JP4483279B2 - Biological substance fluorescent labeling agent, biological substance fluorescent labeling method, and bioassay method and apparatus - Google Patents

Description

  The present invention relates to a biological material fluorescent labeling agent and a biological material fluorescent labeling method useful in the field of bioinformatics (biological information science) such as a DNA chip, a bioassay method using the biological material fluorescent labeling agent, and an apparatus therefor.

  Currently, integrated substrates for bioassays called so-called DNA chips or DNA microarrays (hereinafter collectively referred to as DNA chips), in which predetermined DNA is finely arranged by microarray technology, are used for gene mutations, SNPs (single nucleotide multiples). 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.

  Since this DNA chip has a large number of DNA oligonucleotide chains, cDNA (complementary DNA), etc. integrated on a glass substrate or silicon substrate, it is possible to comprehensively analyze intermolecular interactions such as hybridization. Become.

  The DNA analysis method using a DNA chip is, for example, reverse transcription PCR (Polymerase Chain Reaction) using mRNA (messenger RNA) extracted from cells, tissues, etc. with respect to probe DNA immobilized (immobilized) on a DNA chip. ) A sample DNA generated by PCR amplification while incorporating the fluorescent probe dNTP by reaction or the like is dropped, a hybridization reaction is performed on the DNA chip, and fluorescence detection is performed with a predetermined photodetector.

  In recent years, it has been proposed to apply the substrate technology cultivated in the field of optical discs to DNA analysis by changing the shape of the DNA chip to a disc shape instead of the conventional rectangular shape (see Patent Document 1). .

  When performing DNA analysis using this optical disc technology, for example, while rotating a disk-shaped DNA chip, the fluorescence generated from the double-stranded DNA after the hybridization reaction is detected by a photodetector, and the fluorescence is detected. Thus, it is possible to apply a servo technique such as specifying the light emission position by an addressing technique, thereby increasing the number of sample substances to be processed and improving the detection accuracy and the detection speed.

JP 2001-238664 A Special Table 2002-525394

  By the way, organic fluorescent dyes are generally used as fluorescent labeling agents in DNA analysis so far.

  However, when an organic fluorescent dye is used for fluorescence detection, the transition from LUMO to HOMO of π electrons becomes supersaturated at a light intensity above a certain level, and fluorescence above a certain intensity cannot be obtained. In addition, when an organic fluorescent dye is activated by photoexcitation, it reacts with surrounding water and decomposes, so that the effect of fading is great. Therefore, when an organic fluorescent dye is used for fluorescence detection, it cannot be excited with a large intensity of excitation light or repeatedly detected. Furthermore, since the organic fluorescent dye has a narrow wavelength range that can be excited, there is a problem that the wavelength of the excitation light is limited.

  On the other hand, the use of quantum dots having high fluorescence intensity (also referred to as semiconductor nanocrystals or semiconductor ultrafine particles) as a fluorescent labeling agent has been proposed (see Patent Document 2).

  However, in general, such quantum dots use compound semiconductors such as III-V group compound semiconductors and II-VI group compound semiconductors, and have a large adverse effect on the environment. In addition, since an organic metal solution or the like is also used for crystal growth of quantum dots, there is a problem that it is difficult to handle and manufacturing is not easy.

  The present invention has been proposed in view of such a conventional situation, and has a small influence on the environment and is easy to manufacture, a biological material fluorescent labeling agent and a biological material fluorescent labeling method, and the biological material fluorescent labeling agent It is an object of the present invention to provide a bioassay method and a device therefor.

The biological material fluorescent labeling agent according to the present invention proposed to achieve the above-described object is obtained by annealing SiO _{x formed} on a silicon wafer in an inert gas atmosphere, and then subjecting the silicon wafer to an aqueous hydrofluoric acid solution. in process generated by a core made of silicon crystals of size of 4 times or less of the exciton Bohr radius, is composed of a shell layer made of silicon oxide covering said core, constituting the shell layer Silicon oxide is obtained by bonding organic molecules that bind to a biological substance to the surface of a particle of a quantum dot that is silanized and has a mercapto group introduced therein.

In addition, the biological material fluorescent labeling method according to the present invention proposed to achieve the above-described object is obtained by annealing SiO _x deposited on a silicon wafer in an inert gas atmosphere, and then squeezing the silicon wafer. A core made of a silicon crystal having a crystal diameter of 4 times or less of an exciton Bohr radius generated by treatment with an acid aqueous solution, and a shell layer made of silicon oxide covering the core, the shell layer being The silicon oxide to be formed is one in which a quantum dot in which a mercapto group is introduced by silanization is bonded to a biological substance via an organic molecule.

  Here, examples of the biological material include nucleotide chains or proteins collected from the living body.

