Degital Versatile Disc), or an MD (Mini Disc). For example, it is molded into a desired form using a synthetic resin such as quartz glass, silicon, polycarbonate, polystyrene, etc. having light transmissivity, preferably a synthetic resin that can be injection molded. In addition, by forming the substrate using an inexpensive synthetic resin, a low running cost can be realized as compared with a conventionally used glass chip.

  The carbon compound layer 12 is a layer formed of diamond, diamond-like carbon (hereinafter abbreviated as “DLC”), or a mixture of these, and the layer 12 has an insulating property in addition to light transmittance. (Hereinafter the same). The method for forming the carbon compound layer 12 is not particularly limited. The film thickness of the carbon compound layer 12 is desirably 1 to 100 nm. The light transmittance may be 1% or more. However, when an optical pickup is assumed, higher light transmittance is desirable. Since diamond and DLC have a high refractive index, they can be used when providing a light transmission selectivity by forming a multiple reflection film.

  FIG. 2 is a diagram schematically showing another layer structure of a substrate that can be used in the detection unit according to the present invention and a concept of a suitable optical pickup means for the substrate.

  In the substrate 1 b shown in FIG. 2, a light transmissive conductor layer 14 is formed between the base material layer 11 and the carbon compound layer 12. The conductor layer 14 can be employed as long as it is a light transmissive conductor. For example, ITO (indium-tin-oxide) can be formed by sputtering vapor deposition on the base material layer 11. This conductor layer 14 can be used as one of the counter electrodes used when an electric field is applied to the medium M in the reaction region 2. Moreover, in the board | substrate 1b, a titanium oxide film can be provided between the base material layer 11 and the conductor layer 14, and it can be set as the reflective layer for servo light mentioned later.

In the substrate 1a of FIG. 1, both the base material layer 11 and the carbon compound layer 12 are light transmissive, and in the substrate 1b of FIG. 2, the base material layer 11, the conductor layer 14, and the carbon compound layer 12 are all light transmissive. since it is, the substrate 1a, the excitation light (excitation laser beam) P of a predetermined wavelength from the rear surface 100b side 1b, the through the condensing lens 3 (corresponding to the condensing lens shown by reference numeral L ₂ in FIG. 6) Irradiation is directed toward the reaction region 2 (fluorescent substance in the reaction region 2), whereby the intensity of the fluorescence F generated in the reaction region 2 is detected by the detector 4 (corresponding to the detector indicated by reference numeral 111 in FIG. 6). .

Reference numeral 5 in FIG. 2 shows an arrangement is the condenser lens in front of the detector 4 (corresponding to the condensing lens shown by reference numeral L ₃ of Figure 6). Details of the optical pickup means will be described later.

  FIG. 3 is an enlarged view of a portion X shown in FIG. This enlarged view shows a state in which one end of a detection substance (detection nucleic acid) D such as a DNA probe is fixed to the surface 12a of the carbon compound layer 12 constituting the bottom surface of the reaction region 2 by an amide bond. ing.

  The immobilization of the target substance D on the surface 12a of the carbon compound layer 12 formed of diamond, DLC, or a mixture thereof is, for example, a step of injecting a reactive gas in the gas phase while the surface 12a is irradiated with ultraviolet rays. Then, the functional group is chemically modified and formed, and the reaction is performed with the target substance D modified with the functional group having reactivity with the functional group.

  For example, a carboxyl group is formed on the surface 12a of the carbon compound layer 12 by the chemical modification, the carboxyl group is reacted with an amino group-modified DNA probe, and the DNA probe is formed on the carbon compound layer 12 by an amide bond. Can be fixed.

  In addition, when the functional group which the substance for detection D itself has and the functional group of the surface 12a can be reacted, the substance D for detection may be fixed by reacting these functional groups. it can.

  Further, in FIG. 3, the detection substance D immobilized on the surface of the carbon compound layer 12 is hybridized with the target substance (here, the target nucleic acid) T having a complementary base sequence. And a state where the fluorescent intercalator I is inserted and bonded to the base pair portion of the complementary strand obtained by the hybridization. Although not shown, a target substance (target nucleic acid) T labeled with a fluorescent substance may be used without using the fluorescent intercalator I.

  As shown in FIG. 3, if the surface 12a of the carbon compound layer 12 made of diamond, DLC, or a mixture thereof is modified with a negatively charged functional group such as a carboxyl group, the negative charge is similarly obtained. Non-specific adsorption of the detection or target biological substance (for example, nucleic acid) or fluorescent intercalator I can be effectively prevented by electrical repulsion.

