JP2007137700A - Method for manufacturing fluorescent silicon particle, fluorescent silicon particle and method for observing biological substance by using the particle - Google Patents
Method for manufacturing fluorescent silicon particle, fluorescent silicon particle and method for observing biological substance by using the particle Download PDFInfo
- Publication number
- JP2007137700A JP2007137700A JP2005331172A JP2005331172A JP2007137700A JP 2007137700 A JP2007137700 A JP 2007137700A JP 2005331172 A JP2005331172 A JP 2005331172A JP 2005331172 A JP2005331172 A JP 2005331172A JP 2007137700 A JP2007137700 A JP 2007137700A
- Authority
- JP
- Japan
- Prior art keywords
- silicon
- fluorescent
- mixed solution
- hno
- silicon particles
- 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.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Silicon Compounds (AREA)
Abstract
Description
本発明は、蛍光性シリコン粒子の製造方法、蛍光性シリコン粒子およびそれを用いて生体物質を観察する方法に関する。 The present invention relates to a method for producing fluorescent silicon particles, a fluorescent silicon particle, and a method for observing a biological material using the same.
ナノメートルの径をもつシリコン粒子は、蛍光性／発光性をもつことが知られており、蛍光体として使用する場合、従来の有機蛍光色素と比較して、著しく強固で光分解する確率が低くブリーチングを生じないという特性を持っている。このような蛍光性シリコン粒子は、電気化学的手法により製造できることが知られている（特許文献１）。 Silicon particles with a nanometer diameter are known to have fluorescence / luminescence properties, and when used as phosphors, they are significantly stronger and less prone to photolysis than conventional organic fluorescent dyes. It has the characteristic of not causing bleaching. It is known that such fluorescent silicon particles can be produced by an electrochemical method (Patent Document 1).
しかしながら、特許文献１の方法は操作が複雑であり一般的ではない。そこで、本発明の目的は、より簡便な方法で蛍光性シリコン粒子を製造することにある。また、製造された蛍光性シリコン粒子を用いて、生物標本中の微量な生体物質を観察する方法を提供することにある。 However, the method of Patent Document 1 is not common because the operation is complicated. Accordingly, an object of the present invention is to produce fluorescent silicon particles by a simpler method. Another object of the present invention is to provide a method for observing a trace amount of biological material in a biological specimen using the produced fluorescent silicon particles.
上記課題を解決すべく請求項１に記載の発明によれば、シリコン基体にHFとHNO3の混合溶液を作用させる工程と、前記シリコン基体にHFとHNO3とH2Oとの混合溶液を作用させる工程とを含むことを特徴とする蛍光性シリコン粒子の製造方法が提供される。 In order to solve the above problems, according to the first aspect of the present invention, there is provided a step of allowing a mixed solution of HF and HNO 3 to act on a silicon substrate, and a mixed solution of HF, HNO 3 and H 2 O on the silicon substrate. And a step of allowing the fluorescent silicon particles to be produced.
また、請求項２に記載の発明によれば、シリコン基体をHFとHNO3の混合溶液の蒸気にさらす工程と、前記シリコン基体をHFとHNO3とH2Oとの混合溶液に浸す工程とを含むことを特徴とする蛍光性シリコン粒子の製造方法が提供される。 Further, according to the invention described in claim 2, comprising the steps of immersing the silicon substrate and exposing the vapor of the mixed solution of HF and HNO 3, the silicon substrate in a mixed solution of HF and HNO 3 and H 2 O The manufacturing method of the fluorescent silicon particle characterized by including is provided.
また、請求項３に記載の発明によれば、請求項１または２において、前記シリコン基体にシランカップリング剤を作用させる工程を含むことを特徴とする蛍光性シリコン粒子の製造方法が提供される。 According to a third aspect of the present invention, there is provided a method for producing fluorescent silicon particles according to the first or second aspect, further comprising a step of causing a silane coupling agent to act on the silicon substrate. .
また、請求項４に記載の発明によれば、請求項１〜３のいずれか一項において、
前記シリコン基体からポーラスシリコンを分離する工程を含むことを特徴とする蛍光性シリコン粒子の製造方法が提供される。
Moreover, according to the invention of Claim 4, in any one of Claims 1-3,
There is provided a method for producing fluorescent silicon particles, comprising a step of separating porous silicon from the silicon substrate.
また、請求項５に記載の発明によれば、請求項４において、前記シリコン基体から分離された蛍光性シリコン粒子を液体クロマトグラフィーにより精製する工程を含むことを特徴とする蛍光性シリコン粒子の製造方法が提供される。 According to the invention described in claim 5, the manufacturing of the fluorescent silicon particles according to claim 4, further comprising a step of purifying the fluorescent silicon particles separated from the silicon substrate by liquid chromatography. A method is provided.