Further, in the bioassay method according to the present invention proposed to achieve the above-described object, a plurality of reaction regions are formed on the surface, and a plurality of known detection substances are immobilized in each predetermined reaction region. The bioassay substrate was formed by annealing SiO _x deposited on a silicon wafer in an inert gas atmosphere and then treating the silicon wafer with an aqueous hydrofluoric acid solution. Quantum in which a core made of a silicon crystal having a crystal diameter of less than twice and a shell layer made of silicon oxide covering the core are silanized and mercapto groups are introduced into the silicon oxide constituting the shell layer A reaction step in which a target substance labeled with a dot is dropped to interact with each other, and after the reaction in the reaction step, an unreacted target substance is removed from the bioassay. A removal step of removing from each reaction region of the substrate, and irradiating the bioassay substrate with excitation light of a predetermined wavelength, and determining whether there is fluorescence of a predetermined wavelength generated from the quantum dots in response to the excitation light And an analysis step of performing biochemical analysis of the target substance by detecting.

Moreover, the bioassay device according to the present invention proposed to achieve the above-described object has a plurality of reaction regions formed on the surface, and a plurality of known detection substances are immobilized in each predetermined reaction region. A holding means for holding a bioassay substrate, and annealing the SiO _{x formed} on the silicon wafer on the bioassay substrate in an inert gas atmosphere, and then treating the silicon wafer with a hydrofluoric acid aqueous solution. Formed by a core made of silicon crystal having a crystal diameter equal to or less than four times the exciton Bohr radius and a shell layer made of silicon oxide covering the core, and the silicon oxide constituting the shell layer is silane Drop means for dropping a target substance labeled with a quantum dot into which a mercapto group is introduced, and a target dropped by the detection substance and the drop means After the reaction with the substance, the removing means for removing the unreacted target substance from each reaction region of the bioassay substrate, and the excitation light having a predetermined wavelength are irradiated to the bioassay substrate. And analyzing means for performing biochemical analysis of the target substance by detecting the presence or absence of fluorescence having a predetermined wavelength generated from the quantum dots.

  In this bioassay method and apparatus, the target substance is dropped onto the bioassay substrate and reacted with the detection substance, and then the unreacted target substance is removed from each reaction region of the bioassay board. A removing step (means) to be performed.

  A nucleotide chain can be used as the detection substance and the target substance, and in this case, the interaction is a hybridization reaction.

  Since the quantum dots constituting the biological material fluorescent labeling agent according to the present invention are composed of silicon and silicon oxide, they are easy to handle with little influence on the environment, and particularly silicon oxide around the core made of silicon crystals. As a result, the quantum effect of the silicon crystal forming the core is stabilized. Further, in the biological material fluorescent labeling method according to the present invention, the quantum dots are bonded to the biological material via organic molecules, so that the fluorescent labeling of the biological material is easy and the excitation light is irradiated. The fluorescence intensity increases.

  In addition, since the bioassay method and the device label the target substance using the above-described quantum dots, the fluorescence intensity is large, and further, the photodamage is small and the fading is small compared to the case where an organic dye is used. The accuracy of analysis can be improved.

  Hereinafter, specific embodiments to which the present invention is applied will be described in detail with reference to the drawings. In this embodiment, the present invention is applied to a Si (silicon) quantum dot constituting a biological material fluorescent labeling agent used for fluorescent labeling of DNA, which is a biological material, and a DNA labeling method using the Si quantum dot. It is. In this embodiment, the Si quantum dots indicate whether or not there is a hybridization reaction between a large number of probe DNAs (detection substances) immobilized on a bioassay substrate for DNA analysis and the dropped sample DNA (target substance). It is used when analyzing sample DNA by detecting by fluorescence.

  Here, the “fluorescence labeling of DNA” in the present embodiment is not limited to the case where the Si quantum dots are directly bound to the sample DNA for fluorescence labeling, but the Si quantum dots are bound to the intercalator. This includes the case where the DNA is indirectly fluorescently labeled by insertion and binding to the double-stranded DNA subjected to the hybridization reaction.

  In the following, first, the Si quantum dot and the manufacturing method thereof, and the DNA labeling method using the Si quantum dot will be described, and then the bioassay substrate for DNA analysis onto which the sample DNA solution is dropped, and the bio A bioassay apparatus for performing DNA analysis using an assay substrate will be described. In the present specification, “bioassay” means biochemical analysis based on hybridization and interaction between other substances.

(Si quantum dots)
First, Si quantum dots used for fluorescent labeling of DNA, which is a biological material, and a method for producing the same will be described.