  Without non-specific adsorption to the surface 12a of the carbon compound layer 12, the detection substance D is immobilized, the interaction between the detection substance D and the target substance T (for example, hybridization), the fluorescence intercalator I Assays such as insertion binding reactions are preferred because they can proceed efficiently and reliably under conditions where there is no competition with non-specific adsorption. In addition, fluorescence from non-specifically adsorbed fluorescent intercalator I or non-specifically adsorbed fluorescently labeled target substance T can be extremely reduced, allowing high-precision fluorescence intensity measurement for interaction detection. Can be done.

  Here, FIG. 4 simply shows an example of a preferred embodiment of a detection unit specifically including an electric field applying unit.

4 includes a light-transmitting base material layer 11, a light-transmitting carbon compound layer 12 laminated on the base material layer 11, and a carbon compound layer 12. The synthetic resin layer 13 made of polyimide or the like is laminated, and the well-shaped minute reaction region 2 is formed in the synthetic resin layer 13. Further, a transparent conductor portion 141 such as ITO (indium-tin-oxide) is formed on a part of the carbon compound layer 12 below the reaction region 2. The conductor portion 141 functions as an electrode _{E 1.} In addition, a titanium oxide film can be provided between the base material layer 11 and the carbon compound layer 12 to form a reflective layer for servo light described later.

The electrode E ₂ at a position opposed to the electrodes E ₁ is formed, the upper substrate 15 made by the polyimide is arranged (see FIG. 4). In this case, the reaction region 2 is in a state of being covered except for some of the openings 21 and 22. It is also possible to drop the medium M into the opening 21 or 22 by an ink jet nozzle or the like and send it into the reaction region 2 using a capillary phenomenon.

Here, as shown in FIG. 4, the electrodes E ₁ -E ₂ are opposed to each other so as to sandwich the reaction region 2, and are opposed electrodes that can be energized by the power supply G. By an on / off operation, an electric field can be applied to the medium M in the reaction region 2 or the electric field can be turned off. In addition, by forming the electrode E ₁ with a smaller area than the one electrode E _2, it is devised so that the lines of electric force can be concentrated near the edge of the electrode E ₁ .

  The electric field may be selected from DC, AC, low frequency, and high frequency depending on the purpose. In a DC electric field, electrophoresis is possible. In an AC electric field, for example, by applying a high-frequency AC electric field under a predetermined condition under conditions where electrolysis does not occur, the electric field density is increased by the action of dielectrophoresis. Since the nucleic acid molecule can be moved or extended, it can be effectively used for immobilization of the detection nucleic acid D or a hybridization assay. In addition, at low frequency, it can be used when the hybridized DNA is vibrated and dissociated from the nucleic acid for detection.

In addition, as a suitable condition of the high frequency electric field at this time, 1 MV / m or more and 1 MHz or more are desirable. For example, about 1 × 10 ⁶ V / m and about 1 MHz can be selected (Masao Washizu and Osamu Kurosawa: “Electrostatic Manipulation of DNA in Microfabricated Structures ”, IEEE Transaction on Industrial Application Vol.26, No.26, p.1165-1172 (1990)).

The arrangement configuration of the electrodes E ₁ -E ₂ is not limited to the embodiment shown in FIG. 4, and an arrangement configuration facing each other so as to sandwich at least one of the reaction regions 2 is appropriately selected according to the purpose. it can. The counter electrodes do not necessarily have to be a pair, and depending on circumstances, two or more pairs may be formed. For example, two pairs of counter electrodes may be provided so that the counter axes are orthogonal to each other.

In the present invention, a bioassay substrate having a configuration in which a plurality of the detection units 10 described above are arranged can be provided. As an example, it is also possible to provide a disc-shaped bioassay substrate 100 having a form as shown in FIG. Note that the counter electrodes E ₁ -E ₂ of the detection unit 10 disposed on the bioassay substrate 100 are arranged so as to be laterally distributed so as to sandwich the reaction region 2 (see FIG. 5).

  In the following, the optical pickup means suitable for the present invention will be described in detail by taking the case of using this disk-shaped bioassay substrate 100 as a representative example.

  FIG. 6 is a block diagram showing an example of a preferred embodiment of the optical pickup means. In the following description, it is assumed that the bioassay substrate 100 is a DNA chip.

  First, the bioassay substrate (DNA chip) 100 shown in FIG. 6 is fixed to a spindle (jig) 102 protruding above a disk support base 101 having a rotating means. The spindle 102 is inserted while being positioned in a hole (see FIG. 5) in the center of the substrate 100.

