磁性奈米粒子及其複合材料之製備應用與氧化鋅奈米粒子細胞毒性之研究

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研究生：	李佩玲 Lee, Pei-Ling
論文名稱：	磁性奈米粒子及其複合材料之製備應用與氧化鋅奈米粒子細胞毒性之研究 The preparation and application of magnetic nanoparticles/nanocomposites and the cytotoxicity study of zinc oxide nanoparticles
指導教授：	孫毓璋 Sun, Yuh-Chang 凌永健 Ling, Yong-Chien
口試委員:
學位類別：	博士 Doctor
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	143
中文關鍵詞：	綠色化學、磁性奈米粒子、奈米碳管、氧化鋅奈米粒子、細胞毒性、重金屬、揮發性有機物
外文關鍵詞：	Green chemistry, Magnetic nanoparticles, Carbon nanotubes, Zinc oxide nanoparticles, Cytotoxicity, Heavy metals, VOCs
相關次數：	點閱：4 下載：0
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奈米科技與綠色化學皆為近年新興之科技研發方向，隨著永續概念的深化，結合兩者的優點並加速擴展其研發應用實為當務之急。因此本論文以奈米科技為主軸，結合綠色奈米製程與綠色化學之概念，發展磁性奈米粒子(magnetic nanoparticles)及其奈米複合材料(nanocomposites)之製備與應用於生物與環境中重金屬與揮發性有機物(volatile organic compounds, VOCs)之分析。於重金屬分析(第二章)，本研究子題係利用磁性奈米粒子可藉由外加磁場操控、高比表面積與易修飾之特性，開發一新穎的磁性奈米吸附劑(magnetic nano-adsorbent)結合實驗室閥上(Lab-on-valve)系統串聯感應耦合電漿質譜儀(ICPMS)應用於微量重金屬之快速分析。根據分析效能之結果顯示，本研究子題所發展之分析系統除可成功地應用於生物與環境基質中微量重金屬之快速分析外，因自製的磁性奈米吸附劑具有較高的吸附容量，而有效地降低溶液與吸附劑的使用量，為奈米技術與綠色分析化學整合之實例。於揮發性有機物分析(第三章)，本研究子題係以微波輔助酸化及靜電交互作用法製備一磁性奈米複合材料-磁性奈米碳管吸附劑，續以揮發性有機物進行吸附性能之評估。其結果顯示本研究子題所發展之製備方法不僅可大幅縮短合成時間及降低溶劑與能源的使用而符合綠色奈米製程之概念外，相較於市售商業化之吸附劑，使用磁性奈米碳管吸附劑應用於揮發性有機物之分析具有較低之偵測極限與較大之破出體積，可有效降低吸附劑的使用量，亦為綠色奈米與綠色分析化學整合之另一實例。再者奈米科技也因奈米尺度所帶來的獨特效應而快速地被延伸至日常生活中，然卻也因潛在性之危害問題如:奈米毒性(nanotoxicology)而受到高度的爭議。有鑑於此本論文另一研究子題係針對人體可能藉由皮膚暴露之途徑接觸到添加於防曬霜之奈米材料，以氧化鋅奈米粒子(ZnO NPs)為例，進行材料製備鑑定、細胞毒性與化學分析之系統性探討與評估(第四章)。結果顯示自氧化鋅奈米粒子中解離之高濃度鋅離子及相關之鉀/鈣離子於細胞中的變化說明了氧化鋅奈米粒子的解離行為於細胞毒性之研究扮演一重要角色。同位素標定之氧化鋅奈米粒子(68ZnO NPs)亦呈現相似之研究結果，將有助於未來氧化鋅奈米粒子相關毒性研究示蹤用途之應用。綜合上述，磁性奈米粒子及其複合材料之製備與應用為奈米科學與綠色分析化學整合之實務，細胞毒性之系統性研究平台之發展亦有助於釐清奈米毒性和尺寸、型態、表面化學活性等物理化學特性之間的關係，更有助於未來設計及合成較低毒性的奈米粒子，朝向更綠色的奈米科學。