  A quantum dot is a small lump of about ten and several nanometers in which several hundred to several thousand semiconductor atoms are gathered. A quantum dot is not a continuous band structure of a bulk size, but takes discrete and discontinuous energy levels, so that the energy of electrons is strongly confined in a three-dimensional quantum well. Therefore, the smaller the size of the quantum dot that is the barrier that confines electrons, the higher the energy of the conductor (LUMO), the lower the energy of the valence band (HOMO), and the valence band and conductor. The band gap energy between increases. That is, the energy for exciting electrons from the valence electron body to the conductor increases. The quantum dot has an effect (quantum size effect) that the band gap energy changes according to such a size.

  Furthermore, phosphors generate electron-hole pairs (excitons) in the band when they absorb light, etc., but when the phosphor size is bulk size, the band structure becomes continuous and the generated electrons The exciton generation is unstable because the and holes move separately. On the other hand, when the size of the phosphor is a quantum dot size, electrons and holes are generated in a very narrow space, so that the overlap of wave functions of electrons and holes becomes large, and the generation of excitons is stabilized. Therefore, in the case of a quantum dot, the transition probability of an electron-hole pair, that is, the oscillator strength is much larger than that of a bulk-size direct interband transition semiconductor. That is, in the case of quantum dots, the fluorescence intensity with respect to the excitation light intensity is very high. Quantum dots have such an effect (confinement effect) that the transition probability of such electron-hole pairs is increased and the fluorescence intensity is increased. It is said that such a confinement effect appears when the crystal diameter is not more than four times the exciton Bohr radius.

  Since the quantum dots have the above-described confinement effect, when the quantum dots are used as a fluorescent labeling agent, even if the excitation light intensity is increased, the fluorescence intensity is not saturated and a high fluorescence intensity can be obtained. Therefore, when quantum dots are used as the fluorescent labeling agent, the excitation light to be irradiated can be condensed and irradiated by the objective lens. For this reason, strong fluorescence can be detected using excitation light with lower intensity.

In the present embodiment, Si, which has less influence on the environment and is easy to handle, is used for the quantum dots as compared with compound semiconductors such as III-V group compound semiconductors and II-VI group compound semiconductors. This Si is an indirect interband transition type semiconductor. However, when a Si crystal having an exciton Bohr radius is manufactured, light emission in the visible region is caused by an increase in band gap energy due to the quantum size effect and a change in phonon mode. As shown. The Si quantum dots made of Si are manufactured using a known technique such as anodization of a p-type silicon wafer, decomposition of SiH ₄ in plasma, or dissolution of heat-treated SiO _x (x ^- 1.99) in hydrofluoric acid. be able to.

  Here, the quantum dot has a so-called core-shell structure consisting of an inner core (core) and an outer shell (shell), and a semiconductor that forms the core by selecting a material having a larger band gap energy than the core material as the shell layer material. It is known that the quantum effect of crystals is stabilized.

Therefore, the Si quantum dots in the present embodiment have a core-shell structure in which a shell layer made of SiO ₂ is formed around a core made of Si crystal. In particular, in the present embodiment, since Si having a property of being easily oxidized is used as a core material, it is easy to form a shell layer made of SiO ₂ .

  This Si quantum dot can be manufactured as follows, for example.

When manufacturing Si quantum dots by dissolving heat-treated SiO _x (x ^- 1.999) in hydrofluoric acid, first, SiO _x (x ^- 1.999) formed on a silicon wafer by plasma CVD is used as an inert gas atmosphere. Among them, annealing is performed at 1100 ° C. for about 1 hour. Thereby, Si crystals are deposited in the SiO ₂ film. Next, this silicon wafer is treated with a 1% hydrofluoric acid aqueous solution at room temperature to remove the SiO ₂ film, and several nanometer-sized Si crystals aggregated on the liquid surface are recovered. By this hydrofluoric acid treatment, dangling bonds (unbonded hands) of Si atoms on the crystal surface are terminated with hydrogen, and the Si crystal is stabilized. Thereafter, the surface of the recovered Si crystal is naturally oxidized in an oxygen atmosphere or is thermally oxidized by heating to form a shell layer made of SiO ₂ around the core made of Si crystal.

When Si quantum dots are manufactured by anodizing a p-type silicon wafer, first, hydrofluoric acid (46%), methanol (100%), and hydrogen peroxide solution (30%) are used at a ratio of 1: 2: 2. In the mixed solution, a p-type silicon wafer and platinum are used as a counter electrode and current is applied at 320 mA / cm ² for about 1 hour to precipitate Si crystals. A photoluminescence spectrum of the Si crystal thus obtained is shown in FIG. Thereafter, the surface of the recovered Si crystal is naturally oxidized in an oxygen atmosphere or is thermally oxidized by heating to form a shell layer made of SiO ₂ around the core made of Si crystal.

