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WO2009113375A1 - Silicon oxide film containing silicon nanoparticle phosphor, silicon nanoparticle phosphor, and single molecule observation method - Google Patents

Silicon oxide film containing silicon nanoparticle phosphor, silicon nanoparticle phosphor, and single molecule observation method Download PDF

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WO2009113375A1
WO2009113375A1 PCT/JP2009/053003 JP2009053003W WO2009113375A1 WO 2009113375 A1 WO2009113375 A1 WO 2009113375A1 JP 2009053003 W JP2009053003 W JP 2009053003W WO 2009113375 A1 WO2009113375 A1 WO 2009113375A1
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nanoparticle phosphor
silicon nanoparticle
silicon
oxide film
silicon oxide
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PCT/JP2009/053003
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French (fr)
Japanese (ja)
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一賀 午菴
繁郎 堀田
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コニカミノルタエムジー株式会社
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/113—Silicon oxides; Hydrates thereof
    • C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/59—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing silicon
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/59—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing silicon
    • C09K11/592—Chalcogenides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/60—Particles characterised by their size
    • C01P2004/64—Nanometer sized, i.e. from 1-100 nanometer

Definitions

  • the present invention relates to a silicon nanoparticle phosphor that can be suitably used as a nanoparticle phosphor, a silicon nanoparticle-containing silicon oxide film, and single molecule observation using the silicon nanoparticle phosphor as a biological material labeling agent. Regarding the method.
  • Recent detection equipment enhancements and labeling material enhancements have made it possible to detect single molecules, identify them, and observe movements, and play a major role in analytical chemistry, molecular biology, and analysis of nanostructures. Has been fulfilled.
  • Fluorescent dyes and nanoparticle phosphors have been proposed as labeling materials used for single molecule observation.
  • the nanoparticle phosphor can set the emission peak wavelength relatively freely in the range of about 400 to 2000 nm by selecting the size and material as compared with the fluorescent dye.
  • by taking a wide Stokes shift it is possible to reduce the influence of noise due to overlap with excitation light and background, and to improve the detection capability.
  • since there are very few fadings there are many advantages such as long-term dynamic observation.
  • quantum dot a substance that exhibits a quantum confinement effect in a nanometer-sized semiconductor substance.
  • a quantum dot is a small lump within about 10 and several nanometers in which several hundred to several thousand semiconductor atoms are gathered, but when absorbing energy from an excitation source and reaching an energy excited state, the energy of the quantum dot Releases energy corresponding to the band gap. Therefore, it is considered that by adjusting the size or material composition of the quantum dots, the energy band gap can be adjusted, and energy in various levels of wavelength bands can be used.
  • quantum dots have a crystal structure and the property that the band gap changes depending on the particle size, and the emission wavelength changes as the band gap changes. It leads to variation. In order to avoid this, there is a fundamental problem such as complicated operations such as classifying particles of a single spectrum.
  • the size of the semiconductor nanoparticle phosphor actually used is about 1 to 10 nm, but its size and mass are larger than that of the fluorescent dye, so that it is used as a biological material labeling agent for cells of 10 to 100 ⁇ m. Furthermore, from the viewpoint of ecological imaging, it is considered that there is a problem that the obstacle to cells affects the imaging behavior.
  • An object of the present invention is to improve the biomarker recognition rate by using a silicon nanoparticle phosphor with little variation in emission wavelength and emission intensity, and using the silicon nanoparticle phosphor.
  • the product nd of the thickness ( ⁇ m) d of the silicon nanoparticle phosphor-containing silicon oxide film and the refractive index n is 3 or more and 10 or less.
  • the silicon nanoparticle fluorescent substance containing silicon oxide film characterized by the above-mentioned.
  • a silicon nanoparticle phosphor with little variation in emission wavelength and emission intensity was obtained, and the biomarker recognition rate could be improved by using the silicon nanoparticle phosphor.
  • the present invention is characterized in that, in a silicon nanoparticle phosphor-containing silicon oxide film formed on a semiconductor substrate, the product nd of the film thickness ( ⁇ m) d and the refractive index n is 3 or more and 10 or less. To do.
  • the silicon nanoparticle phosphor-containing silicon oxide film of the present invention can be obtained by heat-treating (annealing) an amorphous silicon oxide film formed on a semiconductor substrate in an inert gas (such as argon).
  • a silicon substrate is mainly used as a semiconductor substrate.
  • the amorphous silicon oxide film is formed by, for example, a high-speed sputtering method (for example, Japanese Patent Application Laid-Open No. 2004-296781).
  • the heat treatment temperature is 900 to 1200 ° C., preferably 1000 to 1100 ° C., and the heat treatment time is 15 to 100 minutes, preferably 30 to 80 minutes, more preferably 50 to 60 minutes.
  • forming an amorphous silicon oxide film on a semiconductor substrate is (1) vaporizing the opposing raw material semiconductor by the first high-temperature plasma generated between the electrodes, and generating it by electrodeless discharge in a reduced pressure atmosphere. It is also possible to pass through the second high-temperature plasma (for example, JP-A-6-279015), (2) laser ablation method (for example, JP-A-2004-356163).
  • the second high-temperature plasma for example, JP-A-6-279015
  • laser ablation method for example, JP-A-2004-356163
  • argon gas is introduced into a vacuum chamber, the argon gas is ionized, and the ionized argon ions are converted into a silicon chip and quartz glass (a silicon chip on quartz glass at a predetermined interval on the quartz glass).
  • the atoms and molecules emitted from the target material are deposited on the semiconductor substrate to form an amorphous silicon oxide (SiO x ) film.
  • Examples of the target material in the high-speed sputtering method include silicon chip and quartz glass. By controlling the area ratio of the silicon chip and quartz glass, silicon nanoparticle phosphors having various particle sizes can be obtained.
  • the area ratio between the silicon chip and the quartz glass is in the range of 1 to 50%.
  • the particle size can also be controlled by changing the high-frequency power and gas pressure, which are sputtering conditions.
  • the high frequency power is 10 to 500 W
  • the gas pressure is 1.33 ⁇ 10 ⁇ 2 to 1.33 ⁇ 10 Pa.