Note that each reaction region 2 of the substrate 100, is the medium M ₁ is dropped or injected containing nucleic acid D for detection in the manufacturing stage of the DNA chip, the hybridization detection step, the medium M ₂ containing the target nucleic acid T is dropped or Injected. The fluorescent intercalator I may be dropped or injected together with the target nucleic acid T.

These media M ₁ and M ₂ are generally in the form of a solution or a viscosity-adjusted gel. In particular, in the case of a solution-like medium, it is desirable to hold the substrate 100 on which the medium M is dropped or injected in order to avoid various problems such as liquid dripping.

In the configuration shown in FIG. 6, a plurality of nozzle heads N are arranged on the upper surface 100 a side (upper side) of the substrate 100. The nozzle head N accurately follows a predetermined medium M (for example, M _{1 to} M ₃ , M ₃ is a medium including an intercalator) following each reaction region 2 of the detection unit 10 arranged at a predetermined position. In addition, it is configured such that it can be dropped or injected at a predetermined timing.

  Reference numeral 103 in FIG. 6 simply indicates the “control unit” in a block diagram. The control unit 103 controls the entire dropping or injection operation of the nozzle head N based on “focus information” with respect to the substrate 100 and “tracking information” obtained from the substrate 100.

Here, the excitation light indicated by symbol P in FIG. 6 is information reading light used in the hybridization detection stage. This excitation light P is emitted from the laser diode 104, converted into parallel light by the collimator lens L _1, and then refracted by 90 ° by the dichroic mirror 105.

Thereafter, the excitation light P, after the mirror 106 disposed ahead in the traveling direction of the light is 90 ° refracted, is incident on the condenser lens L ₂ supported by the actuator 107, is selected from the back 100b of the substrate 100 The detection unit 10 (the reaction region 2) is irradiated. Reference numeral 108 denotes a signal for controlling the laser diode driver 109 transmitted from the control unit 103.

Excitation light P is narrowed down to a size of about several μm on the back surface of the substrate 100 by the condenser lens L _2. In order to make use of this minute excitation light spot diameter, the nozzle head N for dropping or injecting the medium M ₁ containing the detection nucleic acid D, the medium M ₂ containing the target nucleic acid T, etc. on the substrate 100 is in the picoliter order. An ink jet printing nozzle capable of dripping a very small amount of the solution is suitable.

  The number of ink jet printing nozzles, which are an example of the nozzle head N, may be prepared as many as the number and type of the medium to be used. After one type of medium is dropped, the nozzle is once washed and another type of solution is dropped. By doing so, various media may be handled with a smaller number of nozzles.

Incidentally, by using the ink jet printing nozzle, the medium M ₃ including the intercalator I (see Fig. 3), to be dropped or injected into the medium M ₂ simultaneously reaction region 2 containing the target nucleic acid, the predetermined timing after the hybridization Thus, the reaction region 2 can be dropped or injected, or a predetermined cleaning solution can be dropped or injected into the reaction region 2 at a predetermined timing after the immobilization step.

When the media M _{1 to} M ₃ are dropped or injected onto the substrate 100, a desired address is read while reading address mark information recorded in advance on the substrate 100 using servo laser light V (described later). The medium is dropped or injected into (address).

The detection nucleic acid D immobilized on the reaction region 2 (detection surface portion) of the substrate 100 and the target nucleic acid T dropped or injected later showed hybridization, forming complementary double-stranded nucleic acids. Later, or simultaneously with the dropping or injection of the target nucleic acid T, the medium M ₃ containing the fluorescent intercalator I that can be inserted and bonded to the double-stranded nucleic acid is passed through the predetermined nozzle head N to the reaction region 2. Can be dripped or injected.

  When the excitation light P is irradiated, the fluorescence indicated by the symbol F is emitted from the intercalator I (see FIG. 3), and the fluorescence F returns to the back surface 100b side of the substrate 100. The fluorescence F is refracted by 90 ° by the mirror 106 disposed below the substrate 100, passes through the dichroic mirror 105 disposed in the light traveling direction of the fluorescence F, travels straight, and is disposed further forward. The light is refracted by 90 ° by the dichroic mirror 110.

Subsequently, the fluorescence F enters the lens L ₃ above the dichroic mirror 110 and is collected, and then guided to the detector 111. As described above, the dichroic mirror 110 has the property of reflecting the fluorescence F, and has the property of transmitting light to a servo laser beam V described later.