  When DNA is labeled with the Si quantum dots produced as described above, functional groups that bind to each other are introduced into both the Si quantum dots and the DNA.

For example, when bonding Si quantum dots and DNA using the bond between mercapto groups (SH groups), first, the above-mentioned Si quantum dots are dispersed in 30% H ₂ O ₂ for 10 minutes, Hydroxylate. Next, the solvent is replaced with toluene, mercaptopropyltriethoxysilane is added with 2% of toluene, and SiO ₂ on the outermost surface of the Si quantum dots is silanized and a mercapto group is introduced over about 2 hours. Subsequently, the solvent is replaced with pure water, a buffer salt is added, and DNA introduced with a mercapto group is added to one end so as to be 100 nM and left for 1 hour to bond Si quantum dots and DNA. Let FIG. 2 schematically shows a state where the Si quantum dots and single-stranded DNA (ssDNA) are bonded.

  In this example, a silane coupling agent into which a mercapto group is introduced is used, but a silane coupling agent into which an amino group or an aldehyde group is introduced can also be used. In this case, each can be bonded to DNA having an aldehyde group and an amino group introduced at one end.

  In the case where Si quantum dots and DNA are bound using the binding of biotin and avidin, first, the above-mentioned Si quantum dots are placed in a 3: 1 mixture of concentrated sulfuric acid and hydrogen peroxide for about 10 minutes. React and hydroxylate the crystal surface. After washing with water, aminopropyltriethoxysilane is reacted with the hydroxyl group on the outermost surface of the Si quantum dots in a 1: 1 mixed solution of water and ethanol to introduce amino groups into the Si quantum dots. After the reaction, washing with water, replacing the solvent with acetone, adding maleic acid to a concentration of 1 mol / l, heating and refluxing to near the boiling point for about 30 to 45 minutes, the amino group of aminopropyltriethoxysilane And a carboxyl group of maleic acid. After the reaction, it is washed with water, dispersed in a 1: 1 mixture of 869 mM N-hydroxysuccinimide aqueous solution and 522 mM N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide-hydrochloride aqueous solution, and allowed to react for about 10 minutes. . After the reaction, it is washed with water and reacted with 2.21 μM avidin for about 2 hours in an aqueous solution at room temperature, and then 1 mM ethanolamine is added and stirred at room temperature for 10 minutes. As a result, avidin is bonded to the Si quantum dots. After washing with water, the solvent is replaced with pure water, buffer salt is added to 10 mM, and DNA with biotin introduced at one end is added to 1 μM and allowed to stand. Are combined. FIG. 3 schematically shows a state in which the Si quantum dots and single-stranded DNA (ssDNA) are bound to each other.

  It should be noted that the present invention is not limited to an example in which Si quantum dots are directly bonded to DNA for labeling. For example, Si quantum dots may be bonded to an intercalator and inserted into a double-stranded DNA formed by a hybridization reaction. I do not care.

For example, when bonding Si quantum dots and intercalators using bonds between mercapto groups (SH groups), the surface of Si quantum dots is hydroxylated in the same manner as described above, and the outermost SiO ₂ is silanized. In addition, by introducing a mercapto group, for example, it can be combined with an intercalator introduced with a mercapto group as shown in FIG. Also in this case, by using a silane coupling agent into which an amino group or an aldehyde group has been introduced, it can be combined with an intercalator into which an aldehyde group or an amino group has been introduced.

(Bioassay substrate)
Next, the bioassay substrate 1 onto which a solution containing sample DNA is dropped will be described. FIG. 5 schematically shows the upper surface of the bioassay substrate 1.

  A central hole 2 is formed at the center 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 a bioassay apparatus described later.

  As shown in FIG. 6, the bioassay substrate 1 has a layer structure formed of a substrate layer 3, a transparent electrode layer 4, a solid phase layer 5, and a well formation layer 6 from the lower layer side. . The surface on the well formation 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.

  The substrate layer 3 is a material that transmits excitation light and control light, which will be described in detail later, and light having a fluorescence wavelength. For example, the substrate layer 3 is formed of quartz glass, silicon, polycarbonate, polystyrene, or other synthetic resin that can be molded into a disk shape, preferably synthetic resin that can be injection molded. By using an inexpensive synthetic resin substrate, a low running cost can be realized as compared with a conventional glass substrate.

  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 on the substrate layer 3 to a thickness of about 250 nm by, for example, sputtering or electron beam evaporation.

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 24 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.

  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 wells 7 for causing a hybridization reaction between the probe DNA and the sample DNA are formed. The well 7 has a hollow shape in which the upper surface 1a of the bioassay substrate 1 is opened, and has a depth and size that can hold the solution when the solution containing the sample DNA is dropped. It has become. For example, the well 7 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 9. For example, the well forming layer 6 is formed by applying a photosensitive polymer to the thickness of about 5 μm on the solid phase layer 5 by spin coating or the like, and using the photomask in a predetermined pattern. It is formed by exposure and development.

