WO2009113374A1 - Marking method - Google Patents
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- WO2009113374A1 WO2009113374A1 PCT/JP2009/053000 JP2009053000W WO2009113374A1 WO 2009113374 A1 WO2009113374 A1 WO 2009113374A1 JP 2009053000 W JP2009053000 W JP 2009053000W WO 2009113374 A1 WO2009113374 A1 WO 2009113374A1
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- semiconductor nanoparticles
- spectrum
- labeling method
- emission
- labeling
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
Definitions
- the present invention relates to a labeling method using semiconductor nanoparticles.
- nanosilicon having a particle size of 3.5 nm or less is a solution in which single particles are dispersed, and the nanosilicon is formed by heat-treating an amorphous silicon oxide film produced by a high-frequency sputtering method (for example, see Patent Document 1).
- Patent Document 2 obtains light emission of three colors of red, green, and blue, light emission of three colors or more cannot be classified with a wide spectrum.
- An object of the present invention is to perform identification by irradiating a plurality of molecules labeled with semiconductor nanoparticles with excitation light and detecting luminescence.
- the emission spectrum of the semiconductor nanoparticles has at least three types of emission peaks within a wavelength range of 300 nm.
- the present invention is characterized in that a plurality of molecules are labeled and identified by using an emission spectrum that does not overlap within a wavelength range of 300 nm.
- the wavelength range of 300 nm makes it easy to distinguish between 400 to 700 nm of visible light and 800 to 1100 nm of infrared light, and facilitates spectroscopic measurement.
- a method in which a biological sample such as DNA or protein labeled with a fluorescent substance serving as a marker is separated by electrophoresis and detected using fluorescence, or a plurality of types of probe DNAs are arranged as a minute spot on a substrate at high density We know how to use fluorescence for sample analysis and testing, such as pouring target DNA labeled with a fluorescent substance onto a biochip and detecting the presence or absence of hybridization between the target DNA and probe DNA by reading the fluorescence from the fluorescent substance. It has been. In these methods, a labeled fluorescent substance is excited and fluorescence generated from the fluorescent substance is read.
- Fluorescent substances emit fluorescence on the longer wavelength side of the excitation wavelength when irradiated with excitation light, but in most cases, conventional fluorescent substances have different excitation wavelengths when the fluorescence wavelengths are different.
- fluorescent materials in order to increase the fluorescence excitation efficiency of all the fluorescent materials and increase the detection sensitivity, it is necessary to excite each fluorescent material with an excitation wavelength suitable for it.
- Semiconductor nanoparticles have the characteristic that the fluorescence wavelength varies depending on the particle size. Therefore, by producing a plurality of types of semiconductor nanoparticles having different particle sizes, a plurality of types of fluorescent labeling substances having different fluorescence wavelengths can be procured.
- the excitation spectrum of semiconductor nanoparticles is broad on the ultraviolet side. Therefore, when semiconductor nanoparticles are used as the fluorescent labeling substance, only one excitation light source that generates excitation light in the ultraviolet region may be prepared for a plurality of types of fluorescent labeling substances. Further, unlike organic dye-based fluorescent materials, semiconductor nanoparticles can be excited by applying a voltage to generate fluorescence.
- a group of laterally resolved detectors including CCD camera, CCD chip, photodiode array, avalanche diode array, multichannel plate, and multichannel photomultiplier tube are used for signal detection.
- the spectral width is 50 nm or less in order to obtain three or more types of light emission within a range of 300 nm. If it exceeds this, it overlaps with the adjacent spectrum and the emission color becomes blurred.
- no spectrum overlap means that spectra having an intensity of 10% or more do not overlap with the emission peak intensity. Within this range, it is possible to visually recognize other spectra. A case where the portions where the spectrum intensities are 10% or more overlap is regarded as “spectrum overlap”, and it becomes difficult to distinguish other spectra.
- the reason why the emission color is set to three or more colors is that in addition to the conventional three colors of red, green, and blue, intermediate colors thereof can be distinguished.
- the light source of the excitation light is not limited as long as it satisfies the desired wavelength and intensity conditions.
- various lamps such as a high pressure mercury lamp, a low pressure mercury lamp, an ultrahigh pressure mercury lamp, a metal halide lamp, an Ar laser, a Kr laser, and a He—
- Various lasers such as a Ne laser and various LEDs can be used.
- the wavelength of the excitation light depends on the kind of nanoparticles and the particle size, but usually 200 to 1000 nm is used.
- the dispersion medium is not limited as long as the semiconductor nanoparticles are stably dispersed, water, aliphatic alcohols such as ethanol and methanol, aromatic hydrocarbons such as toluene, aliphatic hydrocarbons such as hexane, and the like. A mixture or the like is used. If necessary, a composition comprising an additive such as a surfactant, a dispersion stabilizer, and an antioxidant, and a polymer, a monomer having a polymerizable functional group, and an oligomer can be added.
- an additive such as a surfactant, a dispersion stabilizer, and an antioxidant
- the surface of the semiconductor nanoparticles can be chemically and physically modified as necessary.
- These semiconductor nanoparticles can be synthesized by a known technique such as a reverse micelle method, a hot soap method, or natural oxidation.
- Si semiconductor nanoparticles For production of Si semiconductor nanoparticles according to the present invention, various conventionally known methods can be used. Broadly classified, there are a liquid phase method and a gas phase method. In the present invention, it is preferable to use a gas phase method.
- the opposing raw material semiconductor is evaporated by the first high-temperature plasma generated between the electrodes, and the second high-temperature plasma generated by the non-electric discharge in the reduced pressure atmosphere
- a method for separating and removing nanoparticles made of an anode made of a raw material semiconductor by electrochemical etching (3) A laser ablation method, (4) High-frequency sputtering The law is used.
- Si semiconductor nanoparticles of a silicon oxide film can control the particle size of the Si semiconductor nanoparticles by repeating oxidation of the particle surface and hydrofluoric acid treatment. At that time, more precise control is possible by changing the heating temperature and the heating time. Thereby, the emission spectrum in the present invention can be realized.
- the Si semiconductor nanoparticles according to the present invention can be applied to single molecule analysis in various technical fields by taking out from the film.
- multiple types of molecules may be identified simultaneously by labeling multiple types of molecules with semiconductor nanoparticles having different emission spectra and irradiating the molecules with excitation light. it can.
- the chemical composition is the same as the applicable types of molecules, it includes structural isomers having different chemical structures.
- the semiconductor nanoparticles according to the present invention can be applied to a biological material labeling agent. Further, by adding the biological substance labeling agent according to the present invention to a living cell or living body having a target (tracking) substance, it binds or adsorbs to the target substance, and excitation light having a predetermined wavelength is applied to the conjugate or adsorbent. By irradiating and detecting fluorescence of a predetermined wavelength generated from the fluorescent semiconductor fine particles according to the excitation light, fluorescence dynamic imaging of the target (tracking) substance can be performed. That is, the biomaterial labeling agent according to the present invention can be used for bioimaging methods (technical means for visualizing biomolecules constituting the biomaterial and dynamic phenomena thereof).