  Here, the fluorescence intensity is expected to be very weak compared to a general optical disk RF signal or the like. Therefore, it is preferable to employ a photomultiplier (photoelectric tube) or an avalanche photodiode (APD) having a higher sensitivity than a general photodiode as the detector 111 for detecting fluorescence.

  The fluorescence F detected by the detector 111 is converted into a digital signal 113 having a predetermined number of bits by the AD converter 112. This digital signal 113 is used, for example, for analysis such as creation of a map in which addresses on the substrate 100 are associated with emitted fluorescence intensity.

  Next, the “servo mechanism” related to the present invention will be specifically described.

First, the condenser lens L ₂ which is disposed below the substrate 100 by the actuator 107, are configured to be driven in the focusing direction (vertical direction) and the tracking direction (circumferential direction). The actuator 107 is preferably a biaxial voice coil type of the same type as that used for optical disk pickup.

As described above, substrate 100 is held horizontally, and the condenser lens L ₂ is because it is installed in a vertically downward position when viewed from the substrate 100, it is utilized to the excitation light P as well as focusing servo and tracking servo The servo laser beam V can be irradiated from the back side 100b of the substrate 100.

  By adopting the configuration in which the servo laser light V is irradiated from the back surface 100b side of the substrate 100, the servo laser light V is detected by the detection nucleic acid D and the target existing in the medium of the detection unit 10 of the substrate 100. Without being affected at all by the nucleic acid T, the light is reflected back from the back surface 100b of the substrate 100 by a reflective layer (not shown) formed from a titanium oxide film or the like provided on the substrate 100.

  For this reason, even if the substrate 100 rotates, the direction and intensity of the reflected light are not disturbed by the medium in the substrate 100, which is very suitable. That is, the servo can operate stably without being affected by the disturbance of the focus error and the tracking error.

Specifically, first, reference numeral 114 in FIG. 6 denotes a servo laser diode, and a driver 115 for controlling the diode 114 is arranged behind the servo laser diode. A collimator lens L ₄ is disposed in the light emitting direction of the servo laser diode 114.

This collimator lens L _4, the servo laser beam V is straight is converted into parallel light. Incidentally, the dichroic mirror 116 disposed in front of the collimator lens L ₄ includes the property of having a light-transmitting property with respect to the servo laser beam V.

Here, several methods such as an astigmatism method, a knife edge method, and a skew method are conceivable for the generation of the focus error. In the case of the configuration shown in FIG. 6, non-astigmatism generation lens L ₅ is used. The point aberration method is adopted.

  In addition, the differential push-pull method and the three-spot method are suitable for the generation of tracking errors. Both methods require obtaining three light spots on the disc.

Accordingly, the diffraction grating 117 shown in FIG. 6 is inserted between the collimator lens L ₄ of the servo laser optical system and the dichroic mirror 116 and is diffracted as is common in optical disk pickups. the zero-order and ± 1-order light is irradiated to the substrate 100 via the condenser lens L _2.

  Reference numeral 118 in FIG. 6 indicates a detector of servo laser reflected light. The driver 115 and the detector 1118 described above are controlled by the control unit 103 (see FIG. 6).

  The wavelength of the excitation light P, the wavelength of the fluorescence F, and the wavelength of the servo laser light V may be different from each other. When adopting a configuration in which the wavelengths of these lights P, F, and V are different, the reflective layer provided on the substrate 1 has a certain degree of translucency (transmittance) with respect to the wavelength of the fluorescence F. There is a need for a physical property having a laser beam reflectivity that has a servo operation. That is, the reflectivity / transmittance may have wavelength dependency, and preferably has a low-pass filter-like frequency characteristic. In the case of a single wavelength, it is difficult to improve both the transmittance of the fluorescence F and the reflectance of the laser beam V. However, by providing the wavelength dependence, the transmittance of the fluorescence F and the laser beam V can be improved. The reflectance can be improved.

  First, a 10 nm thick DLC thin film was formed on a silicon substrate, and carboxyl groups were formed on the surface of the DLC thin film by chemical modification. Next, the carboxyl group was reacted with a 30-base single-stranded DNA probe (oligonucleotide) that was amino-modified in an immobilization solution, and the DNA probe was immobilized on the DLC thin film by an amide bond. In addition, the concentration in the solution of a DNA probe prepared three types, 1 micromol, 5 micromol, and 10 micromol, and formed each fixed surface.