また、請求項６に記載の発明によれば、請求項４または５に記載の蛍光性シリコン粒子の製造方法で製造された蛍光性シリコン粒子が提供される。 According to the invention described in claim 6, the fluorescent silicon particles manufactured by the method for manufacturing fluorescent silicon particles according to claim 4 or 5 are provided.
また、請求項７に記載の発明によれば、請求項６の蛍光性シリコン粒子を用いて生体細胞内もしくは細胞膜上の分子の運動を観察する方法が提供される。 According to the seventh aspect of the present invention, there is provided a method for observing the movement of molecules in a living cell or on a cell membrane using the fluorescent silicon particles of the sixth aspect.
１．ポーラスシリコンの製造
＜ステップ１＞ まず、シリコンウエハ等のシリコン基体を用意し、適度な大きさ（例えば、約5cm×5cm）にカットする。次に、図１に示すように、容器２０中で、カットしたシリコン基体１０の端１５を２〜３ｍｍ程、濃フッ酸（HF）と濃硝酸（HNO3）の混合溶液３０に浸ける。
1. Production of porous silicon <Step 1> First, a silicon substrate such as a silicon wafer is prepared and cut into an appropriate size (for example, about 5 cm × 5 cm). Next, as shown in FIG. 1, the
ここで用いる混合溶液中のHFとHNO3の混合比は、体積比において、２：１〜１：２の範囲である。混合溶液中のHNO3の割合が少なすぎると、十分にエッチングができず、多すぎると反応速度が速すぎて均一なエッチングができない。 The mixing ratio of HF and HNO 3 in the mixed solution used here is in the range of 2: 1 to 1: 2 in volume ratio. If the proportion of HNO 3 in the mixed solution is too small, the etching cannot be performed sufficiently, and if it is too large, the reaction rate is too high to perform uniform etching.
また、この段階で、シリコン基体を完全に混合溶液に浸すと、均一にポーラスシリコンが形成されないので、上記の如く、シリコン基体の一部を浸すのが好ましい。 Further, at this stage, if the silicon substrate is completely immersed in the mixed solution, porous silicon is not formed uniformly. Therefore, it is preferable to immerse a part of the silicon substrate as described above.
シリコン基体の端を混合溶液に浸す時間は、０．５〜５秒の範囲である。浸す時間が短いと十分なポーラスシリコンが得られない。一方、浸す時間が長すぎると一旦形成されたポーラスシリコンが溶出してしまう。この処理は、室温で行うことができる。 The time for immersing the edge of the silicon substrate in the mixed solution is in the range of 0.5 to 5 seconds. If the immersion time is short, sufficient porous silicon cannot be obtained. On the other hand, if the immersion time is too long, the porous silicon once formed is eluted. This treatment can be performed at room temperature.
また、HFとHNO3の混合溶液は、蒸気を発生するので、シリコン基体の端を混合溶液に浸すことなく、シリコン基体全体（ポーラスシリコンを形成しようとする面の全体）に蒸気を作用させるようにしてもよい。例えば、図２に示すように、混合溶液３０を入れたシャーレ２５の直ぐ上に、シリコン基体１０を配置させて、０．５〜５秒保持するようにしてもよい。
Moreover, since the mixed solution of HF and HNO 3 generates vapor, the vapor is allowed to act on the entire silicon substrate (the entire surface on which porous silicon is formed) without immersing the edge of the silicon substrate in the mixed solution. It may be. For example, as shown in FIG. 2, the
＜ステップ２＞ 次に、上記ステップ１で処理したシリコン基体を、ＨＦとＨＮＯ３と水の混合溶液に浸す。ここで用いる混合溶液中のＨＦとＨＮＯ３との混合比は、体積比において、ＨＦ：ＨＮＯ３＝１：１〜１：５である。またＨＦと水との混合比は、体積比において、１：３〜１：５である。後述の実施例で説明するように、混合溶液中の組成比を調整することにより、様々な蛍光波長を示すポーラスシリコンを製造することができる。 <Step 2> Next, the silicon substrate treated in Step 1 is immersed in a mixed solution of HF, HNO 3 and water. The mixing ratio of HF and HNO 3 in the mixed solution used here is HF: HNO 3 = 1: 1 to 1: 5 in the volume ratio. The mixing ratio of HF and water is 1: 3 to 1: 5 in the volume ratio. As will be described in Examples described later, porous silicon having various fluorescence wavelengths can be produced by adjusting the composition ratio in the mixed solution.
混合溶液に浸す時間は、目的とするポーラスシリコンや、用いる混合溶液の組成により適宜選択できるが、通常２〜１０分の範囲である。また、この処理は、室温で行うことができる。 The time for dipping in the mixed solution can be appropriately selected depending on the intended porous silicon and the composition of the mixed solution to be used, but is usually in the range of 2 to 10 minutes. This treatment can be performed at room temperature.