- the semiconductor nanoparticles described above are generally hydrophobic, for example, when used as a biological material labeling agent, there are problems such as poor water dispersibility and aggregation of the particles. It is preferable to hydrophilize the surface of the nanoparticle shell.
- hydrophilic treatment method for example, there is a method of chemically and / or physically binding a surface modifier to the particle surface after removing the lipophilic group on the surface with pyridine or the like.
- a surface modifier those having a carboxyl group or an amino group as hydrophilic groups are preferably used, and specific examples include mercaptopropionic acid, mercaptoundecanoic acid, aminopropanethiol and the like.
- 10 ⁇ 5 g of Ge / GeO 2 type semiconductor nanoparticles are dispersed in 10 ml of pure water in which 0.2 g of mercaptoundecanoic acid is dissolved, and stirred for 10 minutes at 40 ° C.
- the surface of the shell of inorganic nanoparticles can be modified with a carboxyl group.
- the biological material labeling agent according to the present invention is obtained by bonding the above-described hydrophilic treatment semiconductor nanoparticles, the molecular labeling substance, and organic molecules.
- the biological material labeling agent according to the present invention enables the biological material to be labeled by specifically binding and / or reacting with the target biological material.
- Examples of the molecular labeling substance include nucleotide chains, antibodies, antigens and cyclodextrins.
- the hydrophilic semiconductor nanoparticles and the molecular labeling substance are bound by organic molecules.
- the organic molecule is not particularly limited as long as it is an organic molecule capable of binding a semiconductor nanoparticle and a molecular labeling substance.
- albumin, myoglobin, casein, etc. among proteins, and avidin, which is a kind of protein, are used together with biotin. It is also preferably used.
- the form of the bond is not particularly limited, and examples thereof include covalent bond, ionic bond, hydrogen bond, coordinate bond, physical adsorption, and chemical adsorption. A bond having a strong bonding force such as a covalent bond is preferable from the viewpoint of bond stability.
- the semiconductor nanoparticles are hydrophilized with mercaptoundecanoic acid
- avidin and biotin can be used as organic molecules.
- the carboxyl group of the semiconductor nanoparticles subjected to the hydrophilic treatment is preferably covalently bonded to avidin
- the avidin is further selectively bonded to biotin
- biotin is further bonded to the biological material labeling agent to thereby bind the biological material labeling agent. It becomes.
- the obtained silicon oxide film containing Si atoms is rapidly heated to a temperature shown in Table 1 in an Ar atmosphere and subjected to heat treatment to aggregate the Si atoms in the film to a nano size.
- the heat treatment time is 50 minutes.
- Hydrofluoric acid treatment Surface treatment is performed by exposing the obtained silicon oxide film containing Si nanoparticles to 40 ° C. hydrofluoric acid vapor.
- Heat oxidation treatment The silicon oxide film containing Si nanoparticles after hydrofluoric acid treatment is subjected to natural oxidation or overheating oxidation treatment.
- the temperature and time of the heat oxidation treatment are shown in Table 1.
- the Si semiconductor nanoparticles dispersed in ethanol obtained were irradiated with excitation light having a wavelength of 280 nm, and the generated fluorescence spectrum was measured.
- the half-value width and peak wavelength of the emission spectrum are shown in Table 1.
- the emission spectrum was obtained using a Hitachi Fluorometer F-7000.
- SWNIR spectrometer
- the obtained avidin-conjugated nanoparticle solution was mixed and stirred with a biotinylated oligonucleotide having a known base sequence to prepare an oligonucleotide labeled (labeled) with semiconductor nanoparticles.
- the oligonucleotide When the above labeled (labeled) oligonucleotide is dropped onto a DNA chip on which oligonucleotides having various base sequences are immobilized and washed, the oligonucleotide has a complementary base sequence to the labeled (labeled) oligonucleotide. It was confirmed that only these spots emitted light of different colors depending on the particle size of the semiconductor nanoparticles by the excitation light of 810 nm.
- the labeling (labeling) of the oligonucleotide with the semiconductor nanoparticle phosphor according to the present invention was possible. That is, it can be seen from this example that the semiconductor nanoparticle phosphor according to the present invention can be applied to a semiconductor nanoparticle label. In addition, high luminescence intensity is obtained in the near-infrared emission region which is excellent in terms of biopermeability, and it can be applied to a semiconductor nanoparticle label for detecting biomolecules.
- Comparative Example 1 With respect to Si semiconductor nanoparticles 1, 4, and 10 subjected to hydrophilic treatment in which the three colors in the visible region overlap each other, a labeled body was prepared as described above, and light was emitted.
- Comparative Example 2 With respect to Si semiconductor nanoparticles 1, 2, 4, 5, 6, 9, and 10 subjected to hydrophilic treatment, in which the seven colors in the visible region overlap, labeled bodies were produced as described above, and light was emitted.
- Comparative Example 3 For Si semiconductor nanoparticles 16, 19, and 25 subjected to hydrophilic treatment, in which the three colors in the near-infrared region do not overlap, a labeled body was prepared as described above, and light was emitted.
- Example 1 For Si semiconductor nanoparticles 17, 18, 20, 23, 24, 26, and 27 that have been subjected to hydrophilic treatment that do not overlap the seven colors in the near-infrared region, labeled bodies were prepared as described above and emitted light.
- Example 2 With respect to Si semiconductor nanoparticles 2, 3, 5, 6, 9, 11, and 12 that were subjected to hydrophilic treatment and in which the seven colors in the visible region did not overlap, a labeled body was prepared as described above, and light was emitted.
Abstract
Provided is a marking method which applies an excitation light to a plurality of molecules marked by semiconductor nano-particles and identifies the molecules by detecting light emission. The semiconductor nano-particles have at least three types of light emission peak in light emission spectra within a wavelength range of 300 nm and the spectra are not overlapped by one another.
Description
本発明は、半導体ナノ粒子による標識方法に関する。
The present invention relates to a labeling method using semiconductor nanoparticles.
粒子サイズ３．５ｎｍ以下のナノシリコンが粒子単体で分散した溶液であり、該ナノシリコンが高周波スパッタリング法で作製したアモルファス酸化ケイ素膜に熱処理を施して形成したものであることが知られている（例えば、特許文献１参照）。
It is known that nanosilicon having a particle size of 3.5 nm or less is a solution in which single particles are dispersed, and the nanosilicon is formed by heat-treating an amorphous silicon oxide film produced by a high-frequency sputtering method ( For example, see Patent Document 1).