  • the product nd of the film thickness ( ⁇ m) d and the refractive index n of the silicon oxide film containing the silicon nanoparticle phosphor formed on the semiconductor substrate is controlled to be 3 or more and 10 or less, It is preferably controlled to 3 or more and 7 or less, particularly preferably 3.5 or more and 5 or less.
  • Nd control can take any means. n may be controlled or d may be controlled, and of course, it is preferable to control them simultaneously.
  • n may be controlled or d may be controlled, and of course, it is preferable to control them simultaneously.
  • n in the high-speed sputtering method, from the viewpoint of controlling n, it is effective to control the amount of silicon chip on the target in the high-speed sputtering apparatus, the internal pressure of an inert gas such as argon, the magnetic flux density, and the temperature and time of heat treatment It is.
  • d it is effective to control the sputtering time and change the distance between the target and the semiconductor substrate.
  • the product nd of refractive index and film thickness of the obtained silicon nanoparticle phosphor-containing silicon oxide film is directly obtained by measuring with a Fourier transform infrared spectrophotometer (FT / IR-6100, manufactured by JASCO Corporation). be able to.
  • FT / IR-6100 Fourier transform infrared spectrophotometer
  • the silicon nanoparticle phosphor in the silicon oxide film of the present invention can be isolated by hydrofluoric acid treatment of the silicon oxide film.
  • the average particle diameter of the silicon nanoparticle phosphor of the present invention is 1 nm or more and 10 nm or less. Preferably they are 3 nm or more and 8 nm or less, Especially preferably, they are 3.5 nm or more and 7 nm or less.
  • the average particle size of the silicon nanoparticle phosphor needs to be determined three-dimensionally, but it is difficult because it is too fine, and in reality it must be evaluated with a two-dimensional image. (TEM) is preferably obtained by changing the shooting scene of the electron micrograph and shooting and averaging.
  • the average particle diameter is obtained by taking an electron micrograph using a TEM, measuring the cross-sectional area of a sufficient number of particles, and measuring the diameter when the measured value is the area of a corresponding circle. Obtained as the diameter, the arithmetic average was taken as the average particle diameter.
  • the number of cluster particles photographed with a TEM is preferably 100 or more, and more preferably 1000 particles.
  • the arithmetic average of 1000 particles is defined as the average particle size.
  • the silicon nanoparticle phosphor of the present invention can be applied to single molecule analysis in various technical fields.
  • the silicon nanoparticle phosphor of the present invention can be applied to single molecule analysis in various technical fields as a biological material labeling agent. That is, the present invention can be used in a single molecule observation method mainly for identifying molecules by irradiating molecules labeled with a silicon nanoparticle phosphor with excitation light and detecting luminescence.
  • 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 single molecule observation method it is also possible to simultaneously identify multiple types of molecules by labeling multiple types of molecules with semiconductor nanoparticles having different emission spectra and irradiating the molecules with excitation light.
  • the applicable types of molecules include structural isomers having the same chemical composition but different chemical structures.
  • the single molecule observation method includes a step of adhering a silicon nanoparticle phosphor to a cell by light irradiation, a step of washing and removing unreacted silicon nanoparticle phosphor, and a cell labeled with a silicon nanoparticle phosphor. It consists of a step of irradiating excitation light to detect luminescence, and a step of separating the silicon nanoparticle phosphor from the cells by light irradiation. By repeating these steps, cell dynamics can be observed.
  • the silicon nanoparticle phosphor of the present invention can be applied to a biological material labeling agent as described above. Further, by adding a biological substance labeling agent to living cells or living bodies having a target (tracking) substance, the target substance is bound or adsorbed, and the conjugate or adsorbent is irradiated with excitation light of a predetermined wavelength, By 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.
  • the biological material labeling agent according to the present invention can be used for a bioimaging method (technical means for visualizing biological molecules constituting the biological material and dynamic phenomena thereof).
  • the above-described silicon nanoparticle phosphor surface is generally hydrophobic, for example, when used as a biological material labeling agent, there are problems such as poor water dispersibility and aggregation of particles. Therefore, it is preferable to hydrophilize the surface of the silicon nanoparticle phosphor.
  • hydrophilization treatment 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.
  • the biological material labeling agent according to the present invention is obtained by bonding the above-described hydrophilic silicon nanoparticle phosphor and the molecular labeling substance via an organic molecule.
  • the biological substance labeling agent according to the present invention can label a biological substance by specifically binding and / or reacting with the target biological substance.
  • the molecular labeling substance include nucleotides, antibodies, antigens, and cyclodextrins.
  • the organic molecule is not particularly limited as long as it is an organic molecule that can bind the silicon nanoparticle phosphor and the molecular labeling substance.
  • 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 silicon nanoparticle phosphor is hydrophilized with mercaptoundecanoic acid
  • avidin and biotin can be used as organic molecules.
  • the carboxyl group of the silicon nanoparticle subjected to the hydrophilization treatment is preferably covalently bonded to avidin
  • the avidin is further selectively bonded to biotin
  • biotin is further bonded to the molecular labeling substance to thereby label the biological material. It becomes.
  • Example 1 ⁇ Production of silicon nanoparticle phosphor-containing silicon oxide film> (Formation of amorphous silicon oxide film by high-speed sputtering)
  • a high-frequency sputtering apparatus is shown in FIG.
  • This apparatus generally includes a vacuum chamber 16 having an argon gas introduction port 14 and an exhaust port 15 at the lower part of the side surface, and a cooling water 19 that is attached to the upper surface of the vacuum chamber 16 via an insulating material 17 and introduced and discharged from a cooling pipe 18.
  • a high-frequency electrode 22 having a cathode shield 21 attached to the lower surface of the vacuum chamber 16 via an insulating material 17 and cooled by cooling water 19 introduced and discharged from the cooling pipe 18. ing.
  • the argon gas is introduced into the vacuum chamber 16 from the argon gas inlet 14 and the argon gas is ionized by the high frequency controller 23 while keeping the argon gas pressure constant, and the ionized argon ions are converted into the high frequency electrode.
  • the target material 24 on 22 is made to collide with the silicon chip 24a and the quartz glass 24b.
  • silicon atoms and silicon oxide molecules released from the target material 24 are deposited on the semiconductor substrate 1 held by the substrate holder 20 to form an amorphous silicon oxide film mixed with silicon atoms.