なお、組成比を変えた複数の混合溶液を用いて、シリコン基体を混合溶液に浸す処理を、複数段階で行ってもよい。 In addition, you may perform the process which immerses a silicon base | substrate in a mixed solution using the several mixed solution which changed composition ratio in a several step.
＜ステップ３＞ その後、シリコン基体をエタノール等の洗浄液で洗い、乾燥させる。本方法によって作成したポーラスシリコンの蛍光スペクトルは、蛍光分光光度計によって測定することができる。 <Step 3> Thereafter, the silicon substrate is washed with a cleaning solution such as ethanol and dried. The fluorescence spectrum of porous silicon prepared by this method can be measured with a fluorescence spectrophotometer.
２．ポーラスシリコンのシラン化
上記で得られたポーラスシリコンの表面のシラン化は、シランカップリング剤を作用させることにより行うことができる。シラン化によって、ポーラスシリコンの表面にシランカップリング剤の有する官能基を導入することができる。シランカップリング剤の有する官能基としては、アミノ基、メルカプト基、ハロゲン基、ビニル基、グリシジル基、メタクリロキシ基、カルボキシル基、カルボニル基、などがあり、中でもアミノ基、メルカプト基が好ましい。例えば、シリコン基体をメタノール等で洗浄し、３−アミノプロピルジメチルエトキシシラン（3-aminopropyldimethylethoxysilane）、３−メルカプトプロピルトリメトキシシラン（3-mercaptopropyltrimethoxysilane）等のシランカップラング剤の酸性メタノール溶液に浸す。
2. Silanization of porous silicon Silanization of the surface of the porous silicon obtained above can be performed by acting a silane coupling agent. By silanization, the functional group of the silane coupling agent can be introduced on the surface of the porous silicon. Examples of the functional group possessed by the silane coupling agent include an amino group, a mercapto group, a halogen group, a vinyl group, a glycidyl group, a methacryloxy group, a carboxyl group, and a carbonyl group. Of these, an amino group and a mercapto group are preferable. For example, the silicon substrate is washed with methanol or the like and immersed in an acidic methanol solution of a silane coupling agent such as 3-aminopropyldimethylethoxysilane or 3-mercaptopropyltrimethoxysilane.
３．シリコン粒子の分離、精製
シラン化したシリコン基体の表面からシリコン粒子を分離する方法としては、カッターナイフのような鋭敏なエッジを持つ装置で、シリコン基体を擦り、ポーラスシリコンをそぎ落とす方法や、超音波洗浄装置によって、シリコン基体からシリコン粒子を分離する方法等を用いることができる。
3. Separation and purification of silicon particles Silicon particles can be separated from the surface of a silanized silicon substrate by using a device with a sharp edge such as a cutter knife to scrape the silicon substrate and scrape the porous silicon. A method of separating silicon particles from a silicon substrate using a sonic cleaning device can be used.
シリコン粒子の精製は、例えば、緩衝液(pH6.0)に混入させた後、遠心分離機で分離し、上澄みを分取し、分取した上澄みをＨＰＬＣ（高速液体クロマトグラフィー）で分離することにより行うことができる。 For purification of silicon particles, for example, after mixing in a buffer solution (pH 6.0), separating with a centrifuge, separating the supernatant, and separating the separated supernatant by HPLC (High Performance Liquid Chromatography). Can be performed.
４．シリコン粒子への生体材料の結合
シラン化されたシリコン粒子への生体分子の結合は、公知の方法により実現することができる。例えば、シラン化されたシリコン粒子の有する官能基と生体分子の官能基とを、必要に応じて他の化合物の存在下または他の化合物を介して結合させる。より具体的には、アミノ基を持つシリコン粒子をカルボキシル基を持つ蛋白質を結合剤 １−エチル−３−（３−ジメチルアミノプロピル）カルボジイミドＨＣｌ（1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, hydrochloride）を用いて結合させる。
4). Bonding of biomaterials to silicon particles The binding of biomolecules to silanized silicon particles can be achieved by known methods. For example, the functional group of the silanized silicon particle and the functional group of the biomolecule are bonded in the presence of another compound or via another compound as necessary. More specifically, a silicon particle having an amino group is bound to a protein having a carboxyl group as a binder 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide HCl (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide , hydrochloride).
以下に本発明を実施例により詳細に説明する。ただし、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to these examples.