また、酸化ケイ素膜内に高周波スパッタリングと熱処理で形成された粒子サイズ１．５～３．５ｎｍの発光ナノシリコンを含有し、紫外線照射により、青、緑、赤色を合わせた白色を鮮明、且つ安定して発光する技術が知られている（例えば、特許文献２参照）。
In addition, it contains light-emitting nanosilicon with a particle size of 1.5 to 3.5 nm formed by high-frequency sputtering and heat treatment in a silicon oxide film, and the combination of blue, green, and red is clear and stable by ultraviolet irradiation. Thus, a technique for emitting light is known (for example, see Patent Document 2).
しかしながら、特許文献２は赤、緑、青の３色の発光を得ているが、スペクトルが広く３色以上の発光を分類することはできない。
However, although Patent Document 2 obtains light emission of three colors of red, green, and blue, light emission of three colors or more cannot be classified with a wide spectrum.
量子ドットを使用することで単一励起波長で多色発光が可能となるが、スペクトル同士の重なりがあるために発光色がぼけてしまい、発光色の選択範囲が狭まってしまうという問題がある。
特開２００６－７００８９号公報
特開２００７－６３３７８号公報
By using quantum dots, multicolor light emission is possible at a single excitation wavelength. However, there is a problem that the emission color is blurred due to the overlap of spectra, and the selection range of the emission color is narrowed.
JP 2006-70089 A JP 2007-63378 A
本発明の目的は、半導体ナノ粒子で標識された複数の分子に励起光を照射し、発光を検出することによりその同定を行うことである。
An object of the present invention is to perform identification by irradiating a plurality of molecules labeled with semiconductor nanoparticles with excitation light and detecting luminescence.
本発明の上記目的は、下記構成により達成される。
The above object of the present invention is achieved by the following configuration.
１．半導体ナノ粒子で標識された分子に励起光を照射し、発光を検出することにより該分子の同定を行う標識方法において、該半導体ナノ粒子の発光スペクトルが波長範囲３００ｎｍ以内に少なくとも３種類の発光ピークを持ち、且つ各スペクトルは重ならないことを特徴とする標識方法。
1. In a labeling method for identifying molecules by irradiating molecules labeled with semiconductor nanoparticles with excitation light and detecting luminescence, the emission spectrum of the semiconductor nanoparticles has at least three types of emission peaks within a wavelength range of 300 nm. A labeling method characterized in that each spectrum does not overlap.
２．前記発光スペクトルの波長範囲が可視領域であることを特徴とする前記１に記載の標識方法。
2. 2. The labeling method according to 1 above, wherein a wavelength range of the emission spectrum is a visible region.
３．前記発光スペクトルの波長範囲が近赤外領域８００～１１００ｎｍであることを特徴とする前記１に記載の標識方法。
3. 2. The labeling method according to 1 above, wherein the wavelength range of the emission spectrum is a near infrared region of 800 to 1100 nm.
４．前記各スペクトルの幅が５０ｎｍ以下であり、隣り合うピーク波長間距離はスペクトル幅よりも大きいことを特徴とする前記１～３のいずれか１項に記載の標識方法。
4. 4. The labeling method according to any one of 1 to 3, wherein a width of each spectrum is 50 nm or less, and a distance between adjacent peak wavelengths is larger than a spectrum width.
５．前記半導体ナノ粒子の平均粒径が１ｎｍ以上１０ｎｍ以下であることを特徴とする前記１～４のいずれか１項に記載の標識方法。
5. 5. The labeling method according to any one of 1 to 4, wherein the semiconductor nanoparticles have an average particle size of 1 nm to 10 nm.
６．前記半導体ナノ粒子がＳｉまたはＧｅであることを特徴とする前記１～５のいずれか１項に記載の標識方法。
6. 6. The labeling method according to any one of 1 to 5, wherein the semiconductor nanoparticles are Si or Ge.
本発明により、半導体ナノ粒子で標識された複数の分子に励起光を照射し、その同定を行うことができた。
According to the present invention, it was possible to identify a plurality of molecules labeled with semiconductor nanoparticles by irradiating them with excitation light.
以下、本発明について詳述する。
Hereinafter, the present invention will be described in detail.
本発明は、３００ｎｍの波長範囲内に重なり合わない発光スペクトルを使用することで、複数の分子を標識し、その同定を行うことを特徴とする。３００ｎｍの波長範囲によって、可視光の４００～７００ｎｍ、赤外光の８００～１１００ｎｍにおいて識別がしやすく、また分光器測定が簡便となる。
The present invention is characterized in that a plurality of molecules are labeled and identified by using an emission spectrum that does not overlap within a wavelength range of 300 nm. The wavelength range of 300 nm makes it easy to distinguish between 400 to 700 nm of visible light and 800 to 1100 nm of infrared light, and facilitates spectroscopic measurement.
マーカーとなる蛍光物質で標識したＤＮＡや蛋白質などの生体試料を電気泳動によって分離し、蛍光を利用して検出する方法、あるいは基板上に複数種類のプローブＤＮＡを微小なスポットとして高密度に配列したバイオチップに蛍光物質で標識したターゲットＤＮＡを注ぎ、蛍光物質からの蛍光を読み取ってターゲットＤＮＡとプローブＤＮＡのハイブリダイゼーションの有無を検出する方法など、試料の分析や試験に蛍光を利用する方法が知られている。これらの方法では標識蛍光物質を励起し、蛍光物質から発生される蛍光を読み取る。
A method in which a biological sample such as DNA or protein labeled with a fluorescent substance serving as a marker is separated by electrophoresis and detected using fluorescence, or a plurality of types of probe DNAs are arranged as a minute spot on a substrate at high density We know how to use fluorescence for sample analysis and testing, such as pouring target DNA labeled with a fluorescent substance onto a biochip and detecting the presence or absence of hybridization between the target DNA and probe DNA by reading the fluorescence from the fluorescent substance. It has been. In these methods, a labeled fluorescent substance is excited and fluorescence generated from the fluorescent substance is read.
蛍光物質は励起光を照射すると励起波長の長波長側に蛍光を発するが、従来の蛍光物質はほとんどの場合、蛍光波長が異なれば励起波長も異なる。複数の蛍光物質を使用した場合に、全ての蛍光物質の蛍光励起効率を上げて検出感度を高めるためには、各蛍光物質をそれに適した励起波長で励起する必要がある。
Fluorescent substances emit fluorescence on the longer wavelength side of the excitation wavelength when irradiated with excitation light, but in most cases, conventional fluorescent substances have different excitation wavelengths when the fluorescence wavelengths are different. When a plurality of fluorescent materials are used, in order to increase the fluorescence excitation efficiency of all the fluorescent materials and increase the detection sensitivity, it is necessary to excite each fluorescent material with an excitation wavelength suitable for it.