Furthermore, the bottom surface 9 of the well 7 is modified with the functional group so that the probe DNA whose one end is modified with the functional group binds to the bottom surface 9 (the portion where the solid-phased layer 5 is exposed). For example, as shown in FIG. 7, the well 7 has the bottom surface 9 (the solid phased layer 5 formed of SiO ₂ ) modified with a silane molecule 11 having a mercapto group (SH group) 10. For this reason, for example, probe DNA whose one end is modified with a mercapto group can be bound to the bottom surface 9 of the well 7. In this way, in the bioassay substrate 1, one end of the probe DNA can be bound to the bottom surface 9 of the well 7, so as shown in FIG. 8, the probe DNA ( P) can be combined.

  Further, in the bioassay substrate 1, as shown in FIG. 5, a plurality of wells 7 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.

  The bioassay substrate 1 is formed with address pits 8 that can be read by irradiating laser light from the lower surface 1 b side of the bioassay substrate 1. The address pit 8 is information for specifying the position of each well 7 on the plane of the bioassay substrate 1. By optically reading information from the address pits 8, it is possible to identify which one of the plurality of wells 7 is currently irradiated with laser light. By providing such address pits 8, it is possible to control the dropping position of the solution by a dropping device, which will be described later, and to specify the fluorescence detection position by the objective lens.

  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, and the radial direction. Positioning servo control for controlling the irradiation position of the laser beam and the dropping position by the dropping device, and information detection processing of the address pits 8 can be performed. That is, by associating the information content recorded in the address pits 8 with the wells 7 in the vicinity of the address pits 8, the information in the address pits 8 is read out, so that one specific well 7 can be read. The position of the well 7 that emits the laser light only to the fluorescent light is specified, or the relative position between the specific one well 7 and the dropping device is controlled to control the specific one well. The solution can be added dropwise to 7.

(Bioassay device)
Next, a bioassay device 21 that performs DNA analysis using the above-described bioassay substrate 1 will be described with reference to FIG.

  As shown in FIG. 9, the bioassay device 21 stores a disk loading unit 22 that holds and rotates the bioassay substrate 1, and stores various solutions for hybridization, and the solution in the well 7 of the bioassay substrate 1. Are provided, a dropping unit 23 for dropping the excitation light, an excitation light detecting unit 24 for detecting excitation light from the well 7 of the bioassay substrate 1, and a control / servo unit 25 for managing and controlling each of the above parts.

  The disc loading unit 22 is inserted into the center hole 2 of the bioassay substrate 1 to hold the bioassay substrate 1 and a spindle that rotates the bioassay substrate 1 by driving the chucking mechanism 31. And a motor 32. The disc loading unit 22 rotationally drives the bioassay substrate 1 in a state where the bioassay substrate 1 is held horizontally so that the upper surface 1a side is upward.

  The dropping unit 23 includes a storage unit 33 that stores the sample solution S, and a dropping head 34 that drops the sample solution S in the storage unit 33 onto the bioassay substrate 1. The drip head 34 is disposed above the upper surface 1a of the bioassay substrate 1 loaded horizontally. Further, the dropping head 34 controls the relative position with the bioassay substrate 1 in the radial direction based on the position information and rotation synchronization information read from the address pits 8 of the bioassay substrate 1 shown in FIG. The sample solution S containing the sample DNA labeled with the above-mentioned Si quantum dots, or the intercalator to which the sample DNA and the Si quantum dots are combined, is precisely dropped following the predetermined well 7. .

  The excitation light detection unit 24 has an optical head 40. The optical head 40 is disposed on the lower side of the horizontally loaded bioassay substrate 1, that is, on the lower surface 1b side. The optical head 40 is movable in the radial direction of the bioassay substrate 1 by, for example, a thread mechanism (not shown).

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

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

  The excitation light detection unit 24 includes an excitation light source 44 that emits the excitation light P, a collimator lens 45 that uses the excitation light P emitted from the excitation light source 44 as a parallel light beam, and an excitation light that is converted into a parallel light beam by the collimator lens 45. A first dichroic mirror 46 that refracts the light P on the optical path X and irradiates the light guide mirror 43.

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

  In addition, the excitation light detection unit 25 refracts the fluorescence F emitted from the optical head 40 onto the optical path X by the avalanche photodiode 47 that detects the fluorescence F, the condenser lens 48 that collects the fluorescence F, and the like. And a second dichroic mirror 49 for irradiating the avalanche photodiode 47.

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

  The avalanche photodiode 47 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 25.