  • the area ratio between the silicon chip and the quartz glass and the sputtering time are adjusted.
  • the argon gas pressure was 0.3 Pa and the high frequency power was 200 W.
  • the obtained amorphous silicon oxide film is rapidly heated to 1100 ° C. in an argon atmosphere and subjected to heat treatment for the heat treatment time shown in Table 1 to agglomerate silicon atoms in the film to nano size, thereby producing a silicon nanoparticle phosphor. To form.
  • the obtained silicon nanoparticle phosphor-containing silicon oxide film is subjected to surface treatment by exposing it to 40 ° C. hydrofluoric acid vapor. Thereafter, the film surface was washed by exposure to water vapor, left in an air atmosphere for 1 hour, and then placed in ethanol for ultrasonic treatment for 10 minutes.
  • the emission spectrum of each particle was observed using a near-field light scanning optical microscope when excited at a wavelength of 280 nm.
  • Table 2 shows the half width of the emission spectrum, the maximum emission wavelength, and the relative emission intensity. The relative emission intensity was determined by setting Sample 1 to 100.
  • the silicon nanoparticle phosphor of the present invention has a small FWHM and a standard deviation of emission intensity for each particle and a small variation. From this, it can be said that the silicon nanoparticle phosphor of the present invention is excellent as a biological material labeling agent for single molecule observation.
  • Example 2 1 ⁇ 10 ⁇ 5 g of the obtained silicon nanoparticle phosphor is redispersed in 10 ml pure water in which 0.2 g of mercaptoundecanoic acid is dissolved, and the surface is hydrophilized by stirring at 40 ° C. for 10 minutes. Thereafter, 25 mg of avidin was added to each of the aqueous solutions of these semiconductor nanoparticles, and the mixture was stirred at 40 ° C. for 10 minutes to produce avidin-conjugated nanoparticles.
  • the target protein in the cell is biotinylated and the avidin-conjugated nanoparticles are dropped and washed on the immobilized chip, one avidin-conjugated nanoparticle binds to the biotin bound to the target protein, and the spot Emits light of the color caused by the silicon nanoparticle phosphor upon ultraviolet irradiation. For 100 spots, the number of emitted spots is counted, and the ratio is shown in Table 3 as the biomarker recognition rate.

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Abstract

Disclosed is a silicon oxide film containing a silicon nanoparticle phosphor having no significant variation in luminescence wavelength and luminescence intensity. The use of the silicon nanoparticle phosphor can improve the biological label recognition rate. The silicon nanoparticle phosphor is obtained by treating a silicon oxide film containing a silicon nanoparticle phosphor with hydrofluoric acid. The silicon oxide film containing a silicon nanoparticle phosphor is provided on a semiconductor substrate and is characterized in that the product (nd) of the film thickness (d) (μm) and the refractive index (n) of the silicon oxide film containing a silicon nanoparticle phosphor is not less than 3 and not more than 10.

Description

シリコンナノ粒子蛍光体含有酸化ケイ素膜、シリコンナノ粒子蛍光体及び単一分子観察方法Silicon nanoparticle phosphor-containing silicon oxide film, silicon nanoparticle phosphor, and single molecule observation method
　本発明は、ナノ粒子蛍光体として好適に用いることができるシリコンナノ粒子蛍光体や、それに係るシリコンナノ粒子含有酸化ケイ素膜、及び該シリコンナノ粒子蛍光体を生体物質標識剤とした単一分子観察方法に関する。 The present invention relates to a silicon nanoparticle phosphor that can be suitably used as a nanoparticle phosphor, a silicon nanoparticle-containing silicon oxide film, and single molecule observation using the silicon nanoparticle phosphor as a biological material labeling agent. Regarding the method.
　近年の検出機材の高感度化や標識材料の高輝度化によって、単一分子の検出、同定、及び運動の観察が可能になり、分析化学、分子生物学及びナノ構造体の解析に大きな役割を果たしてきている。 Recent detection equipment enhancements and labeling material enhancements have made it possible to detect single molecules, identify them, and observe movements, and play a major role in analytical chemistry, molecular biology, and analysis of nanostructures. Has been fulfilled.
　単一分子の観察に使用される標識材料として、蛍光色素やナノ粒子蛍光体が提案されている。特にナノ粒子蛍光体は蛍光色素に比べて、大きさや材質を選択することにより、凡そ４００～２０００ｎｍの範囲で比較的自由に発光ピーク波長を設定することができる。また、ストークスシフトを広くとることで、励起光との重なりやバックグラウンドによるノイズ影響を小さくし、検出能を高めることができる。また、褪色が非常に少ないため、長時間の動態観察が可能であることなど、利点が非常に多い。 Fluorescent dyes and nanoparticle phosphors have been proposed as labeling materials used for single molecule observation. In particular, the nanoparticle phosphor can set the emission peak wavelength relatively freely in the range of about 400 to 2000 nm by selecting the size and material as compared with the fluorescent dye. In addition, by taking a wide Stokes shift, it is possible to reduce the influence of noise due to overlap with excitation light and background, and to improve the detection capability. In addition, since there are very few fadings, there are many advantages such as long-term dynamic observation.
　一般に、ナノメートルサイズの半導体物質で量子閉じ込め（ｑｕａｎｔｕｍ　ｃｏｎｆｉｎｅｍｅｎｔ）効果を示す物質は「量子ドット」と称されている。このような量子ドットは、半導体原子が数百個から数千個集まった１０数ｎｍ程度以内の小さな塊であるが、励起源から光を吸収してエネルギー励起状態に達すると、量子ドットのエネルギーバンドギャップに相当するエネルギーを放出する。従って、量子ドットの大きさまたは物質組成を調節すると、エネルギーバンドギャップを調節することができて、様々な水準の波長帯のエネルギーを利用することができる可能性があると考えられている。 In general, a substance that exhibits a quantum confinement effect in a nanometer-sized semiconductor substance is called a “quantum dot”. Such a quantum dot is a small lump within about 10 and several nanometers in which several hundred to several thousand semiconductor atoms are gathered, but when absorbing energy from an excitation source and reaching an energy excited state, the energy of the quantum dot Releases energy corresponding to the band gap. Therefore, it is considered that by adjusting the size or material composition of the quantum dots, the energy band gap can be adjusted, and energy in various levels of wavelength bands can be used.