＜実施例１−１＞ 605nmの蛍光波長をもつポーラスシリコンの製造
シリコンウエハ（面方位（１００）、p-type、ホウ素ドープ、比抵抗１〜１０Ω・cm)を、約5cm×5cmの大きさにカットした。図１に示したように、カットしたシリコンウエハの端を２〜３mm程HFとHNO3の混合溶液（体積比１：１）に２秒間浸けた。次に、シリコンウエハの全体を、HFとHNO3とH2Oの混合溶液（体積比２：７：８)に3分間浸けた。その後エタノールで洗い、乾燥させた。図３に、本方法によって作成したポーラスシリコンを蛍光分光光度計によって275nm励起で取得した蛍光スペクトルを示す。
<Example 1-1> Production of porous silicon having a fluorescence wavelength of 605 nm A silicon wafer (plane orientation (100), p-type, boron-doped, specific resistance 1 to 10 Ω · cm) is about 5 cm × 5 cm in size. Cut into. As shown in FIG. 1, the edge of the cut silicon wafer was immersed in a mixed solution of HF and HNO 3 (volume ratio 1: 1) for about 2 to 2 mm. Next, the entire silicon wafer was immersed in a mixed solution of HF, HNO 3 and H 2 O (volume ratio 2: 7: 8) for 3 minutes. Thereafter, it was washed with ethanol and dried. FIG. 3 shows a fluorescence spectrum obtained by exciting porous silicon prepared by the present method with a fluorescence spectrophotometer at 275 nm excitation.
＜実施例１−２＞ 650nmの蛍光波長をもつポーラスシリコンの製造
シリコンウエハ（面方位（１００）、p-type、ホウ素ドープ、比抵抗１〜１０Ω・cm)を、約5cm×5cmの大きさにカットした。図１に示したように、カットしたシリコンウエハの端を２〜３mm程度HFとHNO3の混合溶液（体積比１：１）に2秒間浸けた。次に、シリコンウエハの全体を、HFとHNO3とH2O溶液(体積比２：７：８)に3分間浸けた。さらに、シリコンウエハの全体を、HFとHNO3とH2Oの混合溶液(体積比２：５：８)に3分間浸けた。その後エタノールで洗い、乾燥させた。図４に、本方法によって作成したポーラスシリコンを蛍光分光光度計によって275nm励起で取得した蛍光スペクトルを示す。
<Example 1-2> Production of porous silicon having a fluorescence wavelength of 650 nm A silicon wafer (plane orientation (100), p-type, boron doped, specific resistance 1 to 10 Ω · cm) is about 5 cm × 5 cm in size Cut into. As shown in FIG. 1, the edge of the cut silicon wafer was immersed in a mixed solution of HF and HNO 3 (volume ratio 1: 1) for about 2 to 3 mm for 2 seconds. Next, the entire silicon wafer was immersed in a HF, HNO 3, and H 2 O solution (volume ratio 2: 7: 8) for 3 minutes. Further, the entire silicon wafer was immersed in a mixed solution of HF, HNO 3 and H 2 O (volume ratio 2: 5: 8) for 3 minutes. Thereafter, it was washed with ethanol and dried. FIG. 4 shows a fluorescence spectrum obtained by exciting porous silicon prepared by the present method with a fluorescence spectrophotometer at 275 nm excitation.
＜実施例１−３＞ 425nmの蛍光波長をもつポーラスシリコンの製造
シリコンウエハ（面方位（１００）、p-type、ホウ素ドープ、比抵抗１〜１０Ω・cm)を、約5cm×5cmの大きさにカットした。カットしたシリコンウエハの端を２〜３mm程度HFとHNO3の混合溶液（体積比１：１）に2秒間浸けた。次に、シリコンウエハの全体を、HFとHNO3とH2Oの混合溶液(体積比２：５：８)に７分間浸けた。その後エタノールで洗い、乾燥させた。図５に、本方法によって作成したポーラスシリコンを蛍光分光光度計によって275nm励起で取得した蛍光スペクトルを示す。
<Example 1-3> Production of porous silicon having a fluorescence wavelength of 425 nm A silicon wafer (plane orientation (100), p-type, boron-doped, specific resistance 1 to 10 Ω · cm) is about 5 cm × 5 cm in size. Cut into. The edge of the cut silicon wafer was immersed in a mixed solution of HF and HNO 3 (volume ratio 1: 1) for about 2 to 3 mm for 2 seconds. Next, the entire silicon wafer was immersed in a mixed solution of HF, HNO 3 and H 2 O (volume ratio 2: 5: 8) for 7 minutes. Thereafter, it was washed with ethanol and dried. FIG. 5 shows a fluorescence spectrum obtained by exciting porous silicon prepared by the present method with a fluorescence spectrophotometer at 275 nm excitation.