  The excitation light detection unit 24 detects the control light source 50 that emits the control light V, the collimator lens 51 that uses the control light V emitted from the control light source 50 as a parallel light beam, and the reflected light R of the control light V. A photodetection circuit 52; a cylindrical lens 53 that collects reflected light R on the photodetection circuit 52 by causing astigmatism; and an optical separator 54 that separates the control light V and the reflected light R. ing.

  The control light source 50 is a light emitting unit having a laser light source that emits laser light having a wavelength of, for example, 780 nm. The wavelength of the control light V is set to a wavelength at which the address pit 8 can be detected. Further, the wavelength of the control light V is set to a wavelength different from the wavelengths of the excitation light P and the fluorescence F. As long as such a wavelength is used, the wavelength of the control light V is not limited to 780 nm and may be any wavelength. The collimator lens 51 turns the control light V emitted from the control light source 70 into a parallel light beam. The control light V made into a parallel light beam is incident on the optical separator 54.

  The photodetector circuit 52 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 a reproduction signal for the address pit 8 from the detected reflected light R. Since the reflected light R is light generated by reflecting the control light V with the bioassay substrate 1, the wavelength of the reflected light R is 780 nm, which is the same as that of the control light V.

  The focus error signal is an error signal indicating the amount of positional deviation between the focused position of the light collected by the objective lens 41 and the substrate layer 3 of the bioassay substrate 1. When the focus error signal becomes 0, the distance between the objective lens 41 and the bioassay substrate 1 is optimal. The positioning error signal is a signal indicating the amount of displacement between the predetermined well 7 position and the focal position in the disk radial direction. When the positioning error signal becomes 0, the irradiation position of the control light V in the disc radial direction coincides with the predetermined well 7. The reproduction signal of the address pit 8 is a signal indicating the information content described in the address pit 8 recorded on the bioassay substrate 1. By reading this information content, it is possible to identify the well 7 that is currently irradiating the control light V.

  The photodetect circuit 52 supplies the control / servo unit 25 with a focus error signal, a positioning error signal, and a reproduction signal for the address pit 8 generated based on the reflected light R.

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

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

  The control / servo unit 25 performs various servo controls based on the focus error signal, the positioning error signal, and the address pit 8 reproduction signal detected by the excitation light detection unit 24.

  In other words, the control / servo unit 25 controls the distance between the objective lens 41 and the bioassay substrate 1 by driving the biaxial actuator 42 in the optical head 40 based on the focus error signal, and the focus error signal is 0. Servo control is performed so that The control / servo unit 25 drives the biaxial actuator 42 in the optical head 40 based on the positioning error signal to control the movement of the objective lens 41 in the radial direction of the bioassay substrate 1, and the positioning error signal becomes zero. Servo control is performed as follows. Further, the control / servo unit 25 moves the optical head 40 to a predetermined radial position based on the reproduction signal of the address pit 8, and moves the objective lens 41 to the target well position.

  The bioassay device 21 configured as described above performs the following operation when performing a bioassay.

  When the Si quantum dots are directly bonded to the sample DNA and labeled, the bioassay device 21 is labeled with the Si quantum dots (D) on the well 7 as shown in FIG. 10 while rotating the bioassay substrate 1. A solution containing the sample DNA (S) is dropped, and the probe DNA (P) and the labeled sample DNA (S) are allowed to react (hybridize) as shown in FIG.

  When the Si quantum dots are bonded to the intercalator, the bioassay device 21 drops the solution containing the sample DNA (S) on the well 7 as shown in FIG. 12 while rotating the bioassay substrate 1. The probe DNA (P) and the sample DNA (S) are allowed to interact (hybridize). Further, a buffer solution containing an intercalator M having Si quantum dots bonded thereto is dropped on the bioassay substrate 1 after the hybridization reaction, and probe DNA (P) and sample DNA (S ) And an intercalator M in which Si quantum dots are bonded to each other in the double helix.

  Thereafter, the bioassay device 21 rotates the bioassay substrate 1 after the sample DNA labeled with the Si quantum dots or the intercalator in which the sample DNA and the Si quantum dots are combined is dropped, and the excitation light P The fluorescent labeling agent in the well 7 is irradiated from the lower surface 1 b side of the bioassay substrate 1, and the fluorescence F generated from the fluorescent labeling agent is detected from below the bioassay substrate 1 according to the excitation light P.

  Here, in the bioassay device 21, the excitation light P and the control light V are irradiated to the bioassay substrate 1 through the same objective lens 41. Therefore, the bioassay device 21 can specify the irradiation position of the excitation light P, that is, the emission position of the fluorescence F by performing focus control, positioning control, and address control using the control light V. The probe DNA bound to the sample DNA can be identified from the fluorescence emission position.