　しかしながら、量子ドットは結晶構造を持ち、粒径によりバンドギャップが変化するという性質を持ち、バンドギャップの変化に伴い発光波長が変化するため、個々の粒径のばらつきが直接粒子毎の発光スペクトルのばらつきにつながる。これを回避するには、単一スペクトルの粒子を分級するなど煩雑な操作が必要になるなどの原理的な問題を抱えている。 However, quantum dots have a crystal structure and the property that the band gap changes depending on the particle size, and the emission wavelength changes as the band gap changes. It leads to variation. In order to avoid this, there is a fundamental problem such as complicated operations such as classifying particles of a single spectrum.
　また、実際に利用される半導体ナノ粒子蛍光体の大きさは１～１０ｎｍ程度であるが、蛍光色素に比べて大きさ、質量も大きいため、１０～１００μｍの細胞の生体物質標識剤として用いる場合に、生態イメージングの観点で細胞への障害性がイメージング挙動に影響するという問題があると考えられる。 In addition, the size of the semiconductor nanoparticle phosphor actually used is about 1 to 10 nm, but its size and mass are larger than that of the fluorescent dye, so that it is used as a biological material labeling agent for cells of 10 to 100 μm. Furthermore, from the viewpoint of ecological imaging, it is considered that there is a problem that the obstacle to cells affects the imaging behavior.
　また、半導体ナノ粒子蛍光体の中で、半導体基板上にアモルファス酸化ケイ素膜を形成し、熱処理等でシリコンナノ粒子蛍光体とする技術が知られている（例えば、特許文献１～３参照）。しかしながら、これらは形成された酸化ケイ素膜の特性には言及していない。
特開２００４－２９６７８１号公報 特開２００６－７００８９号公報 特開２００７－６３３７８号公報 In addition, among the semiconductor nanoparticle phosphors, a technique is known in which an amorphous silicon oxide film is formed on a semiconductor substrate to form a silicon nanoparticle phosphor by heat treatment or the like (see, for example, Patent Documents 1 to 3). However, they do not mention the properties of the formed silicon oxide film.
Japanese Patent Application Laid-Open No. 2004-296781 JP 2006-70089 A JP 2007-63378 A
　本発明の目的は、発光波長、発光強度にバラツキが少ないシリコンナノ粒子蛍光体、及び該シリコンナノ粒子蛍光体を用いることによって生体標識認識率を向上させることである。 An object of the present invention is to improve the biomarker recognition rate by using a silicon nanoparticle phosphor with little variation in emission wavelength and emission intensity, and using the silicon nanoparticle phosphor.
　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．半導体基板上に成膜されたシリコンナノ粒子蛍光体含有酸化ケイ素膜において、該シリコンナノ粒子蛍光体含有酸化ケイ素膜の膜厚（μｍ）ｄと屈折率ｎの積ｎｄが３以上１０以下であることを特徴とするシリコンナノ粒子蛍光体含有酸化ケイ素膜。 1. In a silicon nanoparticle phosphor-containing silicon oxide film formed on a semiconductor substrate, the product nd of the thickness (μm) d of the silicon nanoparticle phosphor-containing silicon oxide film and the refractive index n is 3 or more and 10 or less. The silicon nanoparticle fluorescent substance containing silicon oxide film characterized by the above-mentioned.
　２．前記１に記載のシリコンナノ粒子蛍光体含有酸化ケイ素膜をフッ酸処理して得られることを特徴とするシリコンナノ粒子蛍光体。 2. 2. A silicon nanoparticle phosphor obtained by treating the silicon nanoparticle phosphor-containing silicon oxide film according to 1 with a hydrofluoric acid treatment.
　３．平均粒径が１ｎｍ以上１０ｎｍ以下であることを特徴とする前記２に記載のシリコンナノ粒子蛍光体。 3. 3. The silicon nanoparticle phosphor as described in 2 above, wherein the average particle size is from 1 nm to 10 nm.
　４．前記２または３に記載のシリコンナノ粒子蛍光体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当該分子の同定を行うことを特徴とする単一分子観察方法。 4. 4. A single molecule observation method, wherein the molecule is identified by irradiating the molecule labeled with the silicon nanoparticle phosphor according to 2 or 3 with excitation light and detecting light emission.
　５．前記４に記載の単一分子観察方法において、異なる発光スペクトルを持つシリコンナノ粒子蛍光体で複数種類の分子をそれぞれ標識し、当該分子に励起光を照射することによって、同時に複数種類の物質の同定を行うことを特徴とする単一分子観察方法。 5. 5. The single molecule observation method according to 4 above, wherein a plurality of types of molecules are labeled with silicon nanoparticle phosphors having different emission spectra, and the plurality of types of substances are identified simultaneously by irradiating the molecules with excitation light. A method for observing single molecules.
　本発明によって、発光波長、発光強度にバラツキが少ないシリコンナノ粒子蛍光体が得られ、該シリコンナノ粒子蛍光体を用いることによって生体標識認識率を向上させることができた。 According to the present invention, a silicon nanoparticle phosphor with little variation in emission wavelength and emission intensity was obtained, and the biomarker recognition rate could be improved by using the silicon nanoparticle phosphor.
本発明で用いる高周波スパッタリング装置の一態様を示す図である。It is a figure which shows the one aspect | mode of the high frequency sputtering device used by this invention.
符号の説明Explanation of symbols
　１４　アルゴンガス導入口
　１５　排気口
　１６　真空チャンバー
　１７　絶縁材料
　１８　冷却管
　１９　冷却水
　２０　基板ホルダー
　２１　陰極シールド
　２２　高周波電極
　２３　高周波コントローラ
　２４　ターゲット材料
　２４ａ　シリコンチップ
　２４ｂ　石英ガラス 14 Argon gas introduction port 15 Exhaust port 16 Vacuum chamber 17 Insulating material 18 Cooling pipe 19 Cooling water 20 Substrate holder 21 Cathode shield 22 High frequency electrode 23 High frequency controller 24 Target material 24a Silicon chip 24b Quartz glass
　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、半導体基板上に成膜されたシリコンナノ粒子蛍光体含有酸化ケイ素膜において、該膜の膜厚（μｍ）ｄと屈折率ｎの積ｎｄが３以上１０以下であることを特徴とする。本発明のシリコンナノ粒子蛍光体含有酸化ケイ素膜は、半導体基板上に形成したアモルファス酸化ケイ素膜を不活性ガス（アルゴン等）中で熱処理（アニール処理）することによって得られる。 The present invention is characterized in that, in a silicon nanoparticle phosphor-containing silicon oxide film formed on a semiconductor substrate, the product nd of the film thickness (μm) d and the refractive index n is 3 or more and 10 or less. To do. The silicon nanoparticle phosphor-containing silicon oxide film of the present invention can be obtained by heat-treating (annealing) an amorphous silicon oxide film formed on a semiconductor substrate in an inert gas (such as argon).