  30-base single-stranded target DNA (oligonucleotide, Comp) having a sequence complementary to the DNA probe, 30-base single-stranded target DNA having no complementary sequence (oligonucleotide, Scramble) ) And those modified with Cy3, a fluorescent dye, were prepared. Using them, hybridization was performed in a buffer solution (citrate buffer solution). After hybridization, the substrate surface was washed with a citrate buffer solution containing SDS (Sodium dodecyl sulfate). In addition, all the oligonucleotides used in this test were contracted synthetic products from Espec Oligo Service.

  In the test group (test group 1) using the target DNA modified with the fluorescent dye Cy3, the fluorescence measurement (570 nm) was performed as it was after the washing operation. That is, “test group 1” assumes a case where hybridization is detected without using an intercalator. The results are shown in FIG. 7 (the vertical axis indicates the amount of fluorescence, and also in FIG. 8 described later). As the spectroscopic system for the fluorescence measurement, a microspectroscopic system (manufactured by Otsuka Electronics Co., Ltd.) that combines a fluorescence microscope (manufactured by Nikon Instick Co., Ltd.) and an optical fiber type instantaneous multiphotometric detection system was used. .

  When the target DNA not modified with Cy3 is used (test group 2), a Tris • EDTA buffer containing SYBERGreen I (Molecular Probes), which is a fluorescent dye that selectively intercalates with double-stranded DNA, is used. After staining, fluorescence measurement (520 nm) was performed. That is, “Test Group 2” is a case where hybridization is detected using an intercalator. The result is shown in FIG.

  First, looking at FIG. 7 showing the results of “Test Group 1”, complementary target DNAs having complementary sequences (indicated by “Comp-Cy3” in FIG. 7) are in the case of 5 μM and 10 μM. It can be seen that almost the same amount of fluorescence is shown. That is, at a concentration of 5 μM or more, the probe fixation amount is considered to be almost saturated.

  On the other hand, at a concentration of 1-5 μM, the amount of DNA probe immobilized on the surface of the DLC thin film increases, and accordingly, the amount of hybridization with the target DNA is considered to increase accordingly.

  On the other hand, in the case of the target DNA having no complementary sequence (shown as “Scramble-Cy3” in FIG. 7) in the “test group 1”, regardless of the concentration of the DNA probe in the solution, Fluorescence was hardly measured and the amount of fluorescence hardly changed.

  From the above results in “Test Zone 1”, it was possible to allow normal hybridization to proceed on a silicon substrate provided with a DLC thin film, since fluorescence was measured only with a target DNA having a complementary sequence. I can understand.

  “Control group X” shown in FIG. 7 shows the result of measuring the fluorescence intensity of the silicon substrate provided with the DLC thin film in a state where no fluorescence staining is performed, and has a complementary sequence. It can be seen that there is almost no difference in the amount of fluorescence in the case of no DNA (see “Scramble-Cy3” in FIG. 7).

  Next, FIG. 8 showing the result of the above-mentioned “test group 2” shows DNA that is not complementary to target DNA that is complementary (indicated by “Comp-SBGr” in FIG. 8) (“Scramble-” in FIG. 8). In this case, the fluorescence intensity according to the concentration of the DNA probe is shown.

  When complementary target DNA is used (see “Comp-SBGr” in FIG. 8), it can be seen that the fluorescence intensity shows saturation at a DNA probe concentration of 5 μM, as in the result of FIG. On the other hand, in the case of DNA having no complementary sequence (see “Scramble-SBGr” in FIG. 8), the fluorescence intensity was measured about one-fifth that of complementary target DNA.

  When DNA that does not have a complementary sequence is used, hybridization is not observed, and the intercalator SYBERGreen I is known to adsorb to single-stranded DNA to some extent and have luminescence. Thus, the fluorescence measured in the case of DNA having no complementary sequence is considered to be fluorescence derived from the intercalator SYBERGreen I adsorbed on a single-stranded DNA probe fixed on the substrate surface.

  In “Test Group 2”, the fluorescence intensity when using non-complementary DNA has the same probe concentration dependency as the result of the target DNA (Comp-Cy3) having a complementary sequence in FIG. (See FIG. 8), it was also found that the amount of single-stranded DNA on the substrate can be measured from the fluorescence intensity using intercalator SYBERGreen I.

  In “Test group 2”, a test was also conducted in which a silicon substrate on which a DLC thin film modified with a carboxyl group was laminated was stained with an intercalator SYBERGreen I (control group B in FIG. 8). In this control group B, almost no fluorescence was observed as in the control group A where no staining was performed. From this, it was confirmed that there was almost no non-specific adsorption of the intercalator SYBERGreen I on the surface of the DLC thin film modified with a carboxyl group.