半導体ナノ粒子は、粒径により蛍光波長が変わるという特徴を有する。従って、粒径の異なる半導体ナノ粒子を複数種類製造することで、蛍光波長の異なる複数種類の蛍光標識物質を調達することができる。一方、半導体ナノ粒子の励起スペクトルは紫外側にブロードに存在する。そのため、蛍光標識物質として半導体ナノ粒子を用いる場合には、複数種類の蛍光標識物質に対して、紫外領域の励起光を発生するただ一つの励起光源を用意すればよい。また、半導体ナノ粒子は有機色素系の蛍光物質と違い、電圧をかけることによって励起し、蛍光を発生させることも可能である。
Semiconductor nanoparticles have the characteristic that the fluorescence wavelength varies depending on the particle size. Therefore, by producing a plurality of types of semiconductor nanoparticles having different particle sizes, a plurality of types of fluorescent labeling substances having different fluorescence wavelengths can be procured. On the other hand, the excitation spectrum of semiconductor nanoparticles is broad on the ultraviolet side. Therefore, when semiconductor nanoparticles are used as the fluorescent labeling substance, only one excitation light source that generates excitation light in the ultraviolet region may be prepared for a plurality of types of fluorescent labeling substances. Further, unlike organic dye-based fluorescent materials, semiconductor nanoparticles can be excited by applying a voltage to generate fluorescence.
ＣＣＤカメラ、ＣＣＤチップ、フォトダイオードアレイ、アバランシェダイオードアレイ、マルチチャネルプレート、及びマルチチャネル光電子増倍管を含む群の横方向分解検出器が信号検出に使用される。
A group of laterally resolved detectors including CCD camera, CCD chip, photodiode array, avalanche diode array, multichannel plate, and multichannel photomultiplier tube are used for signal detection.
これは、３００ｎｍの範囲内で３種類以上の発光を得るためには、スペクトル幅は５０ｎｍ以下であることが好ましい。これ以上になると隣のスペクトルと重なり、発光色がぼやけてしまう。
It is preferable that the spectral width is 50 nm or less in order to obtain three or more types of light emission within a range of 300 nm. If it exceeds this, it overlaps with the adjacent spectrum and the emission color becomes blurred.
本発明において、「スペクトルの重なりのない」とは、発光ピーク強度に対して強度１０％以上のスペクトルが重なっていないことを言う。この範囲内であると他のスペクトルとの視認ができる。スペクトルの強度が１０％以上の部分が重なっている場合を「スペクトルが重なる」とし、他のスペクトルの区別が難しくなる。
In the present invention, “no spectrum overlap” means that spectra having an intensity of 10% or more do not overlap with the emission peak intensity. Within this range, it is possible to visually recognize other spectra. A case where the portions where the spectrum intensities are 10% or more overlap is regarded as “spectrum overlap”, and it becomes difficult to distinguish other spectra.
発光色を３色以上とするのは従来の赤、緑、青の３色に加えて、その中間色を見分けることができるためである。
The reason why the emission color is set to three or more colors is that in addition to the conventional three colors of red, green, and blue, intermediate colors thereof can be distinguished.
励起光の光源は所望の波長と強度の条件を満足するものであれば限定されず、例えば、高圧水銀灯、低圧水銀灯、超高圧水銀灯、メタルハライドランプ等の各種ランプ、Ａｒレーザー、Ｋｒレーザー、Ｈｅ－Ｎｅレーザー等の各種レーザー及び各種ＬＥＤを用いることができる。励起光の波長はナノ粒子の種類及び粒子サイズに依存するが、通常は２００～１０００ｎｍが用いられる。
The light source of the excitation light is not limited as long as it satisfies the desired wavelength and intensity conditions. For example, various lamps such as a high pressure mercury lamp, a low pressure mercury lamp, an ultrahigh pressure mercury lamp, a metal halide lamp, an Ar laser, a Kr laser, and a He— Various lasers such as a Ne laser and various LEDs can be used. The wavelength of the excitation light depends on the kind of nanoparticles and the particle size, but usually 200 to 1000 nm is used.
細胞、生体に適用する場合、それ自身または周囲の細胞に影響を与えないように赤外領域の発光、検出であることが望ましい。
When applying to cells and living organisms, it is desirable to emit and detect in the infrared region so as not to affect itself or surrounding cells.
分散媒は半導体ナノ粒子が安定に分散されるものであれば限定されず、水、エタノール、メタノールなどの脂肪族アルコール、トルエン等の芳香族炭化水素、ヘキサンなどの脂肪族炭化水素など及びこれらの混合物などが用いられる。必要に応じて、界面活性剤、分散安定剤、酸化防止剤などの添加物、並びにポリマーや重合性官能基を有するモノマー、オリゴマーからなる組成物を加えることができる。
The dispersion medium is not limited as long as the semiconductor nanoparticles are stably dispersed, water, aliphatic alcohols such as ethanol and methanol, aromatic hydrocarbons such as toluene, aliphatic hydrocarbons such as hexane, and the like. A mixture or the like is used. If necessary, a composition comprising an additive such as a surfactant, a dispersion stabilizer, and an antioxidant, and a polymer, a monomer having a polymerizable functional group, and an oligomer can be added.
半導体ナノ粒子の表面は必要に応じて化学的、物理的に修飾することができる。これらの半導体ナノ粒子は、逆ミセル法、ホットソープ法、自然酸化などの公知の手法により合成することが可能である。
The surface of the semiconductor nanoparticles can be chemically and physically modified as necessary. These semiconductor nanoparticles can be synthesized by a known technique such as a reverse micelle method, a hot soap method, or natural oxidation.
本発明に係るＳｉ半導体ナノ粒子の製造については、従来公知の種々の方法を用いることができる。大きく分類すると液相法と気相法があるが、本発明においては気相法を用いることが好ましい。
For production of Si semiconductor nanoparticles according to the present invention, various conventionally known methods can be used. Broadly classified, there are a liquid phase method and a gas phase method. In the present invention, it is preferable to use a gas phase method.
気相法の製造方法としては、（１）対向する原料半導体を電極間で発生させた第一の高温プラズマによって蒸発させ、減圧雰囲気中において無電局放電で発生させた第二の高温プラズマ中に通過させる方法（例えば、特開平６－２７９０１５公報）、（２）電気化学的エッチングによって原料半導体からなる陽極からなるナノ粒子を分離・除去する方法、（３）レーザーアブレーション法、（４）高周波スパッタリング法などが用いられる。
As a manufacturing method of the vapor phase method, (1) the opposing raw material semiconductor is evaporated by the first high-temperature plasma generated between the electrodes, and the second high-temperature plasma generated by the non-electric discharge in the reduced pressure atmosphere (2) A method for separating and removing nanoparticles made of an anode made of a raw material semiconductor by electrochemical etching, (3) A laser ablation method, (4) High-frequency sputtering The law is used.
例えば、酸化ケイ素膜のＳｉ半導体ナノ粒子は粒子表面の酸化とフッ酸処理を繰り返すことによりＳｉ半導体ナノ粒子の粒径を制御できる。その際に、加熱温度や加熱時間を変えることによって、更に精密な制御が可能である。これにより本発明における発光スペクトルが実現できる。
For example, Si semiconductor nanoparticles of a silicon oxide film can control the particle size of the Si semiconductor nanoparticles by repeating oxidation of the particle surface and hydrofluoric acid treatment. At that time, more precise control is possible by changing the heating temperature and the heating time. Thereby, the emission spectrum in the present invention can be realized.