＜実施例２−１＞ 3-aminopropyldimethylethoxysilaneを用いたシラン化
実施例１−１で得られたシリコンウエハをメタノールで洗浄した。そして、体積比0.8%の3-aminopropyldimethylethoxysilane酸性メタノール液(500m1のメタノールに4mlの氷酢酸を混入したもの)10mlに1時間浸けた。さらに12.5mlの純水(18Ωcm以上)を加え、さらに1時間浸けた。シリコンウエハを取り出して十分にメタノールで洗った後、真空中で24時間乾燥させた。
<Example 2-1> Silanization using 3-aminopropyldimethylethoxysilane The silicon wafer obtained in Example 1-1 was washed with methanol. Then, it was immersed for 1 hour in 10 ml of 3-aminopropyldimethylethoxysilane acidic methanol solution (500 ml of methanol mixed with 4 ml of glacial acetic acid) with a volume ratio of 0.8%. Further, 12.5 ml of pure water (18 Ωcm or more) was added, and it was further immersed for 1 hour. The silicon wafer was taken out and thoroughly washed with methanol, and then dried in a vacuum for 24 hours.
＜実施例２−２＞ 3-mercaptopropyltrimethoxysilaneを用いたシラン化
実施例１−１で得られたシリコンウエハをメタノールで洗浄した。そして、体積比1.2%の3-mercaptopropyltrimethoxysilane酸性メタノール液(500mlのメタノールに4mlの氷酢酸を混入したもの)10mlに1時間浸けた。さらに12.5mlの純水(18Ωcm以上)を加え、さらに1時間浸けた。シリコンウエハを取り出して十分にメタノールで洗った後、真空中で24時間乾燥させた。
<Example 2-2> Silanation using 3-mercaptopropyltrimethoxysilane The silicon wafer obtained in Example 1-1 was washed with methanol. Then, it was immersed for 1 hour in 10 ml of a 3-mercaptopropyltrimethoxysilane acidic methanol solution having a volume ratio of 1.2% (500 ml of methanol mixed with 4 ml of glacial acetic acid). Further, 12.5 ml of pure water (18 Ωcm or more) was added, and the mixture was further immersed for 1 hour. The silicon wafer was taken out and thoroughly washed with methanol, and then dried in a vacuum for 24 hours.
＜実施例３＞ 分離と精製
実施例２−１でシラン化したシリコンウエハを、カッターナイフのような鋭敏なエッジを持つ装置で擦り、ポーラスシリコンをそぎ落とした。
<Example 3> Separation and purification The silicon wafer silanized in Example 2-1 was rubbed with a device having a sharp edge such as a cutter knife to scrape porous silicon.
得られたシリコンナノ粒子を0.025M 2-morpholinoethanesulfonic acid緩衝液(pH6.0)に混入し、遠心分離機（遠心加速度7000×ｇ）で10分間遠心し、シリコン粒子が含まれている上澄みを分取した。 The obtained silicon nanoparticles are mixed in 0.025M 2-morpholinoethanesulfonic acid buffer (pH 6.0) and centrifuged with a centrifuge (centrifugal acceleration 7000 × g) for 10 minutes to separate the supernatant containing silicon particles. I took it.
分取した上澄みに含まれているシリコン粒子を、ＨＰＬＣ（高速液体クロマトグラフィー）により精製した。使用したカラムはSuperdex75ge1で緩衝液は0.05Mリン酸pH7 0.15M NaCl 流速は0.4ml/minである。図６、７に、溶出したシリコン粒子を示す。図６は、シラン化したあとのシリコン粒子である。図７は、参照用のシラン化する前のシリコン粒子である。各々、右側のピークは約直径3nmの粒子のピークである。左側のピークは対照用の分子であるRNase直径4nmである。 Silicon particles contained in the collected supernatant were purified by HPLC (high performance liquid chromatography). The column used was Superdex75ge1, the buffer was 0.05M phosphate pH7 0.15M NaCl, and the flow rate was 0.4 ml / min. 6 and 7 show the eluted silicon particles. FIG. 6 shows silicon particles after silanization. FIG. 7 shows silicon particles before silanization for reference. In each case, the peak on the right is the peak of a particle having a diameter of about 3 nm. The peak on the left is RNase 4 nm in diameter as a control molecule.
＜実施例４＞ 生体細胞の観察
実施例３で得られたシリコン粒子を、0.6mM 1-ethyl-3-(3-dimethylaminopropy1)-carbodiimide, hydrochlorideに混入させ、15分後に、ターゲットとなる分子を混入させた。この操作によって、シリコン粒子上のアミノ基とタンパク質中のカルボキシル基が縮合し、シリコン粒子とタンパク質の複合体が形成された。
<Example 4> Observation of living cells The silicon particles obtained in Example 3 were mixed in 0.6 mM 1-ethyl-3- (3-dimethylaminopropy1) -carbodiimide, hydrochloride, and after 15 minutes, the target molecule was It was mixed. By this operation, the amino group on the silicon particle and the carboxyl group in the protein were condensed to form a complex of the silicon particle and the protein.