　半導体基板として、主に用いられるのはシリコン基板である。 As a semiconductor substrate, a silicon substrate is mainly used.
　アモルファス酸化ケイ素膜の形成は、例えば、高速スパッタリング法（例えば、特開２００４－２９６７８１号公報）によって行われる。 The amorphous silicon oxide film is formed by, for example, a high-speed sputtering method (for example, Japanese Patent Application Laid-Open No. 2004-296781).
　熱処理温度は９００～１２００℃、好ましくは１０００～１１００℃であり、熱処理時間は１５～１００分、好ましくは３０～８０分、より好ましくは５０～６０分である。 The heat treatment temperature is 900 to 1200 ° C., preferably 1000 to 1100 ° C., and the heat treatment time is 15 to 100 minutes, preferably 30 to 80 minutes, more preferably 50 to 60 minutes.
　なお、半導体基板上にアモルファス酸化ケイ素膜を形成することは、（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極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号公報）、（２）レーザーアブレーション法（例えば、特開２００４－３５６１６３号公報）によっても可能である。 In addition, forming an amorphous silicon oxide film on a semiconductor substrate is (1) vaporizing the opposing raw material semiconductor by the first high-temperature plasma generated between the electrodes, and generating it by electrodeless discharge in a reduced pressure atmosphere. It is also possible to pass through the second high-temperature plasma (for example, JP-A-6-279015), (2) laser ablation method (for example, JP-A-2004-356163).
　高速スパッタリング装置において、アルゴンガスを真空チャンバーに導入し、アルゴンガスをイオン化し、イオン化されたアルゴンイオンを高周波電極のターゲット材料であるシリコンチップと石英ガラス（石英ガラス上にシリコンチップが所定の間隔で配列されている。）へ衝突させ、ターゲット材料から放出された原子や分子を半導体基板上に堆積させ、アモルファス酸化ケイ素（ＳｉＯx）膜を形成する。 In a high-speed sputtering apparatus, argon gas is introduced into a vacuum chamber, the argon gas is ionized, and the ionized argon ions are converted into a silicon chip and quartz glass (a silicon chip on quartz glass at a predetermined interval on the quartz glass). The atoms and molecules emitted from the target material are deposited on the semiconductor substrate to form an amorphous silicon oxide (SiO x ) film.
　高速スパッタリング法におけるターゲット材料としては、シリコンチップと石英ガラスが挙げられ、シリコンチップと石英ガラスの面積比を制御することによって、様々な粒子サイズのシリコンナノ粒子蛍光体が得られる。シリコンチップと石英ガラスの面積比は１～５０％の範囲である。 Examples of the target material in the high-speed sputtering method include silicon chip and quartz glass. By controlling the area ratio of the silicon chip and quartz glass, silicon nanoparticle phosphors having various particle sizes can be obtained. The area ratio between the silicon chip and the quartz glass is in the range of 1 to 50%.
　スパッタリング条件である高周波電力やガス圧を変化させても粒子サイズを制御することができる。高周波電力は１０～５００Ｗ、ガス圧は１．３３×１０-2～１．３３×１０Ｐａの範囲である。 The particle size can also be controlled by changing the high-frequency power and gas pressure, which are sputtering conditions. The high frequency power is 10 to 500 W, and the gas pressure is 1.33 × 10 −2 to 1.33 × 10 Pa.
　本発明においては、半導体基板上に成膜されたシリコンナノ粒子蛍光体を含有する酸化ケイ素膜の膜厚（μｍ）ｄと屈折率ｎの積ｎｄが３以上１０以下に制御することであり、好ましくは３以上７以下、特に好ましくは３．５以上５以下に制御することである。 In the present invention, the product nd of the film thickness (μm) d and the refractive index n of the silicon oxide film containing the silicon nanoparticle phosphor formed on the semiconductor substrate is controlled to be 3 or more and 10 or less, It is preferably controlled to 3 or more and 7 or less, particularly preferably 3.5 or more and 5 or less.
　ｎｄが３未満の場合、膜内に形成されたＳｉ粒子の分布が不均一であり、粒径分布も広がってしまう。１０を超える場合、膜内から取り出す際に凝集したり、均一な発光が得られないなどの不具合が生じる。これらのことから、ｎｄを３～１０にすることで、高い発光効率、生体標識として優れるなどの本発明の効果に結びついたものと考えられる。 When nd is less than 3, the distribution of Si particles formed in the film is not uniform, and the particle size distribution is also widened. If it exceeds 10, problems such as agglomeration when the film is taken out from the film and uniform light emission cannot be obtained. From these facts, it is considered that by setting nd to 3 to 10, the effects of the present invention, such as high luminous efficiency and excellent biolabeling, were associated.
　ｎｄの制御はあらゆる手段をとることができる。ｎを制御しても、ｄを制御してもよく、もちろん同時に制御することが好ましい。例えば、高速スパッタリング法において、ｎを制御する観点から、高速スパッタリング装置内でのターゲット上のシリコンチップの量とアルゴン等不活性ガスの内圧、磁束密度の制御、熱処理の温度や時間の制御が有効である。また、ｄを制御する観点から、スパッタリング時間の制御やターゲットと半導体基板間の距離の変更が有効である。 Nd control can take any means. n may be controlled or d may be controlled, and of course, it is preferable to control them simultaneously. For example, in the high-speed sputtering method, from the viewpoint of controlling n, it is effective to control the amount of silicon chip on the target in the high-speed sputtering apparatus, the internal pressure of an inert gas such as argon, the magnetic flux density, and the temperature and time of heat treatment It is. From the viewpoint of controlling d, it is effective to control the sputtering time and change the distance between the target and the semiconductor substrate.
　得られたシリコンナノ粒子蛍光体含有酸化ケイ素膜の屈折率と膜厚の積ｎｄは、フーリエ変換赤外分光光度計（ＦＴ／ＩＲ－６１００、日本分光株式会社製）で測定することで直接求めることができる。 The product nd of refractive index and film thickness of the obtained silicon nanoparticle phosphor-containing silicon oxide film is directly obtained by measuring with a Fourier transform infrared spectrophotometer (FT / IR-6100, manufactured by JASCO Corporation). be able to.
　本発明の酸化ケイ素膜中のシリコンナノ粒子蛍光体は、該酸化ケイ素膜をフッ酸処理して単離することができる。 The silicon nanoparticle phosphor in the silicon oxide film of the present invention can be isolated by hydrofluoric acid treatment of the silicon oxide film.
　本発明のシリコンナノ粒子蛍光体の平均粒径は、１ｎｍ以上１０ｎｍ以下である。好ましくは３ｎｍ以上８ｎｍ以下、特に好ましくは３．５ｎｍ以上７ｎｍ以下である。なお、本発明において、シリコンナノ粒子蛍光体の平均粒径は本来３次元で求める必要があるが、微粒子過ぎるため難しく、現実には二次元画像で評価せざるを得ないため、透過型電子顕微鏡（ＴＥＭ）を用いて電子顕微鏡写真の撮影シーンを変えて数多く撮影し、平均化することで求めることが好ましい。 The average particle diameter of the silicon nanoparticle phosphor of the present invention is 1 nm or more and 10 nm or less. Preferably they are 3 nm or more and 8 nm or less, Especially preferably, they are 3.5 nm or more and 7 nm or less. In the present invention, the average particle size of the silicon nanoparticle phosphor needs to be determined three-dimensionally, but it is difficult because it is too fine, and in reality it must be evaluated with a two-dimensional image. (TEM) is preferably obtained by changing the shooting scene of the electron micrograph and shooting and averaging.
　従って、本発明において、当該平均粒径はＴＥＭを用いて電子顕微鏡写真を撮影し、十分な数の粒子について断面積を計測し、その計測値を相当する円の面積としたときの直径を粒径として求めて、その算術平均を平均粒径とした。ＴＥＭで撮影するクラスター粒子数としては１００個以上が好ましく、１０００個の粒子を撮影するのが更に好ましい。本発明においては、１０００個の粒子の算術平均を平均粒径とした。 Therefore, in the present invention, the average particle diameter is obtained by taking an electron micrograph using a TEM, measuring the cross-sectional area of a sufficient number of particles, and measuring the diameter when the measured value is the area of a corresponding circle. Obtained as the diameter, the arithmetic average was taken as the average particle diameter. The number of cluster particles photographed with a TEM is preferably 100 or more, and more preferably 1000 particles. In the present invention, the arithmetic average of 1000 particles is defined as the average particle size.
　本発明のシリコンナノ粒子蛍光体は、種々の技術分野における単一分子分析に応用できる。 The silicon nanoparticle phosphor of the present invention can be applied to single molecule analysis in various technical fields.
　本発明のシリコンナノ粒子蛍光体は、生体物質標識剤として種々の技術分野における単一分子分析に応用することができる。即ち、シリコンナノ粒子蛍光体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当該分子の同定を行うことを主眼とする単一分子観察方法に利用することができる。 The silicon nanoparticle phosphor of the present invention can be applied to single molecule analysis in various technical fields as a biological material labeling agent. That is, the present invention can be used in a single molecule observation method mainly for identifying molecules by irradiating molecules labeled with a silicon nanoparticle phosphor with excitation light and detecting luminescence.
　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.
　また、単一分子観察方法において、異なる発光スペクトルを持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。 In the single molecule observation method, it is also possible to simultaneously identify multiple types of molecules by labeling multiple types of molecules with semiconductor nanoparticles having different emission spectra and irradiating the molecules with excitation light. . The applicable types of molecules include structural isomers having the same chemical composition but different chemical structures.
　更に当該単一分子観察方法は、光照射によりシリコンナノ粒子蛍光体を細胞に接着させる工程、未反応のシリコンナノ粒子蛍光体を洗浄、除去する工程、シリコンナノ粒子蛍光体で標識された細胞に励起光を照射し発光を検出する工程、光照射によりシリコンナノ粒子蛍光体を細胞から切り離す工程からなり、これらの工程を繰り返し行うことにより、細胞の動態観察を行うこともできる。 Furthermore, the single molecule observation method includes a step of adhering a silicon nanoparticle phosphor to a cell by light irradiation, a step of washing and removing unreacted silicon nanoparticle phosphor, and a cell labeled with a silicon nanoparticle phosphor. It consists of a step of irradiating excitation light to detect luminescence, and a step of separating the silicon nanoparticle phosphor from the cells by light irradiation. By repeating these steps, cell dynamics can be observed.
　以下においては、その他の代表的な応用例等について説明する。 In the following, other typical application examples will be described.
　本発明のシリコンナノ粒子蛍光体は、上述のように生体物質標識剤に適用することができる。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。 The silicon nanoparticle phosphor of the present invention can be applied to a biological material labeling agent as described above. Further, by adding a biological substance labeling agent to living cells or living bodies having a target (tracking) substance, the target substance is bound or adsorbed, and the conjugate or adsorbent is irradiated with excitation light of a predetermined wavelength, By 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.
　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。 That is, the biological material labeling agent according to the present invention can be used for a bioimaging method (technical means for visualizing biological molecules constituting the biological material and dynamic phenomena thereof).
　上述したシリコンナノ粒子蛍光体表面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、シリコンナノ粒子蛍光体の表面を親水化処理することが好ましい。 Since the above-described silicon nanoparticle phosphor surface is generally hydrophobic, for example, when used as a biological material labeling agent, there are problems such as poor water dispersibility and aggregation of particles. Therefore, it is preferable to hydrophilize the surface of the silicon nanoparticle phosphor.
　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 method of hydrophilization treatment, 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.
　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シリコンナノ粒子蛍光体と分子標識物質とを有機分子を介して結合させて得られる。 The biological material labeling agent according to the present invention is obtained by bonding the above-described hydrophilic silicon nanoparticle phosphor and the molecular labeling substance via an organic molecule.
　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。該分子標識物質としては、例えば、ヌクレオチド、抗体、抗原及びシクロデキストリン等が挙げられる。 The biological substance labeling agent according to the present invention can label a biological substance by specifically binding and / or reacting with the target biological substance. Examples of the molecular labeling substance include nucleotides, antibodies, antigens, and cyclodextrins.
　当該有機分子としては、シリコンナノ粒子蛍光体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。 The organic molecule is not particularly limited as long as it is an organic molecule that can bind the silicon nanoparticle phosphor and the molecular labeling substance. For example, among proteins, albumin, myoglobin, casein, etc., and avidin, which is a kind of protein, are also included. It is also preferably used with biotin. 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、シリコンナノ粒子蛍光体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シリコン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、ビオチンが更に分子標識物質と結合することにより生体物質標識剤となる。 Specifically, when the silicon nanoparticle phosphor is hydrophilized with mercaptoundecanoic acid, avidin and biotin can be used as organic molecules. In this case, the carboxyl group of the silicon nanoparticle subjected to the hydrophilization treatment is preferably covalently bonded to avidin, the avidin is further selectively bonded to biotin, and biotin is further bonded to the molecular labeling substance to thereby label the biological material. It becomes.
　以下、実施例により本発明をより詳細に説明するが、本発明はこれに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
　実施例１
　《シリコンナノ粒子蛍光体含有酸化ケイ素膜の作製》
　（高速スパッタリングによるアモルファス酸化ケイ素膜の形成）
　高周波スパッタリング装置の一態様を図１に示す。この装置は、概略、側面下部にアルゴンガス導入口１４と排気口１５を備える真空チャンバー１６、真空チャンバー１６の上面に絶縁材料１７を介して取り付けられ冷却管１８から導入、排出される冷却水１９で冷却される基板ホルダー２０、及び真空チャンバー１６の下面に絶縁材料１７を介して取り付けられ冷却管１８から導入、排出される冷却水１９で冷却される陰極シールド２１を備える高周波電極２２から構成されている。 Example 1
<Production of silicon nanoparticle phosphor-containing silicon oxide film>
(Formation of amorphous silicon oxide film by high-speed sputtering)
One mode of a high-frequency sputtering apparatus is shown in FIG. This apparatus generally includes a vacuum chamber 16 having an argon gas introduction port 14 and an exhaust port 15 at the lower part of the side surface, and a cooling water 19 that is attached to the upper surface of the vacuum chamber 16 via an insulating material 17 and introduced and discharged from a cooling pipe 18. And a high-frequency electrode 22 having a cathode shield 21 attached to the lower surface of the vacuum chamber 16 via an insulating material 17 and cooled by cooling water 19 introduced and discharged from the cooling pipe 18. ing.
　そして、上記装置において、アルゴンガスを真空チャンバー１６内にアルゴンガス導入口１４から導入し、アルゴンガス圧を一定に保ちながら、高周波コントローラ２３によりアルゴンガスをイオン化し、イオン化されたアルゴンイオンを高周波電極２２上のターゲット材料２４である、シリコンチップ２４ａと石英ガラス２４ｂへ衝突させる。 In the above apparatus, the argon gas is introduced into the vacuum chamber 16 from the argon gas inlet 14 and the argon gas is ionized by the high frequency controller 23 while keeping the argon gas pressure constant, and the ionized argon ions are converted into the high frequency electrode. The target material 24 on 22 is made to collide with the silicon chip 24a and the quartz glass 24b.
　この衝突により、ターゲット材料２４から放出されたシリコン原子や酸化ケイ素分子を基板ホルダー２０に保持した半導体基板１上に堆積させ、シリコン原子が混入したアモルファス酸化ケイ素膜を形成する。 By this collision, silicon atoms and silicon oxide molecules released from the target material 24 are deposited on the semiconductor substrate 1 held by the substrate holder 20 to form an amorphous silicon oxide film mixed with silicon atoms.
　このとき、表１に示すように、シリコンチップと石英ガラスの面積比、スパッタリング時間を調整する。また、アルゴンガスの圧は０．３Ｐａ、高周波電力は２００Ｗとした。 At this time, as shown in Table 1, the area ratio between the silicon chip and the quartz glass and the sputtering time are adjusted. The argon gas pressure was 0.3 Pa and the high frequency power was 200 W.
　（アニール処理）
　得られたアモルファス酸化ケイ素膜を、アルゴン雰囲気中において１１００℃まで急速に昇温して表１に示す熱処理時間で熱処理を行い、膜中のシリコン原子をナノサイズまで凝集させ、シリコンナノ粒子蛍光体を形成させる。 (Annealing treatment)
The obtained amorphous silicon oxide film is rapidly heated to 1100 ° C. in an argon atmosphere and subjected to heat treatment for the heat treatment time shown in Table 1 to agglomerate silicon atoms in the film to nano size, thereby producing a silicon nanoparticle phosphor. To form.
　《屈折率と膜厚の積ｎｄの測定》
　得られたシリコンナノ粒子蛍光体含有酸化ケイ素膜の屈折率と膜厚の積ｎｄは、フーリエ変換赤外分光光度計（ＦＴ／ＩＲ－６１００、日本分光株式会社製）で測定することで直接求めることができる。得られた値を表１に示す。 << Measurement of product nd of refractive index and film thickness >>
The product nd of refractive index and film thickness of the obtained silicon nanoparticle phosphor-containing silicon oxide film is directly obtained by measuring with a Fourier transform infrared spectrophotometer (FT / IR-6100, manufactured by JASCO Corporation). be able to. The obtained values are shown in Table 1.
　《平均粒径測定》
　得られたシリコンナノ粒子蛍光体含有酸化ケイ素膜のＴＥＭ像を撮影し、各１０００個以上の粒子を実測して、シリコンナノ粒子蛍光体の平均粒径を求めた。測定結果を表１に示す。 <Measurement of average particle size>
TEM images of the obtained silicon nanoparticle phosphor-containing silicon oxide film were taken, and 1000 or more particles were measured, and the average particle size of the silicon nanoparticle phosphor was determined. The measurement results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　《シリコンナノ粒子蛍光体の分離及び発光スペクトル》
　得られたシリコンナノ粒子蛍光体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。その後、水蒸気に曝して膜面を洗浄し、大気雰囲気下で１時間放置した後、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 and emission spectrum of silicon nanoparticle phosphor >>
The obtained silicon nanoparticle phosphor-containing silicon oxide film is subjected to surface treatment by exposing it to 40 ° C. hydrofluoric acid vapor. Thereafter, the film surface was washed by exposure to water vapor, left in an air atmosphere for 1 hour, and then placed in ethanol for ultrasonic treatment for 10 minutes.
　それぞれの溶液について、近接場光走査型光学顕微鏡を用いて、波長２８０ｎｍで励起させたときの一粒子毎の発光スペクトルを観察した。発光スペクトルの半値幅、極大発光波長、相対発光強度を表２に示す。相対発光強度は、試料１を１００とすることにより求めた。 For each solution, the emission spectrum of each particle was observed using a near-field light scanning optical microscope when excited at a wavelength of 280 nm. Table 2 shows the half width of the emission spectrum, the maximum emission wavelength, and the relative emission intensity. The relative emission intensity was determined by setting Sample 1 to 100.
　また、各溶液で１００個の粒子の発光スペクトルを測定し、半値幅及び発光ピーク強度の標準偏差を算出した。測定結果を表２に示した。 In addition, the emission spectrum of 100 particles in each solution was measured, and the standard deviation of the half width and emission peak intensity was calculated. The measurement results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　本発明のシリコンナノ粒子蛍光体は、粒子毎の半値幅及び発光強度の標準偏差が小さくバラツキが少ない。このことから、本発明のシリコンナノ粒子蛍光体は単一分子観察の生体物質標識剤として優れていると言える。 The silicon nanoparticle phosphor of the present invention has a small FWHM and a standard deviation of emission intensity for each particle and a small variation. From this, it can be said that the silicon nanoparticle phosphor of the present invention is excellent as a biological material labeling agent for single molecule observation.
　実施例２
　得られたシリコンナノ粒子蛍光体を、メルカプトウンデカン酸０．２ｇを溶解した１０ｍｌ純水中に１×１０-5ｇ再分散させ、４０℃、１０分間攪拌することで表面を親水化する。その後、これら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。 Example 2
1 × 10 −5 g of the obtained silicon nanoparticle phosphor is redispersed in 10 ml pure water in which 0.2 g of mercaptoundecanoic acid is dissolved, and the surface is hydrophilized by stirring at 40 ° C. for 10 minutes. Thereafter, 25 mg of avidin was added to each of the aqueous solutions of these semiconductor nanoparticles, and the mixture was stirred at 40 ° C. for 10 minutes to produce avidin-conjugated nanoparticles.
　細胞中の標的タンパク質をビオチン化して、固定化したチップ上に上記のアビジンコンジュゲートナノ粒子を滴下、洗浄すると、標的タンパク質に結合したビオチンに１個のアビジンコンジュゲートナノ粒子が結合し、そのスポットが紫外線照射によりシリコンナノ粒子蛍光体に起因する色の発光をする。スポット１００個について、発光したスポットの数をカウントし、その比率を生体標識認識率として表３に示す。 When the target protein in the cell is biotinylated and the avidin-conjugated nanoparticles are dropped and washed on the immobilized chip, one avidin-conjugated nanoparticle binds to the biotin bound to the target protein, and the spot Emits light of the color caused by the silicon nanoparticle phosphor upon ultraviolet irradiation. For 100 spots, the number of emitted spots is counted, and the ratio is shown in Table 3 as the biomarker recognition rate.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表３より、本発明のシリコンナノ粒子蛍光体からは高い生体標識認識率が得られることが分かる。 From Table 3, it can be seen that a high biomarker recognition rate can be obtained from the silicon nanoparticle phosphor of the present invention.

Claims (5)

  1. 半導体基板上に成膜されたシリコンナノ粒子蛍光体含有酸化ケイ素膜において、該シリコンナノ粒子蛍光体含有酸化ケイ素膜の膜厚（μｍ）ｄと屈折率ｎの積ｎｄが３以上１０以下であることを特徴とするシリコンナノ粒子蛍光体含有酸化ケイ素膜。 In a silicon nanoparticle phosphor-containing silicon oxide film formed on a semiconductor substrate, the product nd of the thickness (μm) d of the silicon nanoparticle phosphor-containing silicon oxide film and the refractive index n is 3 or more and 10 or less. The silicon nanoparticle fluorescent substance containing silicon oxide film characterized by the above-mentioned.
  2. 請求の範囲第１項に記載のシリコンナノ粒子蛍光体含有酸化ケイ素膜をフッ酸処理して得られることを特徴とするシリコンナノ粒子蛍光体。 A silicon nanoparticle phosphor obtained by treating the silicon nanoparticle phosphor-containing silicon oxide film according to claim 1 with hydrofluoric acid.
  3. 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２項に記載のシリコンナノ粒子蛍光体。 3. The silicon nanoparticle phosphor according to claim 2, wherein the average particle diameter is 1 nm or more and 10 nm or less.
  4. 請求の範囲第２項または第３項に記載のシリコンナノ粒子蛍光体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当該分子の同定を行うことを特徴とする単一分子観察方法。 A single molecule characterized by irradiating the molecule labeled with the silicon nanoparticle phosphor according to claim 2 or 3 with excitation light and detecting the light emission to identify the molecule. Observation method.
  5. 請求の範囲第４項に記載の単一分子観察方法において、異なる発光スペクトルを持つシリコンナノ粒子蛍光体で複数種類の分子をそれぞれ標識し、当該分子に励起光を照射することによって、同時に複数種類の物質の同定を行うことを特徴とする単一分子観察方法。 The single molecule observation method according to claim 4, wherein a plurality of types of molecules are labeled with silicon nanoparticle phosphors having different emission spectra, and the molecules are irradiated with excitation light at the same time. A single molecule observation method characterized by performing identification of a substance.
PCT/JP2009/053003 2008-03-14 2009-02-20 Silicon oxide film containing silicon nanoparticle phosphor, silicon nanoparticle phosphor, and single molecule observation method WO2009113375A1 (en)

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