(DNA analysis method)
Next, a DNA analysis method will be described.

  First, the bioassay substrate 1 is horizontally loaded on the disk loading unit 22 of the bioassay device 21.

  Subsequently, the bioassay substrate 21 is rotated by the bioassay device 21 while performing position control based on the address pits 8, and a solution containing a probe DNA whose one end is modified with a mercapto group or the like is contained from the dropping head 34 in a predetermined manner. Drip into well 7. At this time, a plurality of types of probe DNAs are dropped on one bioassay substrate 1. However, one type of probe DNA is allowed to enter one well 7. It should be noted that which type of probe DNA is dropped into each well 7 is prepared by previously creating an arrangement map or the like indicating the correspondence between the well and the probe DNA, and dropping is controlled based on the arrangement map.

  Subsequently, an electrode is inserted into the solution in the well 7 from the upper surface 1 a side of the bioassay substrate 1, and an alternating electric field of about 1 MV / m and 1 MHz is applied to each well 7. When the alternating electric field is applied in this manner, the probe DNA extends in the vertical direction with respect to the bioassay substrate 1 and the probe DNA is moved in the vertical direction to the bioassay substrate 1 to be subjected to surface modification treatment in advance. On the other hand, the modified end of the probe DNA can be bound to immobilize (immobilize) the probe DNA in the well 7 (Masao Washizu and Osamu Kurosawa: “Electrostatic Manipulation of DNA in Microfabricated Structures”, IEEE Transaction on Industrial Application Vol.26, No.26, P.1165-1172 (1900)).

  Subsequently, a solution containing sample DNA labeled with Si quantum dots is dropped from the dropping head 34 to the wells 7 on the bioassay substrate 1 together with a solution containing a buffer salt by the bioassay device 21. When Si quantum dots are bonded to an intercalator, a solution containing only sample DNA can be dropped into each well 7 on the bioassay substrate 1 together with a solution containing a buffer salt at this time.

  Subsequently, after the sample DNA is dropped, the bioassay substrate 1 is transferred to a constant temperature layer or the like, the inside of the well 7 is heated to several tens of degrees, and an AC electric field of about 1 MV / m and 1 MHz is applied in the heated state. 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 corresponding to each other in the base sequence are in the same well 7, they cause a hybridization reaction.

  Here, when the Si quantum dots are coupled to the intercalator, the bioassay device 21 subsequently drops the intercalator coupled with the Si quantum dots into the well 7 of the bioassay substrate 1. Such an intercalator is inserted and bonded between the double helix of the probe DNA that has undergone the hybridization reaction and the sample DNA.

  Subsequently, the surface 1a of the bioassay substrate 1 is washed with pure water or the like, and the sample DNA labeled with the Si quantum dots in the well 7 where the hybridization reaction has not occurred (or the sample DNA and the Si quantum dots are combined). Remove the intercalator). As a result, Si quantum dots remain only in the well 7 in which the hybridization reaction has occurred.

  Subsequently, the bioassay apparatus 21 rotates the bioassay substrate 1 while performing focus servo control, positioning servo control, and address control using the control light F, and irradiates the predetermined well 7 with the excitation light P. Along with the irradiation of the excitation light P, whether or not the fluorescence F is generated is detected while detecting the address information.

  Subsequently, the bioassay device 21 creates a map indicating the position of each well 7 on the bioassay substrate 1 and the intensity of fluorescence F emission. 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 7.

  Although the best mode for carrying out the present invention has been described above, the present invention is not limited to the above-described embodiment, and various modifications, substitutions, or replacements can be made without departing from the gist of the present invention. It will be apparent to those skilled in the art that the equivalent can be done.

  For example, in the above-described embodiment, the bioassay substrate is formed in a disk shape. However, the present invention is not limited to this shape, and an elliptical shape, a rectangular shape, or other flat plate shape can be appropriately selected and determined.

  Since the quantum dots constituting the biological material fluorescent labeling agent according to the present invention described above are composed of silicon and silicon oxide, they are easy to handle and have little influence on the environment and are easy to manufacture. This quantum dot has the characteristics that the fluorescence intensity is high and the photodamage is small and the fading is small compared to the case where an organic dye is used, so that it can be used for various applications. For example, biochemical analysis is performed. By using it as a fluorescent labeling agent for labeling the target substance, the accuracy of the analysis can be improved.