生細胞の表面で、単一シリコン粒子が結合したものを観察する方法は以下のとおりである。 A method for observing the surface of a living cell where single silicon particles are bonded is as follows.
シリコン粒子をレセプターと特異的に結合する分子と共有結合させ、あらかじめ生細胞の培養液(growth mediumもしくはbalanced salt solution)に混入して培養しておく。実施例としては、上述したようなアミノ基を持ったシリコン粒子をトランスフェリン(鉄イオンを細胞内に輸送するタンパク質)に結合させた。トランスフェリンとシリコンナノ粒子の複合体は細胞表面にあるトランスフェリン受容体に結合した。この際細胞はラットの腎臓由来の株細胞であるNRK細胞を使用した。培養にはFCS（ウシ胎児血清、fetal calf serum）10%を含むHamF12を使用した。細胞は最初にハンクス液中で37度で30分培養したあとで、トランスフェリンとシリコン粒子の複合体を混入した。この複合体は、NRK細胞表面のトランフェリン受容体に結合した。 Silicon particles are covalently bound to molecules that specifically bind to the receptor, and are mixed in advance in a culture medium (growth medium or balanced salt solution) of living cells and cultured. As an example, silicon particles having an amino group as described above were bound to transferrin (a protein that transports iron ions into cells). The complex of transferrin and silicon nanoparticles bound to the transferrin receptor on the cell surface. In this case, NRK cells, which are cell lines derived from rat kidney, were used. For the culture, HamF12 containing 10% FCS (fetal calf serum) was used. The cells were first incubated in Hanks' solution at 37 degrees for 30 minutes and then mixed with transferrin and silicon particle complexes. This complex bound to the transferrin receptor on the NRK cell surface.
観察は倒立顕微鏡を用い、全反射照明条件で観察した。開口数1.45油浸対物レンズを用い、落射照明装置の光路より543nmのHeNeレーザを対物レンズ内へ光軸と平行に導入し、対物レンズの後焦点面にレーザの焦点を結び、その焦点の位置をレンズの周辺部近くになるように調整することで、対物内全反射照明状態になるようにした。粒子像の撮影には、高感度CCDカメラもしくはSITカメラにイメージインテンシファイアーを設置したものを使用した。 Observation was performed using an inverted microscope under total reflection illumination conditions. Using a 1.45 oil immersion objective lens, a HeNe laser of 543 nm is introduced into the objective lens parallel to the optical axis from the optical path of the epi-illuminator, and the focal point of the laser is focused on the back focal plane of the objective lens. Was adjusted to be close to the periphery of the lens so that it would be in the state of total internal reflection illumination. A particle image was captured using a high-sensitivity CCD camera or SIT camera with an image intensifier.
その結果、細胞膜上のトランスフェリン受容体の運動の様子を明確に観察することができた。 As a result, the movement of the transferrin receptor on the cell membrane could be clearly observed.
以上、実施例により本願発明を詳細に説明した。 As described above, the present invention has been described in detail by way of examples.
本願発明の蛍光性シリコン粒子の製造方法では、電気化学的手法を用いないので、操作が簡便である。また、近年、生細胞内もしくは細胞表面における単一の生体分子の挙動を観察することによって、分子の機能を解明し、これらの分子の機能の異常が原因となっているさまざまな病気の探求や治療法の開発がなされている。本願発明で製造された蛍光性シリコン粒子は、生物材料の標識として使用することができる。 In the method for producing fluorescent silicon particles of the present invention, since an electrochemical method is not used, the operation is simple. In recent years, by observing the behavior of single biomolecules in living cells or on the cell surface, the functions of the molecules have been elucidated, and various diseases caused by abnormal functions of these molecules have been investigated. Therapies are being developed. The fluorescent silicon particles produced in the present invention can be used as a label for biological materials.
また、上記実施例では、シリコン基体の表面からシリコン粒子を分離した後にシラン化するのではなく、生成したポーラスシリコンをシラン化した後にシリコン粒子を分離している。こうすることで、粒子のそれぞれに対して均一にシラン化を行うことができる。 Further, in the above embodiment, the silicon particles are not separated from the surface of the silicon substrate and then silanized, but the produced porous silicon is silanized and then the silicon particles are separated. By doing so, silanization can be performed uniformly on each of the particles.
なお、上記実施形態および実験例は、本願発明の技術的思想の範囲内で様々な変形が可能である。 The above embodiment and experimental examples can be variously modified within the scope of the technical idea of the present invention.
