Chapter 1 (Introduction)

Photoluminescent semiconductor nanoparticles can show excellent optical properties compared with organic dyes or fluorescent proteins. There is increasing attention being focused on nanoscale materials in the fields of optoelectronics and biolabeling. Chalcogenide (e.g. CdSe, PbSe) semiconductor nanoparticles have been the subject of much fundamental and applied research. However, the presence of heavy metals and the consequent toxicity of these materials has restricted their application. In the past years, photoluminescent nanometer-scale silicon was reported and has been closely studied based on its potential novel optical properties. Synthesis of silicon nanoparticles (Si-NPs) have also been reported by different methods, like solid, liquid or gas phase reactions. However, particle synthesis with full-color of photoluminescence (PL) is still a challenge. Of those methods, gas phase synthesis has the advantages of rapid and continuous production. However, the main precursor for gas phase synthesis is silane, and the preparation has to be followed by surface modification using wet chemistry to ensure stability of the resultant nanocrystals. It is worth examining other precursors for the synthesis of Si-NPs to assess their practicality. Furthermore, due to the outstanding optical property and low cytotoxicity, Si-NP shows promising application for bio-imaging agent and the in vitro/vivo imaging is undergoing. However, there is not yet sufficient understanding of the properties of Si-NPs as selective bio-labels. There is therefore a need for new simple synthetic routes to water-dispersible Si-NPs and for further study of the fundamental properties of these NPs as bio-labeling probes.

In this thesis, we will focus on synthesis and application of photoluminescent Si-NPs. RF-plasma was used to decompose SiBr4. Firstly, the research background and current progresses are introduced (Chapter 1). The synthesis procedure was listed after the general introduction and the synthesized product was characterized from the aspect of surface component, size, crystallinity, optical properties and stability (Chapter 2). The parameters in terms of particle formation, which may affect crystal size and optical properties, were discussed in Chapter 3. In Chapter 4, cell-imaging application of Si-NPs was investigated after surface modification process to obtain water-dispersible Si-NPs. Finally, conclusion and future prospects are given in Chapter 5.

Chapter 2 (Synthesis of silicon nanoparticles using plasma-CVD)

Si-NPs were synthesized from rf (13.56 MHz radio frequency) plasma. In brief, the precursor material was introduced into the chamber via an Ar gas carrier passing through liquid SiBr4. H2 was also introduced as a reducing agent along with the inert argon carrier gas. The plasma power density was fixed at 1.25 W/cm2. The flow rate of SiBr4 and H2 were 1-3 and 20 sccm respectively. To obtain tunable PL colors, the total pressure in the chamber was controlled in 2-4 Torr. Nanoparticles exhibiting blue emission, which were obtained at 3 Torr, were used for characterization. The synthesized particles are considered to be surface oxidized and the surface Si-O-Si bonding was confirmed by FTIR. High magnification images of crystals and the electron-diffraction patterns are observed from TEM.

The optical properties were investigated. UV-vis absorption shows a shoulder near 360 nm, whereas the PL spectrum shows emission with a peak at 470 nm when excited at 340 nm. The optical stability is another important issue which directly relates to the applications. We took a number of PL measurements using a single sample over half a year. From the integrated peak areas of the PL spectra, we found that the PL intensity declined by 20% in the first 14 days and then remained almost constant for the following 200 days. We consider that the observed PL stability is caused by the protected surfaces of the silicon nanoparticles. The photostability of the nanoparticles-ethanol suspension was also checked. The PL retains 80% of the initial intensity after 3 hours irradiation. This result is quite important for cell-imaging applications, especially for those long-term measurements such as observation of the cell division. The quantum yield was also measured by compared with Rhodanmine 6G under the same excitation wavelength and showed as high as 24%.