本発明に係るＳｉ半導体ナノ粒子は、膜中から取り出すことにより種々の技術分野における単一分子分析に応用できる。例えば、上記単一分子観察方法において、異なる発光スペクトルをもつ半導体ナノ粒子で複数種類の分子をそれぞれ標識し、該分子に励起光を照射することによって、同時に複数種類の分子の同定を行うこともできる。なお、適用可能な複数種類の分子としては化学組成は同じであるが、化学構造の異なる構造異性体等も含む。
The Si semiconductor nanoparticles according to the present invention can be applied to single molecule analysis in various technical fields by taking out from the film. For example, in the single molecule observation method, multiple types of molecules may be identified simultaneously by labeling multiple types of molecules with semiconductor nanoparticles having different emission spectra and irradiating the molecules with excitation light. it can. In addition, although the chemical composition is the same as the applicable types of molecules, it includes structural isomers having different chemical structures.
本発明に係る半導体ナノ粒子は、生体物質標識剤に適応することができる。また、標的（追跡）物質を有する生細胞もしくは生体に本発明に係る生体物質標識剤を添加することで、標的物質と結合もしくは吸着し、該結合体もしくは吸着体に所定の波長の励起光を照射し、当該励起光に応じて蛍光半導体微粒子から発生する所定の波長の蛍光を検出することにより、上記標的（追跡）物質の蛍光動態イメージングを行うことができる。即ち、本発明に係る生体物質標識剤は、バイオイメージング法（生体物質を構成する生体分子やその動的現象を可視化する技術手段）に利用することができる。
The semiconductor nanoparticles according to the present invention can be applied to a biological material labeling agent. Further, by adding the biological substance labeling agent according to the present invention to a living cell or living body having a target (tracking) substance, it binds or adsorbs to the target substance, and excitation light having a predetermined wavelength is applied to the conjugate or adsorbent. By irradiating and detecting fluorescence of a predetermined wavelength generated from the fluorescent semiconductor fine particles according to the excitation light, fluorescence dynamic imaging of the target (tracking) substance can be performed. That is, the biomaterial labeling agent according to the present invention can be used for bioimaging methods (technical means for visualizing biomolecules constituting the biomaterial and dynamic phenomena thereof).
上述した半導体ナノ粒子は一般的には疎水性であるため、例えば、生体物質標識剤として使用する場合は、このままでは水分散性が悪く、粒子が凝集してしまう等の問題があるため、半導体ナノ粒子のシェルの表面を親水化処理することが好ましい。
Since the semiconductor nanoparticles described above are generally hydrophobic, for example, when used as a biological material labeling agent, there are problems such as poor water dispersibility and aggregation of the particles. It is preferable to hydrophilize the surface of the nanoparticle shell.
親水化処理の方法としては、例えば、表面の親油性基をピリジン等で除去した後に粒子表面に表面修飾剤を化学的及び／または物理的に結合させる方法がある。表面修飾剤としては、親水基としてカルボキシル基、アミノ基を持つものが好ましく用いられ、具体的にはメルカプトプロピオン酸、メルカプトウンデカン酸、アミノプロパンチオールなどが挙げられる。
As a hydrophilic treatment method, for example, there is a method of chemically and / or physically binding a surface modifier to the particle surface after removing the lipophilic group on the surface with pyridine or the like. As the surface modifier, those having a carboxyl group or an amino group as hydrophilic groups are preferably used, and specific examples include mercaptopropionic acid, mercaptoundecanoic acid, aminopropanethiol and the like.
具体的には、例えば、Ｇｅ／ＧｅＯ2型半導体ナノ粒子１０-5ｇをメルカプトウンデカン酸０．２ｇが溶解した純水１０ｍｌ中に分散させて、４０℃、１０分間攪拌し、シェルの表面を処理することで無機ナノ粒子のシェルの表面をカルボキシル基で修飾することができる。
Specifically, for example, 10 −5 g of Ge / GeO 2 type semiconductor nanoparticles are dispersed in 10 ml of pure water in which 0.2 g of mercaptoundecanoic acid is dissolved, and stirred for 10 minutes at 40 ° C. By processing, the surface of the shell of inorganic nanoparticles can be modified with a carboxyl group.
本発明に係る生体物質標識剤は、上述した親水化処理された半導体ナノ粒子と分子標識物質と有機分子を介して結合させて得られる。本発明に係る生体物質標識剤は、分子標識物質が目的とする生体物質と特異的に結合及び／または反応することにより、生体物質の標識が可能となる。
The biological material labeling agent according to the present invention is obtained by bonding the above-described hydrophilic treatment semiconductor nanoparticles, the molecular labeling substance, and organic molecules. The biological material labeling agent according to the present invention enables the biological material to be labeled by specifically binding and / or reacting with the target biological material.
該分子標識物質としては、例えば、ヌクレオチド鎖、抗体、抗原及びシクロデキストリン等が挙げられる。
Examples of the molecular labeling substance include nucleotide chains, antibodies, antigens and cyclodextrins.
本発明に係る生体物質標識剤は、親水化処理された半導体ナノ粒子と分子標識物質とが有機分子により結合されている。該有機分子としては、半導体ナノ粒子と分子標識物質とを結合できる有機分子であれば特に制限はないが、例えば、タンパク質中でもアルブミン、ミオグロビン及びカゼイン等、またタンパク質の一種であるアビジンをビオチンと共に用いることも好適に用いられる。上記結合の態様としては特に限定されず、共有結合、イオン結合、水素結合、配位結合、物理吸着及び化学吸着等が挙げられる。結合の安定性から共有結合などの結合力の強い結合が好ましい。
In the biological material labeling agent according to the present invention, the hydrophilic semiconductor nanoparticles and the molecular labeling substance are bound by organic molecules. The organic molecule is not particularly limited as long as it is an organic molecule capable of binding a semiconductor nanoparticle and a molecular labeling substance. For example, albumin, myoglobin, casein, etc. among proteins, and avidin, which is a kind of protein, are used together with biotin. It is also preferably used. The form of the bond is not particularly limited, and examples thereof include covalent bond, ionic bond, hydrogen bond, coordinate bond, physical adsorption, and chemical adsorption. A bond having a strong bonding force such as a covalent bond is preferable from the viewpoint of bond stability.