例えば、上記で説明した方法でポーラスシリコンを生成し、シラン化し、さらに生体分子を結合させた後に、シリコン基体からシリコン粒子を分離するようにしてもよい。こうすれば、均一に生体分子が結合したシリコン粒子を得ることができる。 For example, porous silicon may be generated by the method described above, silanized, and after biomolecules are bonded, the silicon particles may be separated from the silicon substrate. In this way, silicon particles in which biomolecules are uniformly bonded can be obtained.
１０…シリコン基体（シリコンウエハ）
２０…容器、２５…シャーレ
３０…混合溶液
10 ... Silicon substrate (silicon wafer)
20 ... container, 25 ...
Claims (7)
前記シリコン基体にHFとHNO3とH2Oとの混合溶液を作用させる工程と
を含むことを特徴とする蛍光性シリコン粒子の製造方法。 A step of allowing a mixed solution of HF and HNO 3 to act on a silicon substrate;
And a step of allowing a mixed solution of HF, HNO 3 and H 2 O to act on the silicon substrate.
前記シリコン基体をHFとHNO3とH2Oとの混合溶液に浸す工程とを含む
ことを特徴とする蛍光性シリコン粒子の製造方法。 Exposing the silicon substrate to a vapor mixture of HF and HNO 3 ;
And a step of immersing the silicon substrate in a mixed solution of HF, HNO 3 and H 2 O.
前記シリコン基体にシランカップリング剤を作用させる工程を含む
ことを特徴とする蛍光性シリコン粒子の製造方法。 In claim 1 or 2,
A method for producing fluorescent silicon particles, comprising a step of applying a silane coupling agent to the silicon substrate.
前記シリコン基体からポーラスシリコンを分離する工程を含む
ことを特徴とする蛍光性シリコン粒子の製造方法。 In any one of Claims 1-3,
A method for producing fluorescent silicon particles, comprising a step of separating porous silicon from the silicon substrate.
前記シリコン基体から分離された蛍光性シリコン粒子を液体クロマトグラフィーにより精製する工程を含む
ことを特徴とする蛍光性シリコン粒子の製造方法。 In claim 4,
A method for producing fluorescent silicon particles, comprising a step of purifying the fluorescent silicon particles separated from the silicon substrate by liquid chromatography.
A method for observing the movement of a molecule in a living cell or on a cell membrane using the fluorescent silicon particles according to claim 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005331172A JP4721340B2 (en) | 2005-11-16 | 2005-11-16 | Method for producing fluorescent silicon particles, fluorescent silicon particles and method for observing biological material using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005331172A JP4721340B2 (en) | 2005-11-16 | 2005-11-16 | Method for producing fluorescent silicon particles, fluorescent silicon particles and method for observing biological material using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2007137700A true JP2007137700A (en) | 2007-06-07 |
| JP4721340B2 JP4721340B2 (en) | 2011-07-13 |
Family
ID=38201026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2005331172A Active JP4721340B2 (en) | 2005-11-16 | 2005-11-16 | Method for producing fluorescent silicon particles, fluorescent silicon particles and method for observing biological material using the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4721340B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009043903A (en) * | 2007-08-08 | 2009-02-26 | Stanley Electric Co Ltd | Led light source |
| JP2009091168A (en) * | 2007-10-04 | 2009-04-30 | Akihiro Kusumi | Fluorescent nanoparticle and method of observing biological material using the same |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06144822A (en) * | 1992-10-30 | 1994-05-24 | Tonen Chem Corp | Production of highly pure fine silicon particle and production of highly pure polycrystalline silicon using the same particle |
| JPH06268255A (en) * | 1993-03-11 | 1994-09-22 | Rikagaku Kenkyusho | Porous silicon and manufacture thereof |
| JPH09169513A (en) * | 1995-11-22 | 1997-06-30 | Dow Corning Corp | Silicon microparticle |
| JP2002068885A (en) * | 2000-08-28 | 2002-03-08 | Shin Etsu Chem Co Ltd | Silicon component and method of measuring amount of metal impurity on its surface |
| JP2004024953A (en) * | 2002-06-21 | 2004-01-29 | Kiyoshi Yatsui | Fine particle and fine particle production method |
| JP2005170749A (en) * | 2003-12-12 | 2005-06-30 | Morita Kagaku Kogyo Kk | Fine semiconductor particle having ultrafine pores on its surface, its forming method and device using it |
| JP2006071330A (en) * | 2004-08-31 | 2006-03-16 | Tokyo Denki Univ | Nanosilicon fluorescence element for detecting/visual sensing cancerous cells and its manufacturing method |
| WO2007026533A1 (en) * | 2005-08-30 | 2007-03-08 | Tokyo Denki University | Soluble tablet containing nanosilicon particles and process for production thereof |
-
2005
- 2005-11-16 JP JP2005331172A patent/JP4721340B2/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06144822A (en) * | 1992-10-30 | 1994-05-24 | Tonen Chem Corp | Production of highly pure fine silicon particle and production of highly pure polycrystalline silicon using the same particle |
| JPH06268255A (en) * | 1993-03-11 | 1994-09-22 | Rikagaku Kenkyusho | Porous silicon and manufacture thereof |