  From the above, if a substrate having a DLC thin film surface chemically modified with a carboxyl group is used, nonspecific adsorption of the fluorescent intercalator to the DLC thin film surface does not occur. It was confirmed that can be easily realized using a fluorescent intercalator.

  ITO was provided on a quartz substrate by sputtering deposition, and a DLC thin film was formed thereon. The wall structure was formed of polyimide, and a well structure was provided on the substrate (see the substrate configuration in FIG. 2).

  Next, the DLC thin film was subjected to a surface treatment to modify the carboxyl group. To this, a nucleic acid molecule modified with an amino group and labeled with Cy3 was treated with a fixing solution and dropped into a well. Then, an aluminum substrate was placed so as to cover the well, and using an inverted fluorescence microscope, the light from the mercury lamp was irradiated from the back surface of the substrate, and the fluorescence of the return light was measured.

  As a result, the light transmittance was 90% or more, and the fluorescence in the well could be observed. As a result, it has been found that the fluorescent substance existing in the well on the substrate on which the fine structure is formed can be optically measured from the back side of the substrate by the light irradiation means.

  The present invention is useful as a technique for detecting an interaction between substances. In particular, it can be used as a technique for detecting an interaction such as hybridization based on measurement of fluorescence intensity of a fluorescently labeled target substance or fluorescent intercalator.

It is a figure which shows simply the basic layer structure of the board | substrate which can be used for the interaction detection part between the substances concerning this invention. It is a figure which shows simply the concept of the suitable optical pick-up means with respect to the other layer structure of the board | substrate which can be used for the detection part which concerns on this invention, and this board | substrate. FIG. 3 is an enlarged view of a portion X shown in FIG. 2. It is a figure which shows simply an example of suitable embodiment of a detection part provided with an electric field application means concretely. It is a figure which shows embodiment which is an example of the board | substrate for bioassays concerning this invention. It is a block diagram which shows an example of suitable embodiment of the optical pick-up means suitable for this invention. It is a figure (graph) which shows the result of the test section 1 of an Example. It is a figure (graph) which shows the result of the test section 2 of an Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1a, 1b Substrate 2 Reaction area | region 3, 5 Condensing lens 4 Detector 10 Interaction detection part between substances (abbreviation "detection part")
DESCRIPTION OF SYMBOLS 11 Base material layer 12 Carbon compound layer 13 Synthetic resin layer 14 Conductor layer 100 (Disc-shaped) bioassay substrate 100b Back surface 141 Narrow conductor layer (electrode)
D Substance for detection (eg, DNA probe)
E ₁ , E ₂ Counter electrode F Fluorescence G Power source M Medium P Excitation light S for reading S Switch T Target substance

Claims (9)

A light-transmitting substrate; a light-transmitting and insulating carbon compound layer laminated on the substrate; and a reaction region formed on an upper surface side of the carbon compound layer to provide an interaction field between substances. And a counter electrode capable of applying an electric field to a medium stored or held in the reaction region, wherein the carbon compound layer is modified with a negatively charged functional group and / or the modified diamond. An interaction detector between substances formed from like carbon. The interaction detection unit according to claim 1, wherein the functional group is a carboxyl group. The interaction detection unit according to claim 1, wherein the interaction is hybridization. The interaction detection unit according to claim 1, wherein the medium includes a fluorescence intercalator for detecting the hybridization. The interaction may be detected by measuring fluorescence intensity obtained by irradiating excitation light of a predetermined wavelength from the back surface side of the substrate toward the reaction region. The interaction detection unit according to claim 1. A bioassay substrate comprising the interaction detection unit according to claim 1. The bioassay substrate according to claim 6, which is a DNA chip. A light-transmitting substrate, a carbon compound layer formed of light-transmitting and insulating diamond or / and diamond-like carbon laminated on the substrate, and an upper surface side of the carbon compound layer; Using an interaction detection unit between substances comprising a reaction region that provides an interaction field, and a counter electrode capable of applying an electric field to a medium stored or held in the reaction region,
Detection of an interaction between substances, wherein the interaction is detected by measuring fluorescence intensity obtained by irradiating excitation light of a predetermined wavelength from the back side of the substrate toward the reaction region. Method. 9. The method for detecting an interaction according to claim 8, wherein the fluorescence intensity of a fluorescence intercalator that inserts and binds the hybridization that is the interaction to a complementary strand is measured.

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