本研究使用水果萃取物合成不同型態的奈米金粒子(Gold nanoparticles)，優點為不需使用任何有機溶劑，可以避免因有機溶劑產生的細胞毒性。奈米金粒子使用穿透式電子顯微鏡（TEM）、動態散射光譜儀（DLS）、界達電位儀（Zeta potential）及紫外光-可見光光譜儀（UV-VIS Spectrophotometer）鑑定其性質。奈米金粒子分為兩種型態，球型及三角型。球型奈米金粒子平均尺寸為102.1 nm，為三角形奈米金粒子的兩倍。球型奈米金粒子的表面負電荷低於三角形奈米金粒子。而低濃度與帶有較低負電荷的奈米金粒子，有助於提升細胞攝入量。因此細胞內球型奈米金粒子高於三角形。對類骨細胞及骨母細胞而言，隨著細胞內奈米金粒子數量增加而活性上升。
對骨類細胞而言，攝入奈米金粒子可增進細胞的活性，而表面電荷及濃度會影響細胞對奈米金粒子的攝入。藉由實驗結果發現，奈米金粒子於生醫材料上有高度的發展潛力。

In this research, gold nanoparticles (GNPs) with different morphologies were synthesized by using fruit extract without any organic solvents. Their properties were characterized with transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, and ultraviolet spectrophotometer (UV-vis). The TEM results indicated there were two different shapes of GNPs spherical and triangle GNPs. Spherical GNPs had average size of 102.1 nm, twice larger than triangle. They also had less negative surface charge and less absorption uv-vis peak compared to triangle ones. Rat osteosarcoma cells (UMR) and rat osteoblasts (7F2) were cultured with GNPs. Their cellular uptake and cell’s behavior were then further analyzed. Higher GNPs cellular uptake was obtained when GNPs were less negative and with low concentration for both of UMR and 7F2 cells. Therefore, the intracellular amounts of spherical GNPs were higher, compared to triangle ones. Furthermore, the activity of UMR and 7F2 cells was increased with intracellular amount of GNPs, no matter spherical or triangle ones. In conclusions, the present research revealed that GNPs cellular uptake was influenced by their surface charge and concentration. Moreover, the uptake of GNPs was able to promote cellular activity of osteoblastic cell. The results in this study supported that biosynthesis GNPs are highly potential in biomedical applications.

Acknowledgment i
Abstract (in Chinese) ii
Abstract (in English) iii
Contents iii
Figure List vii
Table List xi
Chapter 1. Introduction 1
Chapter 2. Literatures Review 1
2.1. Nanoparticles 1
2.1.1. Nanoparticles introduction 1
2.1.2. Nanoparticle applications 2
2.1.2. Nanoparticle features 4
2.2. Gold nanoparticle 4
2.2.1. Gold nanoparticle properties 4
2.2.2. Gold nanoparticle synthesis 5
2.2.2. Gold nanoparticle limitation 9
2.3. Raman 11
2.3.1. Raman definition 11
2.3.2. Surface-enhanced Raman spectroscopy (SERS) 12
2.3.3. SERS substrate and benefit 12
2.3.4. Relation between gold nanoparticles and Raman 13
2.3.4.1. The optical properties of noble metals 13
2.3.4.2. Application of gold nanoparticles in life science 14
Chapter 3. Material and Experimental Procedure 27
3.1. Chemicals and materials 27
3.2. Experimental apparatus 28
3.3. Gold nanoparticle 30
3.3.1. Gold nanoparticle synthesis 30
3.4. Cells 32
3.4.1. Cells type 32
3.4.2. Cell culture 34
3.4.3. Cell counting 35
3.5. Experimental design 37
3.6. Cell attachment and spreading area 37
3.7. Cell number staining assay for cell proliferation 38
3.8. MTT assay for mitochondria activity 38
3.9. Alkaline phosphatase (ALPase) quantification assay for ALPase expression 39
3.10. TEM sample preparation 40
3.11. Raman sample preparation 42
3.12. Raman spectroscopy 42
3.13. Zeta Potential analysis 43
3.14. Preparation of medium 43
3.15. Cell freezing 43
3.16. Cell de-freezing 44
Chapter 4. Results and Discussions 45
4.1. Gold nanoparticles shapes and properties 45
4.1.1. Characterization of gold nanoparticles 45
4.1.2. Gold nanoparticles pre-treatment 48
4.1.3. Gold nanoparticles sterilization 50
4.1.4. Gold nanoparticle surface charge 53
4.2. Transmission electron microscopy (TEM) 55
4.3. Cell behaviors with addition of gold nanoparticles 64
4.3.1. Cell attachment 64
4.3.2. Cell spreading area and elongation 67
4.3.3. Cell proliferation 73
4.3.3.1. UMR cell proliferation 73
4.3.3.2. 7F2 cell proliferation 78
4.3.4. Cell activity 81
4.3.4.1. UMR cell activity 81
4.3.4.2. 7F2 cell activity 85
4.3.5. ALPase expression 88
4.3.5.1. UMR cell ALPase expression 88
4.3.6. Cell behaviors influenced by gold nanoparticles 94
4.4. Surface-enhanced Raman signal (SERS) analysis 99
Chapter 5. Conclusions 102
5.1. Conclusions 102
References 105
Appendix 117

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