It is a figure which shows the photoluminescence spectrum of Si crystal | crystallization produced | generated by anodizing of the p-type silicon wafer. It is a figure which shows an example of the coupling | bonding state in the case of coupling | bonding Si quantum dot and DNA using the coupling | bonding of mercapto groups. It is a figure which shows an example of the binding state in the case of making Si quantum dot and DNA couple | bond using the coupling | bonding of biotin and avidin. It is a figure which shows an example of the intercalator into which the mercapto group was introduce | transduced. It is a top view of the bioassay board | substrate used for description of this Embodiment. It is sectional drawing of the bioassay board | substrate. It is a figure which shows the silane molecule | numerator which has SH group modified on the bottom face of a well. It is a figure which shows the probe DNA couple | bonded with the bottom face of the well. It is a block block diagram of the bioassay apparatus provided for description of this Embodiment. It is a figure which shows the operation | movement which is dripping the solution which the sample DNA labeled with Si quantum dot contained with respect to the well. It is a figure which shows the state which the probe DNA and the sample DNA labeled with Si quantum dot have couple | bonded. It is a figure which shows the operation | movement which is dripping the solution containing the intercalator with which sample DNA and Si quantum dot were combined with respect to the well. It is a figure which shows the state in which the intercalator is inserted and bonded in the double helix of probe DNA and sample DNA.

Explanation of symbols

  1 Bioassay Substrate, 3 Substrate Layer, 7 Well, 21 Bioassay Device, 22 Disc Loading Unit, 23 Dropping Unit, 24 Excitation Light Detection Unit, 25 Control / Servo Unit

Claims (8)

Silicon having a crystal diameter equal to or less than four times the exciton Bohr radius generated by annealing SiO _{x formed} on a silicon wafer in an inert gas atmosphere and then treating the silicon wafer with an aqueous hydrofluoric acid solution It is composed of a core made of crystal and a shell layer made of silicon oxide covering the core, and the silicon oxide constituting the shell layer is silanized to introduce a living body on the surface of the quantum dot particles to which mercapto groups are introduced. A biological substance fluorescent labeling agent to which organic molecules that bind to a substance are bound.   The biological material fluorescent labeling agent according to claim 1, wherein the biological material is a nucleotide chain or a protein collected from a living body. Silicon having a crystal diameter equal to or less than four times the exciton Bohr radius generated by annealing SiO _{x formed} on a silicon wafer in an inert gas atmosphere and then treating the silicon wafer with an aqueous hydrofluoric acid solution It is composed of a core made of crystal and a shell layer made of silicon oxide covering the core, and the silicon oxide constituting the shell layer is silanized to introduce a quantum dot into which a mercapto group is introduced via an organic molecule. A biological material fluorescent labeling method for binding to biological material.   4. The biological material fluorescent labeling method according to claim 3, wherein the biological material is a nucleotide chain or protein collected from a living body. An inert gas atmosphere of SiO _{x formed} on a silicon wafer is applied to a bioassay substrate in which a plurality of reaction regions are formed on the surface and a plurality of known detection substances are immobilized in each predetermined reaction region. After being annealed in the core, the silicon wafer is formed by treating the silicon wafer with an aqueous hydrofluoric acid solution, and includes a core made of silicon crystal having a crystal diameter of four times or less of an exciton Bohr radius and silicon oxide covering the core. A reaction step in which a silicon oxide constituting the shell layer is silanized and a target substance labeled with a quantum dot having a mercapto group introduced therein is dropped to interact with each other;
After the mutual reaction in the reaction step, a removal step of removing the unreacted target substance from each reaction region of the bioassay substrate;
Biochemical analysis of the target substance by irradiating the bioassay substrate with excitation light of a predetermined wavelength and detecting the presence or absence of fluorescence of a predetermined wavelength generated from the quantum dots in response to the excitation light A bioassay method comprising the steps of:   The bioassay method according to claim 5, wherein the detection substance and the target substance are nucleotide chains, and the interaction is a hybridization reaction. A holding means for holding a bioassay substrate in which a plurality of reaction regions are formed on the surface and a plurality of known detection substances are immobilized in each predetermined reaction region;
An exciton Bohr radius of the bioassay substrate generated by annealing SiO _{x formed} on a silicon wafer in an inert gas atmosphere and then treating the silicon wafer with an aqueous hydrofluoric acid solution . It is composed of a core made of silicon crystal having a crystal diameter of 4 times or less and a shell layer made of silicon oxide covering the core, and the silicon oxide constituting the shell layer is silanized to introduce a mercapto group. Dropping means for dropping a target substance labeled with quantum dots;
A removing means for removing the unreacted target substance from each reaction region of the bioassay substrate after the detection substance and the target substance dropped by the dropping means are reacted with each other;
Biochemical analysis of the target substance by irradiating the bioassay substrate with excitation light of a predetermined wavelength and detecting the presence or absence of fluorescence of a predetermined wavelength generated from the quantum dots in response to the excitation light And a bioassay device comprising:   The bioassay device according to claim 7, wherein the detection substance and the target substance are nucleotide chains, and the interaction is a hybridization reaction.

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