具体的には、半導体ナノ粒子をメルカプトウンデカン酸で親水化処理した場合は、有機分子としてアビジン及びビオチンを用いることができる。この場合、親水化処理された半導体ナノ粒子のカルボキシル基はアビジンと好適に共有結合し、アビジンが更にビオチンと選択的に結合し、ビオチンが更に生体物質標識剤と結合することにより生体物質標識剤となる。
Specifically, when the semiconductor nanoparticles are hydrophilized with mercaptoundecanoic acid, avidin and biotin can be used as organic molecules. In this case, the carboxyl group of the semiconductor nanoparticles subjected to the hydrophilic treatment is preferably covalently bonded to avidin, the avidin is further selectively bonded to biotin, and biotin is further bonded to the biological material labeling agent to thereby bind the biological material labeling agent. It becomes.
以下、実施例を挙げて本発明を具体的に説明するが、本発明はこれらに限定されない。
Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
〔Ｓｉ半導体ナノ粒子１～２７の作製〕
（スパッタリングによる成膜）
真空チャンバー内にＡｒガスを導入し、高周波コントローラによりイオン化されたＡｒガスイオンをＳｉチップと石英ガラスからなるターゲット材料に衝突させる。これらの放出された原子及び分子を半導体基板上に堆積し、酸化ケイ素膜内にＳｉ原子が混ざった膜を形成する。このとき表１に示すようにＳｉ／ＳｉＯ2量を調整する。 [Preparation of Si semiconductor nanoparticles 1-27]
(Film formation by sputtering)
Ar gas is introduced into the vacuum chamber, and Ar gas ions ionized by the high-frequency controller are collided with a target material made of Si chip and quartz glass. These released atoms and molecules are deposited on a semiconductor substrate to form a film in which Si atoms are mixed in the silicon oxide film. At this time, the amount of Si / SiO 2 is adjusted as shown in Table 1.
（スパッタリングによる成膜）
真空チャンバー内にＡｒガスを導入し、高周波コントローラによりイオン化されたＡｒガスイオンをＳｉチップと石英ガラスからなるターゲット材料に衝突させる。これらの放出された原子及び分子を半導体基板上に堆積し、酸化ケイ素膜内にＳｉ原子が混ざった膜を形成する。このとき表１に示すようにＳｉ／ＳｉＯ2量を調整する。 [Preparation of Si semiconductor nanoparticles 1-27]
(Film formation by sputtering)
Ar gas is introduced into the vacuum chamber, and Ar gas ions ionized by the high-frequency controller are collided with a target material made of Si chip and quartz glass. These released atoms and molecules are deposited on a semiconductor substrate to form a film in which Si atoms are mixed in the silicon oxide film. At this time, the amount of Si / SiO 2 is adjusted as shown in Table 1.
（アニール処理）
得られたＳｉ原子を含有した酸化ケイ素膜をＡｒ雰囲気中で表１に示す温度まで急速に昇温し熱処理を行い、膜中のＳｉ原子をナノサイズまで凝集させる。熱処理時間は５０分とする。 (Annealing treatment)
The obtained silicon oxide film containing Si atoms is rapidly heated to a temperature shown in Table 1 in an Ar atmosphere and subjected to heat treatment to aggregate the Si atoms in the film to a nano size. The heat treatment time is 50 minutes.
得られたＳｉ原子を含有した酸化ケイ素膜をＡｒ雰囲気中で表１に示す温度まで急速に昇温し熱処理を行い、膜中のＳｉ原子をナノサイズまで凝集させる。熱処理時間は５０分とする。 (Annealing treatment)
The obtained silicon oxide film containing Si atoms is rapidly heated to a temperature shown in Table 1 in an Ar atmosphere and subjected to heat treatment to aggregate the Si atoms in the film to a nano size. The heat treatment time is 50 minutes.
（フッ酸処理）
得られたＳｉナノ粒子含有酸化ケイ素膜を４０℃のフッ酸蒸気に曝すことで、表面処理を行う。 (Hydrofluoric acid treatment)
Surface treatment is performed by exposing the obtained silicon oxide film containing Si nanoparticles to 40 ° C. hydrofluoric acid vapor.
得られたＳｉナノ粒子含有酸化ケイ素膜を４０℃のフッ酸蒸気に曝すことで、表面処理を行う。 (Hydrofluoric acid treatment)
Surface treatment is performed by exposing the obtained silicon oxide film containing Si nanoparticles to 40 ° C. hydrofluoric acid vapor.
（加熱酸化処理）
フッ酸処理後のＳｉナノ粒子含有酸化ケイ素膜を自然酸化、または過熱酸化処理を行う。加熱酸化処理の温度と時間は表１に示した。 (Heat oxidation treatment)
The silicon oxide film containing Si nanoparticles after hydrofluoric acid treatment is subjected to natural oxidation or overheating oxidation treatment. The temperature and time of the heat oxidation treatment are shown in Table 1.
フッ酸処理後のＳｉナノ粒子含有酸化ケイ素膜を自然酸化、または過熱酸化処理を行う。加熱酸化処理の温度と時間は表１に示した。 (Heat oxidation treatment)
The silicon oxide film containing Si nanoparticles after hydrofluoric acid treatment is subjected to natural oxidation or overheating oxidation treatment. The temperature and time of the heat oxidation treatment are shown in Table 1.
（Ｓｉナノ粒子の分離、液中への分散）
自然酸化または過熱酸化したＳｉナノ粒子含有酸化ケイ素膜を、エタノール中に投入して１０分間の超音波処理を行った。 (Separation of Si nanoparticles, dispersion in liquid)
The silicon oxide film containing Si nanoparticles that had been naturally oxidized or superheated was placed in ethanol and subjected to ultrasonic treatment for 10 minutes.
自然酸化または過熱酸化したＳｉナノ粒子含有酸化ケイ素膜を、エタノール中に投入して１０分間の超音波処理を行った。 (Separation of Si nanoparticles, dispersion in liquid)
The silicon oxide film containing Si nanoparticles that had been naturally oxidized or superheated was placed in ethanol and subjected to ultrasonic treatment for 10 minutes.
〔発光スペクトル測定〕
得られたエタノール中に分散したＳｉ半導体ナノ粒子に波長２８０ｎｍの励起光を照射し、発生する蛍光スペクトルを測定した。発光スペクトルの半値幅、ピーク波長を表１に示した。発光スペクトルは日立製蛍光光度計Ｆ－７０００を用いて行った。発光波長７００ｎｍ以上の近赤外発光に関しては、Ｈａｍａｍａｔｓｕ社のＵＶ－ＶＩＳを光源としてスペクトロメータ（ＳＷＮＩＲ）を使用して測定を行った。 (Measurement of emission spectrum)
The Si semiconductor nanoparticles dispersed in ethanol obtained were irradiated with excitation light having a wavelength of 280 nm, and the generated fluorescence spectrum was measured. The half-value width and peak wavelength of the emission spectrum are shown in Table 1. The emission spectrum was obtained using a Hitachi Fluorometer F-7000. For near-infrared light emission with an emission wavelength of 700 nm or more, measurement was performed using a spectrometer (SWNIR) with UV-VIS from Hamamatsu as a light source.