| JPH09169513A (en) * | 1995-11-22 | 1997-06-30 | Dow Corning Corp | Silicon microparticle |
| JP2002068885A (en) * | 2000-08-28 | 2002-03-08 | Shin Etsu Chem Co Ltd | Silicon component and method of measuring amount of metal impurity on its surface |
| JP2004024953A (en) * | 2002-06-21 | 2004-01-29 | Kiyoshi Yatsui | Fine particle and fine particle production method |
| JP2005170749A (en) * | 2003-12-12 | 2005-06-30 | Morita Kagaku Kogyo Kk | Fine semiconductor particle having ultrafine pores on its surface, its forming method and device using it |
| JP2006071330A (en) * | 2004-08-31 | 2006-03-16 | Tokyo Denki Univ | Nanosilicon fluorescence element for detecting/visual sensing cancerous cells and its manufacturing method |
| WO2007026533A1 (en) * | 2005-08-30 | 2007-03-08 | Tokyo Denki University | Soluble tablet containing nanosilicon particles and process for production thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009043903A (en) * | 2007-08-08 | 2009-02-26 | Stanley Electric Co Ltd | Led light source |
| JP2009091168A (en) * | 2007-10-04 | 2009-04-30 | Akihiro Kusumi | Fluorescent nanoparticle and method of observing biological material using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4721340B2 (en) | 2011-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Diatoms: self assembled silica nanostructures, and templates for bio/chemical sensors and biomimetic membranes | |
| Acres et al. | Mechanisms of aggregation of cysteine functionalized gold nanoparticles | |
| Ivanova‐Mitseva et al. | Cubic molecularly imprinted polymer nanoparticles with a fluorescent core | |
| JP4567627B2 (en) | Nucleic acid separation method and solid substrate for nucleic acid separation | |
| Jantschke et al. | Decoration of diatom biosilica with noble metal and semiconductor nanoparticles (< 10 nm): assembly, characterization, and applications | |
| JP2004150797A (en) | Extraction method for nucleic acid and protein by dendrimer and dendrimer composition | |
| Sweetman et al. | Porous silicon films micropatterned with bioelements as supports for mammalian cells | |
| FR2737574A1 (en) | APPARATUS FOR PARALLEL MACROMOLECULE ALIGNMENT AND USE | |
| Marsico et al. | Enhanced laser desorption/ionization mass spectrometric detection of biomolecules using gold nanoparticles, matrix, and the coffee ring effect | |
| Kasyanenko et al. | DNA modified with metal nanoparticles: preparation and characterization of ordered metal-DNA nanostructures in a solution and on a substrate | |
| JP5100291B2 (en) | Fluorescent nanoparticles and method for observing biological material using the same | |
| Yuan et al. | A fluorimetric study on the interaction between a Trp‐containing beta‐strand peptide and amphiphilic polymer‐coated gold nanoparticles | |
| JP4721340B2 (en) | Method for producing fluorescent silicon particles, fluorescent silicon particles and method for observing biological material using the same | |
| CN104020147A (en) | Preparation method for fluorescent molecular imprinted probe | |
| JP2007275064A (en) | Method for cytoclasis and nucleic acid purification using single chamber and apparatus therefor | |
| WO2016157220A1 (en) | A process for the preparation of bio-organic coated gold and silver nanoparticles using blue light | |
| JP7192783B2 (en) | Aggregated nanoparticles and fluorescent labels | |
| Kim et al. | Stabilization of α-helices by the self-assembly of macrocyclic peptides on the surface of gold nanoparticles for molecular recognition | |
| Lu et al. | Highly uniform SERS-active microchannel on hydrophobic PDMS: a balance of high reproducibility and sensitivity for detection of proteins | |
| Borberg et al. | Depletion of highly abundant protein species from biosamples by the use of a branched silicon nanopillar on-chip platform | |
| CA3001637A1 (en) | Method for producing erythrocyte proteins | |
| Zissi et al. | The Generation and Study of a Gold‐Based Chemobrionic Plant‐Like Structure | |
| JP2010229440A (en) | Water-dispersible metal nanoparticle | |
| Psarouli et al. | Covalent binding vs. adsorption of biomolecules on silicon nitride planar waveguides | |
| WO2020262617A1 (en) | Cell fusion-accelerating agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20081010 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20081201 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20110215 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20110308 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20110401 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140415 Year of fee payment: 3 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |