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WO2010004774A1 - Suspension de nanoparticules de silicium et agent de marquage pour une substance biologique - Google Patents

Suspension de nanoparticules de silicium et agent de marquage pour une substance biologique Download PDF

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Publication number
WO2010004774A1
WO2010004774A1 PCT/JP2009/052764 JP2009052764W WO2010004774A1 WO 2010004774 A1 WO2010004774 A1 WO 2010004774A1 JP 2009052764 W JP2009052764 W JP 2009052764W WO 2010004774 A1 WO2010004774 A1 WO 2010004774A1
Authority
WO
WIPO (PCT)
Prior art keywords
silicon
nanoparticle suspension
silicon nanoparticle
oxide film
temperature
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2009/052764
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English (en)
Japanese (ja)
Inventor
優 高橋
久美子 西川
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Konica Minolta Medical and Graphic Inc
Original Assignee
Konica Minolta Medical and Graphic Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Konica Minolta Medical and Graphic Inc filed Critical Konica Minolta Medical and Graphic Inc
Publication of WO2010004774A1 publication Critical patent/WO2010004774A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • 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
    • 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
    • C01B33/02—Silicon

Definitions

  • the present invention relates to a silicon nanoparticle suspension and a biological material labeling agent that is safe for a living body using the silicon nanoparticle suspension.
  • the silicon nanoparticle phosphor is manufactured by performing high-frequency sputtering, and then performing heat treatment and HF treatment (see, for example, Patent Documents 1 and 2).
  • a method of immersing in a solution is employed.
  • quantum dots compound semiconductors such as III-IV compound semiconductors and II-IV compound semiconductors are widely known.
  • these compounds have a great impact on the environment, and it is necessary to give sufficient consideration to handling and disposal during use.
  • silicon quantum dots are used that are superior in safety and have less environmental impact than these compound semiconductors.
  • the fluoride adsorbed physically on the surface can be sufficiently removed, but the fluorine that has penetrated into the film.
  • the chemicals are not removed. Therefore, after that, the silicon nanoparticle is taken out as a suspension, and a dangerous level of fluoride is left to be used for biolabeling. In other words, it is extremely dangerous for a living body to use a silicon nanoparticle suspension synthesized by a high-frequency sputtering method as a fluorescent substance for biomarking.
  • JP 2006-70089 A Japanese Patent Application Laid-Open No. 2004-296781 JP 2005-172429 A
  • an object of the present invention is to provide a silicon nanoparticle suspension that can be applied in vivo by removing the fluoride and has excellent emission luminance, and further uses the silicon nanoparticle suspension.
  • a biological material labeling agent that is safe for the living body is provided.
  • a process of producing a silicon atom-containing silicon oxide film on a substrate subjecting the silicon atom-containing silicon oxide film to heat treatment (annealing), hydrofluoric acid treatment, removing fluorine ions from the hydrofluoric acid, and separating the substrate
  • the silicon nanoparticle suspension manufactured in (1) wherein the total content of fluoride in the silicon nanoparticle suspension based on fluorine ions is 100 ppm or less.
  • a biological material labeling agent wherein the silicon nanoparticle suspension according to any one of 1 to 7 and a molecular labeling material are bonded via an organic molecule.
  • the present invention it is possible to provide a silicon nanoparticle suspension that can be applied in vivo by removing the fluoride and has excellent emission luminance, and further to the living body using the silicon nanoparticle suspension. It was possible to provide a safe biological material labeling agent.
  • the silicon nanoparticle suspension of the present invention is characterized in that the total content of fluoride in the silicon nanoparticle suspension based on fluorine ions is 100 ppm or less.
  • the silicon nanoparticle suspension of the present invention is generally obtained through the following steps.
  • a silicon atom-containing silicon oxide film is formed on a semiconductor substrate (for example, a silicon substrate) using a high-frequency sputtering method, (B) heat-treating (annealing) the silicon atom-containing silicon oxide film to form silicon nanoparticles in the silicon oxide film; (C) The silicon oxide film is treated with hydrofluoric acid to remove the silicon oxide to expose the silicon nanoparticles, (D) The exposed silicon nanoparticles are immersed in pure water and subjected to a stirring process to remove adhering hydrofluoric acid and separate the semiconductor substrate to obtain a suspension of silicon nanoparticles.