Chapter 3 (Formation mechanism of silicon nanoparticles with different fluorescent colors)

The synthesis parameters were investigated to reveal the key factors for particles formation. Plasma power was firstly examined. As increasing of the power, increasing of the crystallinity was observed in our system. When the power during synthesis is set at 50 W, the products are amorphous silicon. Whereas the synthesized particles show clear electron diffraction pattern observed from TEM when power increases up to 250 W. Raman was also used to macroscopic analyze the crystallnity of the sample. In addition, the particles synthesized at low power condition show higher Br residue content, which would be one of the reasons for amorphous silicon formation.

In addition to the power effect, pressure, which includes partial pressure and total pressure, was found direct affect the color of PL. In the synthesis condition with low total and partial pressure, the size of crystal can be as large as ~20 nm. After HF/HNO3 mixed acid etching, the sample shows a red PL under UV irradiation. Whereas high pressure condition form nanoparticles with visible blue to green PL. TEM analysis also suggested that the red PL sample (~5.4 nm) has larger size than the blue one (~1.8 nm). The nucleation theory is considered to be one of the reasons, the higher partial pressures the smaller critical sizes in supersaturation condition.

Chapter 4 (Practical application of fluorescent silicon nanoparticles: cell-imaging)

The study of intracellular compartments is an important area of biological chemistry. Among intracellular organelles, endoplasmic reticulum (ER) plays a critical role in protein synthesis and transport in a cell. Once the function of ER is interfered, some serious diseases, such as diabetes and Alzheimer's disease, would be caused. Therefore direct visualization of ER to understand how ER acts in a certain environment is quite important both scientifically and practically. As introduced before, fluorescent Si-NPs are expected as a new, less toxic bio-label for long-term observation. Similar to other inorganic semiconductor nanoparticles, the intrinsic surface of silicon is hydrophobic which limits the water-dispersibility for the further biological applications. Thus, the surface modification process for water-dispersible is quite important to Si-NPs.

For the surface modification, we select using block copolymer (F127) as non-covalent bond coverage. F127 is a kind of surfactant with hydrophobic and hydrophilic blocks. Nanoparticles after modification with F127 show water-dispersibility. TEM image of Si-NPs dispersed in water after F127 modification showing that Si-NPs formed aggregates with small size (20-40 nm). We also measured size distribution by DLS, which is in agreement with the size of aggregates observed from TEM results. Zeta potential was measured and it reveals that copolymer-coated Si-NPs have a neutral surface charge in water. The F127-modified Si-NPs also show good photostability, retaining 80% of the initial PL intensity after UV irradiation for 3 hours. Furthermore, UV irradiation did not induce any increase in aggregation size of dispersed Si-NPs.

Then we studied the utility of our block copolymer functionalized Si-NPs for cell imaging. We firstly measure the cell viability in terms of the concentration of Si-NPs. The result shows our Si-NPs are low-toxic. A concentration of 0.1 mg/ml was used for cell imaging, with which cytotoxicity to human umbilical vein endothelial cells (HUVECs) was negligible. In the low-magnification microscope images of the cells, we confirmed that HUVECs were successfully labeled. High-magnification microscopy revealed that a network structure in the cell is selectively labeled. To test our hypothesis, HUVECs were co-stained with Si-NPs and ER-tracker red (a commercial molecule used to selectively labels the ER). The intracellular localization of ER-tracker red and F127-modified Si-NPs is almost identical, thus F127-modified nanoparticles do selectively label the ER in live HUVECs. As recently reported by Kabanov et al.,[1] hydrophobic poly-propylene oxide (PPO) block contained copolymer plays an important role for the specific pathway via caveolae mediated endocytosis transfer to the mitochondria through ER. This is one of the main reasons that our F127-modified Si-NPs selectively localize at ER, because the F127 also contain PPO block.

General conclusion

This thesis reported synthesis and application of silicon nanoparticles, which were obtained from plasma decomposition of SiBr4. The size and optical properties of the as-synthesized nanoparticles were characterized. The PL colors can be tuned by changing the pressure during synthesis. Furthermore, the particle formation and possible PL mechanism was also discussed. Surface modification of the nanoparticles was done with the assistance of block copolymer F127. The water dispersed nanoparticles show excellent optical stability and small aggregation size after modification. The surface modified nanoparticles can be used to selectively label the endoplasmic reticulum in live cells with low cytotoxicity.

「プラズマCVDによる蛍光シリコンナノ粒子の合成」と題した本論文は、低圧高周波プラズマCVDでの四臭化珪素ガス分解反応を用いることで、シリコンナノ粒子の合成や応用について、極めて安定な蛍光特性を示すシリコンナノ粒子を気相プラズマ中で製造する方法と水分散化のための表面修飾方法を確立し、粒子径の制御と細胞染色への応用を検討することを目的とした研究であり、5章から構成されている。

第1章は序論であり、研究背景および研究目的を述べている。冒頭では、将来期待されるナノテクノロジーの応用例を紹介し、特に光学特性を示す半導体ナノ粒子の合成と応用について総説を述べている。その中で、CdSeナノ粒子は、生体に対する毒性が強く実際の応用は困難であると述べている。毒性低いシリコンナノ粒子は、他の半導体粒子に比べ、研究が十分進んでいるとは言えない。シリコンで量子サイズ効果を出現させるためには、5nm以下の結晶を合成しなければならないが、容易に酸化されるため、その合成は難しい。また、シリコンナノ粒子で最重要課題の一つである水分散化に注目し、バイオイメージングへの展開を示し、本論文の具体的な研究目的を示している。

第2章では、シリコンナノ粒子の合成に関して述べている。安全性と反応性の観点から、新たな前駆体としてSiBr4を用いており、高周波プラズマ場でガス分解反応を用いることで、連続合成に成功している。得られたシリコンナノ粒子は粒径に応じた蛍光を示している。長時間経ても蛍光強度は80%程度を維持し、長期安定性も持ち合わせている。粒子のサイズ、結晶性または光学特性などの基本物性をまとめている。

第3章では、プラズマが引き起こすシリコンナノ粒子の生成メカニズムを扱っている。プラズマパワーによる、プラズマ状態の変化を観察している。プラズマパワーの大小に応じて、プラズマ空間のモード変化が起こる。合成圧力による粒径の変化と蛍光スベクトルのシフトも観察されている。低い圧力の場合は、粒子径は大きくなる。滞留時間の明確な依存性が観察されなかったことから、古典的核発生理論を用いて原料分圧の粒径への依存性を説明している。ナノ粒子の発光メカニズムも検討し、量子サイズ効果による蛍光発現であるとしている。

第4章では、第2章で合成されたシリコンナノ粒子の水分散化のための表面修飾とバイオイメージングの応用に関して論じている。本論文では、二種類の表面修方法を用いており、水分散化やバイオ応用を確認している。まずは、既往の研究で開発されたアミン修飾方法を用いており、水分散できる粒子の光学特性が既往の研究と一致していることを確認している。さらに、両親媒性ブロックコポリマー(F127)を用いて、水分散化を施し、数十ナノメートルの凝集サイズを持ったシリコンナノ粒子水分散液を得ている。この粒子を用いて、例えば、生細胞内の小胞体を選択的に染色することを明らかにしている。また、励起光の照射時間に対する蛍光強度変化を測定し、表面修飾したシリコンナノ粒子の蛍光はほとんど褪色せず、従来の有機色素に比べて、優れた安定性を持つことを明らかにしている。

以上要するに、本論文は化学工学および材料化学の考え方に基づき、安定性に優れた蛍光特性、また可視光全域の蛍光色を発する示すシリコンナノ粒子の製造方法を開発およびその生成メカニズムを明らかにし、水分散化の表面修飾方法を開発し、細胞イメージング応用を狙っている。機能性ナノ材料の生成メカニズムから応用までを検討した本論文は、化学システム工学への貢献が大きいと考えられる。また、一般に学術的研究対象である材料合成と機能化の実例を示している点は工学への貢献が大きいものと考えられる。

よって本論文は博士(工学)の学位請求論文として合格と認められる。