得られたエタノール中に分散したＳｉ半導体ナノ粒子に波長２８０ｎｍの励起光を照射し、発生する蛍光スペクトルを測定した。発光スペクトルの半値幅、ピーク波長を表１に示した。発光スペクトルは日立製蛍光光度計Ｆ－７０００を用いて行った。発光波長７００ｎｍ以上の近赤外発光に関しては、Ｈａｍａｍａｔｓｕ社のＵＶ－ＶＩＳを光源としてスペクトロメータ（ＳＷＮＩＲ）を使用して測定を行った。 (Measurement of emission spectrum)
The Si semiconductor nanoparticles dispersed in ethanol obtained were irradiated with excitation light having a wavelength of 280 nm, and the generated fluorescence spectrum was measured. The half-value width and peak wavelength of the emission spectrum are shown in Table 1. The emission spectrum was obtained using a Hitachi Fluorometer F-7000. For near-infrared light emission with an emission wavelength of 700 nm or more, measurement was performed using a spectrometer (SWNIR) with UV-VIS from Hamamatsu as a light source.
上記において作製した各種Ｓｉ半導体ナノ粒子、１×１０-5ｇをメルカプトウンデカン酸０．２ｇを溶解した１０ｍｌ純水中に再分散させ、４０℃、１０分間攪拌することで表面が親水化処理された各種Ｓｉ半導体ナノ粒子を得た。
The various Si semiconductor nanoparticles prepared above, 1 × 10 −5 g, were redispersed in 10 ml pure water in which 0.2 g of mercaptoundecanoic acid was dissolved, and the surface was hydrophilized by stirring at 40 ° C. for 10 minutes. Various Si semiconductor nanoparticles were obtained.
その後、表面が親水化処理された各種半導体ナノ粒子の水溶液それぞれにアビジン２５ｍｇを添加し、４０℃で１０分間攪拌を行い、アビジンコンジュゲートナノ粒子を作製した。
Thereafter, 25 mg of avidin was added to each of the aqueous solutions of various semiconductor nanoparticles whose surfaces were hydrophilized, and the mixture was stirred at 40 ° C. for 10 minutes to prepare avidin-conjugated nanoparticles.
得られたアビジンコンジュゲートナノ粒子溶液にビオチン化された塩基配列が既知であるオリゴヌクレオチドを混合攪拌し、半導体ナノ粒子で標識（ラベリング）されたオリゴヌクレオチドを作製した。
The obtained avidin-conjugated nanoparticle solution was mixed and stirred with a biotinylated oligonucleotide having a known base sequence to prepare an oligonucleotide labeled (labeled) with semiconductor nanoparticles.
様々な塩基配列を持つオリゴヌクレオチドを固定化したＤＮＡチップ上に上記の標識（ラベリング）したオリゴヌクレオチドを滴下、洗浄したところ、標識（ラベリング）されたオリゴヌクレオチドと相補的な塩基配列をもつオリゴヌクレオチドのスポットのみが、８１０ｎｍの励起光により半導体ナノ粒子の粒径依存して、異なる色の発光をすることが確認された。
When the above labeled (labeled) oligonucleotide is dropped onto a DNA chip on which oligonucleotides having various base sequences are immobilized and washed, the oligonucleotide has a complementary base sequence to the labeled (labeled) oligonucleotide. It was confirmed that only these spots emitted light of different colors depending on the particle size of the semiconductor nanoparticles by the excitation light of 810 nm.
このことより、本発明に係る半導体ナノ粒子蛍光体でのオリゴヌクレオチドの標識（ラベリング）が可能なことを確認することができた。即ち、この実施例により、本発明に係る半導体ナノ粒子蛍光体は、半導体ナノ粒子標識体に適応できることが分かる。なお、生体透過性の観点で優れる近赤外発光領域で高い発光強度を得ており、生体分子を検出するための半導体ナノ粒子標識体に適応できる。
From this, it was confirmed that the labeling (labeling) of the oligonucleotide with the semiconductor nanoparticle phosphor according to the present invention was possible. That is, it can be seen from this example that the semiconductor nanoparticle phosphor according to the present invention can be applied to a semiconductor nanoparticle label. In addition, high luminescence intensity is obtained in the near-infrared emission region which is excellent in terms of biopermeability, and it can be applied to a semiconductor nanoparticle label for detecting biomolecules.
比較例１
上記可視領域の３色が重なる、親水化処理されたＳｉ半導体ナノ粒子１、４、１０について、上記のように標識体を作製し、発光させた。 Comparative Example 1
With respect to Si semiconductor nanoparticles 1, 4, and 10 subjected to hydrophilic treatment in which the three colors in the visible region overlap each other, a labeled body was prepared as described above, and light was emitted.
上記可視領域の３色が重なる、親水化処理されたＳｉ半導体ナノ粒子１、４、１０について、上記のように標識体を作製し、発光させた。 Comparative Example 1
With respect to Si semiconductor nanoparticles 1, 4, and 10 subjected to hydrophilic treatment in which the three colors in the visible region overlap each other, a labeled body was prepared as described above, and light was emitted.
比較例２
上記可視領域の７色が重なる、親水化処理されたＳｉ半導体ナノ粒子１、２、４、５、６、９、１０について、上記のように標識体を作製し、発光させた。 Comparative Example 2
With respect to Si semiconductor nanoparticles 1, 2, 4, 5, 6, 9, and 10 subjected to hydrophilic treatment, in which the seven colors in the visible region overlap, labeled bodies were produced as described above, and light was emitted.
上記可視領域の７色が重なる、親水化処理されたＳｉ半導体ナノ粒子１、２、４、５、６、９、１０について、上記のように標識体を作製し、発光させた。 Comparative Example 2
With respect to Si semiconductor nanoparticles 1, 2, 4, 5, 6, 9, and 10 subjected to hydrophilic treatment, in which the seven colors in the visible region overlap, labeled bodies were produced as described above, and light was emitted.
比較例３
上記近赤外領域の３色が重ならない、親水化処理されたＳｉ半導体ナノ粒子１６、１９、２５について、上記のように標識体を作製し、発光させた。 Comparative Example 3
For Si semiconductor nanoparticles 16, 19, and 25 subjected to hydrophilic treatment, in which the three colors in the near-infrared region do not overlap, a labeled body was prepared as described above, and light was emitted.
上記近赤外領域の３色が重ならない、親水化処理されたＳｉ半導体ナノ粒子１６、１９、２５について、上記のように標識体を作製し、発光させた。 Comparative Example 3
For Si semiconductor nanoparticles 16, 19, and 25 subjected to hydrophilic treatment, in which the three colors in the near-infrared region do not overlap, a labeled body was prepared as described above, and light was emitted.
実施例１
上記近赤外領域の７色が重ならない、親水化処理されたＳｉ半導体ナノ粒子１７、１８、２０、２３、２４、２６、２７について、上記のように標識体を作製し、発光させた。 Example 1
For Si semiconductor nanoparticles 17, 18, 20, 23, 24, 26, and 27 that have been subjected to hydrophilic treatment that do not overlap the seven colors in the near-infrared region, labeled bodies were prepared as described above and emitted light.
上記近赤外領域の７色が重ならない、親水化処理されたＳｉ半導体ナノ粒子１７、１８、２０、２３、２４、２６、２７について、上記のように標識体を作製し、発光させた。 Example 1
For Si semiconductor nanoparticles 17, 18, 20, 23, 24, 26, and 27 that have been subjected to hydrophilic treatment that do not overlap the seven colors in the near-infrared region, labeled bodies were prepared as described above and emitted light.
実施例２
上記可視領域の７色が重ならない、親水化処理されたＳｉ半導体ナノ粒子２、３、５、６、９、１１、１２について、上記のように標識体を作製し、発光させた。 Example 2
With respect to Si semiconductor nanoparticles 2, 3, 5, 6, 9, 11, and 12 that were subjected to hydrophilic treatment and in which the seven colors in the visible region did not overlap, a labeled body was prepared as described above, and light was emitted.
上記可視領域の７色が重ならない、親水化処理されたＳｉ半導体ナノ粒子２、３、５、６、９、１１、１２について、上記のように標識体を作製し、発光させた。 Example 2
With respect to Si semiconductor nanoparticles 2, 3, 5, 6, 9, 11, and 12 that were subjected to hydrophilic treatment and in which the seven colors in the visible region did not overlap, a labeled body was prepared as described above, and light was emitted.
〔評価〕
＋：発光色がぼやけてしまう
＋＋：色の違いは見えるが三色
＋＋＋：発光色の違いがよくみえた。 [Evaluation]
+: The emission color is blurred ++: The color difference is visible, but three colors +++: The difference in the emission color is visible.
＋：発光色がぼやけてしまう
＋＋：色の違いは見えるが三色
＋＋＋：発光色の違いがよくみえた。 [Evaluation]
+: The emission color is blurred ++: The color difference is visible, but three colors +++: The difference in the emission color is visible.
評価結果を表２に示す。
Evaluation results are shown in Table 2.
本発明の実施例１、２より、７種類を優れた感度で検出できることが分かる。
It can be seen from Examples 1 and 2 of the present invention that seven types can be detected with excellent sensitivity.
Claims (6)
- 半導体ナノ粒子で標識された分子に励起光を照射し、発光を検出することにより該分子の同定を行う標識方法において、該半導体ナノ粒子の発光スペクトルが波長範囲３００ｎｍ以内に少なくとも３種類の発光ピークを持ち、且つ各スペクトルは重ならないことを特徴とする標識方法。 In a labeling method for identifying molecules by irradiating molecules labeled with semiconductor nanoparticles with excitation light and detecting luminescence, the emission spectrum of the semiconductor nanoparticles has at least three types of emission peaks within a wavelength range of 300 nm. A labeling method characterized in that each spectrum does not overlap.
- 前記発光スペクトルの波長範囲が可視領域であることを特徴とする請求の範囲第１項に記載の標識方法。 The labeling method according to claim 1, wherein a wavelength range of the emission spectrum is a visible region.
- 前記発光スペクトルの波長範囲が近赤外領域８００～１１００ｎｍであることを特徴とする請求の範囲第１項に記載の標識方法。 2. The labeling method according to claim 1, wherein the wavelength range of the emission spectrum is a near infrared region of 800 to 1100 nm.
- 前記各スペクトルの幅が５０ｎｍ以下であり、隣り合うピーク波長間距離はスペクトル幅よりも大きいことを特徴とする請求の範囲第１項～第３項のいずれか１項に記載の標識方法。 The labeling method according to any one of claims 1 to 3, wherein the width of each spectrum is 50 nm or less, and the distance between adjacent peak wavelengths is larger than the spectrum width.
- 前記半導体ナノ粒子の平均粒径が１ｎｍ以上１０ｎｍ以下であることを特徴とする請求の範囲第１項～第４項のいずれか１項に記載の標識方法。 The labeling method according to any one of claims 1 to 4, wherein an average particle size of the semiconductor nanoparticles is 1 nm or more and 10 nm or less.
- 前記半導体ナノ粒子がＳｉまたはＧｅであることを特徴とする請求の範囲第１項～第５項のいずれか１項に記載の標識方法。 The labeling method according to any one of claims 1 to 5, wherein the semiconductor nanoparticles are Si or Ge.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002539454A (en) * | 1999-03-01 | 2002-11-19 | ザ・リージェンツ・オブ・ザ・ユニバーシティー・オブ・カリフォルニア | Semiconductor nanocrystal probes for biological applications |
| JP2003028797A (en) * | 2001-07-11 | 2003-01-29 | Hitachi Software Eng Co Ltd | Fluorescence reader |
| JP2004340663A (en) * | 2003-05-14 | 2004-12-02 | Yokogawa Electric Corp | Confocal optical scanner |
| JP2005538353A (en) * | 2002-09-06 | 2005-12-15 | カイロン コーポレイション | Method for verifying fluid movement |
| JP2006077124A (en) * | 2004-09-09 | 2006-03-23 | Nard Inst Ltd | Silicon nanoparticle fluorescent substance |
| WO2007125812A1 (en) * | 2006-04-28 | 2007-11-08 | Konica Minolta Medical & Graphic, Inc. | Inorganic nanoparticle, process for producing the same, and biosubstance labeling agent having inorganic nanoparticle linked thereto |
-
2009
- 2009-02-20 WO PCT/JP2009/053000 patent/WO2009113374A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002539454A (en) * | 1999-03-01 | 2002-11-19 | ザ・リージェンツ・オブ・ザ・ユニバーシティー・オブ・カリフォルニア | Semiconductor nanocrystal probes for biological applications |
| JP2003028797A (en) * | 2001-07-11 | 2003-01-29 | Hitachi Software Eng Co Ltd | Fluorescence reader |
| JP2005538353A (en) * | 2002-09-06 | 2005-12-15 | カイロン コーポレイション | Method for verifying fluid movement |
| JP2004340663A (en) * | 2003-05-14 | 2004-12-02 | Yokogawa Electric Corp | Confocal optical scanner |
| JP2006077124A (en) * | 2004-09-09 | 2006-03-23 | Nard Inst Ltd | Silicon nanoparticle fluorescent substance |
| WO2007125812A1 (en) * | 2006-04-28 | 2007-11-08 | Konica Minolta Medical & Graphic, Inc. | Inorganic nanoparticle, process for producing the same, and biosubstance labeling agent having inorganic nanoparticle linked thereto |
Non-Patent Citations (1)
| Title |
|---|
| H P WU ET AL.: "Size Bunsan Saseta Silicon Nano Biryushimaku kara no Kashi Hakko", THE REVIEW OF LASER ENGINEERING, vol. 28, no. 6, 15 June 2000 (2000-06-15), pages 354 - 358 * |
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