  • high-temperature and high-pressure treatment of pure water is preferably performed in place of the immersion and stirring treatment in pure water in (d) above.
  • an autoclave it is preferable to use an autoclave.
  • the temperature is preferably 100 to 300 ° C, more preferably 200 to 250 ° C.
  • the pressure is preferably from 0.1 to 10 MPa, more preferably from 1 to 5 MPa.
  • this treatment is not limited to the inside of the autoclave as long as it can be immersed in high-temperature and high-pressure water.
  • High-temperature, high-pressure water includes so-called subcritical and supercritical water, regardless of gas or liquid state.
  • the temperature and time of the heat treatment (annealing) in the above (b) is preferably 1000 to 1150 ° C. for 45 to 90 minutes, more preferably 1000 to 1100 ° C. for 50 to 80 minutes.
  • hydrofluoric acid treatment in the above (c) a hydrofluoric acid aqueous solution or hydrofluoric acid vapor is used, but hydrofluoric acid vapor is preferred, and the temperature and time of the hydrofluoric acid vapor to be exposed are preferably 3 to 30 at 30 to 60 ° C. Minutes, more preferably at 35 to 50 ° C. for 5 to 15 minutes.
  • the formation of the silicon atom-containing silicon oxide film is performed by, for example, a high-speed sputtering method (for example, Japanese Patent Application Laid-Open No. 2004-296781).
  • forming a silicon atom-containing silicon oxide film on a semiconductor substrate is generated by (1) evaporating the opposing raw material semiconductor by the first high-temperature plasma generated between the electrodes and generating an electrodeless discharge in a reduced-pressure atmosphere. It is also possible to pass through the second high temperature plasma (for example, Japanese Patent Laid-Open No. 6-279015) and (2) laser ablation method (for example, Japanese Patent Laid-Open No. 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 is formed on a quartz glass as a predetermined material).
  • the atoms and molecules emitted from the target material are deposited on the semiconductor substrate to form a silicon atom-containing silicon oxide (SiO x ) film.
  • Examples of the target material in the high-speed sputtering method include silicon chip and quartz glass, and silicon nanoparticles having various particle sizes can be obtained by controlling the area ratio of the silicon chip and quartz glass.
  • 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 average particle size of the silicon nanoparticles according to the present invention is preferably 1 nm or more and 10 nm or less. More preferably, they are 3 nm or more and 8 nm or less, Especially preferably, they are 3.5 nm or more and 6 nm or less.
  • the content of fluoride in the silicon nanoparticle suspension based on fluorine ions is 100 ppm or less, preferably 50 ppm or less, More preferably, it is 15 ppm or less.
  • the content of the fluoride is 100 ppm or less, the biological safety of the biological material labeling agent prepared from the silicon nanoparticle suspension is improved, and the emission luminance of the silicon nanoparticle suspension itself is also improved.
  • Fluoride content of the silicon nanoparticle suspension is measured using a fluorine ion meter (manufactured by Toko Chemical Co., Ti-5101).
  • the silicon nanoparticle suspension of the present invention can be applied to a biological material labeling agent. Further, by adding the silicon nanoparticle labeling agent (biological material labeling agent) of the present invention to a living cell or living body having a target (tracking) substance, the target substance binds or adsorbs to the conjugate or adsorbent. By irradiating excitation light of a predetermined wavelength 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 of the present invention can be used for bioimaging methods (technical means for visualizing biomolecules constituting the biomaterial and dynamic phenomena thereof).
  • the surface of the above-mentioned silicon nanoparticles is generally hydrophobic, for example, when used as a biological material labeling agent, the water dispersibility is poor as it is, and there is a problem that particles aggregate, It is preferable to hydrophilize the surface of the silicon nanoparticles.
  • 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.
  • the surface modifier those having a carboxyl group and 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 Si / SiO 2 type nanoparticles are dispersed in 10 ml of pure water in which 0.2 g of mercaptoundecanoic acid is dissolved, and stirred at 40 ° C. for 10 minutes to treat the surface of the shell. By doing so, the surface of the shell of the inorganic nanoparticles is modified with a carboxyl group.
  • the biosubstance labeling agent is obtained in a structure in which at least the above-described hydrophilized silicon nanoparticles and the molecular labeling substance that binds to the biomolecule at the end are bound.
  • An organic molecule having a functional group that becomes a linking group and binds to the molecular labeling substance may be interposed between the hydrophilic silicon nanoparticles and the molecular labeling substance.
  • the biological material labeling agent can label the biological material by specifically binding and / or reacting with the target biological material.
  • the molecular labeling substance include nucleotide chains, antibodies, proteins, and cyclodextrins.
  • silicon nanoparticles subjected to hydrophilization treatment and a 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 silicon nanoparticles and a molecular labeling substance.
  • albumin, myoglobin, casein, and the like are part of the protein, and are a kind of protein. It is also preferable to use avidin together with biotin.
  • a binding functional group for example, an amino group or a carboxyl group may have a mercapto group or a maleimide group at the terminal.
  • the link group is not particularly limited as long as it is organic, but preferably has hydrophilicity, may have a hydrophilic functional group, and an ethylene glycol chain is also preferably used.
  • the form of bonding is not particularly limited, and examples thereof include covalent bonding, ionic bonding, hydrogen bonding, coordination bonding, 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.
  • silicon nanoparticles are hydrophilized with mercaptoundecanoic acid and biotin is used as an organic molecule.
  • the carboxyl groups of the hydrophilized silicon nanoparticles are preferably covalently bonded to biotin, Is further selectively bound to a molecular labeling substance (such as an antibody) to which avidin is bound to obtain a biological substance labeling agent.
  • Example 1 Fluorescence 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 emitted atoms and molecules are deposited on a semiconductor substrate to form a silicon oxide film in which silicon atoms are mixed in the silicon oxide film.
  • the obtained silicon atom-containing silicon oxide film containing silicon atoms is rapidly heated to 1000 ° C. in an Ar atmosphere and subjected to heat treatment to agglomerate the silicon atoms in the film to nano size to oxidize silicon nanoparticles.
  • a silicon film is formed.
  • the heat treatment time is 60 minutes, and the temperature is 1000 ° C.
  • Example 2-7 cleaning treatment was performed in the same manner except that the temperature and pressure described in Table 1 were used.
  • Example 8 Residual fluorine ions were removed by immersing the silicon oxide film containing silicon nanoparticles after hydrofluoric acid treatment in pure water at 25 ° C. for 10 minutes.
  • a silicon nanoparticle suspension can be obtained by introducing a silicon oxide film containing naturally oxidized silicon nanoparticles into a solution and performing ultrasonic treatment for 10 minutes.
  • luminance of the silicon nanoparticle suspension shown in Table 1 is the brightness
  • a 146 nm vacuum ultraviolet lamp manufactured by Ushio was used as a light source, a sample was set in a vacuum chamber, irradiated from a fixed distance at a vacuum degree of 1.33 ⁇ 10 Pa, and excitation luminescence was measured with a luminance meter.
  • the luminance value is shown as a relative value when the sample of Example 8 is 100.
  • Silicon nanoparticle suspension prepared in Example 1-8, to 10ml of pure water were dissolved mercaptoundecanoic acid 0.2 g, redispersed 1 ⁇ 10 -5 g min (equivalent amount), 40 ° C., By stirring for 10 minutes, nanoparticles having a hydrophilic surface were obtained.
  • the obtained avidin-conjugated nanoparticle solution was mixed with a biotinylated oligonucleotide with a known base sequence and stirred to prepare an oligonucleotide labeled with the nanoparticle.
  • the biosafety of the silicon nanoparticle labeling agent is evaluated using trypan blue staining.
  • a 0.3% trypan blue solution is diluted with PBS ( ⁇ ) and placed in a test board together with the silicon nanoparticle labeling agent and HERA cells. By counting the number of cells stained blue, the ratio of viable cells after trypan blue staining is calculated. The higher the percentage of living cells, the higher the safety.
  • the silicon nanoparticle suspension having the structure of the present invention As shown in Table 1, by obtaining the silicon nanoparticle suspension having the structure of the present invention, it is excellent in luminance and high detectability is obtained as a biological material labeling agent. Moreover, since the proportion of living cells is high in the biological safety test, an excellent biological substance labeling agent can be provided.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Nanotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Inorganic Chemistry (AREA)
  • Microbiology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Silicon Compounds (AREA)

Abstract

L'invention porte sur une suspension de nanoparticules de silicium qui est applicable in vivo en raison du fait que tout composé fluoré en est éliminé, et qui a une excellente brillance. L'invention porte également sur un agent de marquage pour une substance biologique, qui est sans danger pour les corps vivants et peut être obtenu à l'aide de la suspension de nanoparticules de silicium. La suspension de nanoparticules de silicium est obtenue par les étapes consistant à : produire un film d'oxyde de silicium contenant un atome de silicium sur une plaque de base ; soumettre le film d'oxyde de silicium à un traitement de chauffage (recuit) ; soumettre le film d'oxyde de silicium résultant au traitement par de l'acide fluorhydrique ; et éliminer les ions fluor de l'acide fluorhydrique et séparer la plaque de base, la teneur totale en composés fluorés dans la suspension de nanoparticules de silicium étant de 100 ppm ou moins en termes d'une teneur en ions fluor.
PCT/JP2009/052764 2008-07-07 2009-02-18 Suspension de nanoparticules de silicium et agent de marquage pour une substance biologique Ceased WO2010004774A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2008-176749 2008-07-07
JP2008176749 2008-07-07

Publications (1)

Publication Number Publication Date
WO2010004774A1 true WO2010004774A1 (fr) 2010-01-14

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006070089A (ja) * 2004-08-31 2006-03-16 Tokyo Denki Univ 環境保全性ナノシリコン溶液及びナノシリコンパウダーとそれらの製造方法
JP2008013432A (ja) * 2006-07-05 2008-01-24 Wacker Chemie Ag ポリシリコン破砕物を清浄化する方法
JP2008019114A (ja) * 2006-07-11 2008-01-31 Catalysts & Chem Ind Co Ltd シリコン微粒子の製造方法
WO2008032599A1 (fr) * 2006-09-14 2008-03-20 Konica Minolta Medical & Graphic, Inc. agrégat de nanoparticules semi-conductrices, processus de fabrication de l'agrégat de nanoparticules semi-conductrices, et agent de marquage de substance biologique utilisant l'agrégat de nanoparticules semi-conductrices
WO2009051016A1 (fr) * 2007-10-17 2009-04-23 Konica Minolta Medical & Graphic, Inc. Points quantiques en silicium et agent de marquage biologique les utilisant

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006070089A (ja) * 2004-08-31 2006-03-16 Tokyo Denki Univ 環境保全性ナノシリコン溶液及びナノシリコンパウダーとそれらの製造方法
JP2008013432A (ja) * 2006-07-05 2008-01-24 Wacker Chemie Ag ポリシリコン破砕物を清浄化する方法
JP2008019114A (ja) * 2006-07-11 2008-01-31 Catalysts & Chem Ind Co Ltd シリコン微粒子の製造方法
WO2008032599A1 (fr) * 2006-09-14 2008-03-20 Konica Minolta Medical & Graphic, Inc. agrégat de nanoparticules semi-conductrices, processus de fabrication de l'agrégat de nanoparticules semi-conductrices, et agent de marquage de substance biologique utilisant l'agrégat de nanoparticules semi-conductrices
WO2009051016A1 (fr) * 2007-10-17 2009-04-23 Konica Minolta Medical & Graphic, Inc. Points quantiques en silicium et agent de marquage biologique les utilisant

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