Nanoscience possesses advantageous properties owing to their small size and large surface area. The aim of green chemistry is to reduce waste and hazard in chemical products and process. The potential benefits of merging cross disciplines have been realized and produce innovative techniques in recent years. Herein, we prepared novel magnetic nanoparticles (MNPs) and their nanocomposites, which were applied to analyze trace multiple heavy metals and volatile organic compounds (VOCs), demonstrating successful coupling of green analytical chemistry and nanoscience. For heavy metals analysis (Chapter 2), a hyphenated system consisting of a lab-on-valve system integrated with a micro-column packed with magnetic nano-adsorbent (MNPs-PAA) for on-line pre-treatment followed by ICPMS determination was designed and built. The accuracy was evaluated by analyzing certified reference materials of SRM 2670a (trace elements in urine, low level) and CASS-2 (nearshore seawater reference material for trace metals). Good agreement between the measured and certified values demonstrates that the system is useful for trace analysis of multiple heavy metals in environmental and biological aqueous samples. In conjunction with the reduced use of solvent and the greenness of preparing MNPs-PAA adsorbent makes the proposed method a green analytical chemistry method. For VOCs analysis (Chapter 3), a magnetic nanocomposite (MCNT) consisting of MNPs and multi-walled carbon nanotubes (MWCNTs) was prepared based on their electrostatic interactions. The MCNT integrates the advantages of controllable immobilization provided by MNP’s superparamagnetism and excellent adsorption property provided by the large specific surface area of MWCNTs, rendering it being a preferred gas adsorbent of VOCs followed by TD/GC-MS determination. The lower detection limit by MCNT adsorbent demonstrated its potential use in green analytical chemistry. A wide range of nanomaterials with different types and properties has been examined for their potential use in various applications. The inevitable questions regarding the nanotechnology risk to the environment and human health were highly concerned. Using zinc oxide nanoparticles (ZnO NPs, Chapter 4) as an example, an integrated analytical platform for cytotoxicity assessment was proposed based on the material characterization, in vitro toxicological assessment, and chemical analysis. Elevated levels of dissolved 64Zn from ZnO NPs and variation of intracellular 39K/40Ca were observed in ZnO NPs treated HaCaT cells, indicating that the dissolving behavior of ZnO NPs played an important role in inducing cytotoxicity. The isotope-labeled 68ZnO NPs possess similar trend as ZnO NPs, demonstrating its potential use as tracer in nanotoxicological study of ZnO NPs.
Green nanoscience practice is readily realized via successful integration of green synthesis and green analytical chemistry as demonstrated by the preparation and applications of MNPs and its nanocomposites reported in this thesis. The integration of material characterization, in vitro toxicological assessment, and chemical analysis for nanotoxicology risk assessment as demonstrated by the cytotoxicity study of ZnO NPs might help clarify the doubt about nanotechnology. The platform proposed is potentially useful for future design and synthesis of nanomaterials with lower nanotoxicity, a step forward toward the aim of green nanoscience.

CHAPTER 1  INTRODUCTION…………………...........................................    1-1
 1.1  NANO-MATERIALS……………………………………………………..    1-1
  1.1.1  Magnetic nanoparticles…...…………………………………………….........    1-1
        (1) The basic concepts……………………………………...…………………    1-2
        (2) Synthesis methods………………………………………………………….    1-3
          - Coprecipitation…………………………………………………...……….    1-3
          - Microemulsions..................................................................................    1-3
          - Polyols...............................................................................................    1-3
          - Hydrothermal and high-temperature reactions.................................    1-4
          - Aerosol/Vapor Methods…………………………………………………..    1-4
          - Sonolysis……………………………………………………………………    1-5
  1.1.2  Carbon nanotubes.........…...……………………………………………........    1-5
        (1) The basic concepts………………………………………………………...    1-6
          - Mechanical properties……………………………………………………    1-6
          - Chemical reactivity……………………………………………………….    1-6
          - Vibration property…………………………………………………………    1-7
        (2) Synthesis methods………………………………………………………….    1-7
          - Arc discharge method…………………………………………………….    1-8
          - Laser ablation method……………………………………………………    1-8
          - Chemical vapor decomposition (CVD) method……….………………    1-9
  1.1.3  Nanocomposites……………………………………………….……………….    1-9
 1.2  NANOTOXICOLOGY…………..……………………………………….    1-10
 1.3  GREEN ANALYTICAL CHEMISTRY/NANOSCIENCE…………….    1-13
  1.3.1  Green analytical chemistry………...…………………………………………    1-13
  1.3.2  Green nanoscience……….……………………………………………………    1-14
 1.4  REFERENCES……………………………………………………………    1-17
 1.5  TABLES…………………………………...………………………............    1-20
 1.6  FIGURES……...…………………………………………………………..    1-22
CHAPTER 2  MAGNETIC NANO-ADSORBENT INTEGRATED WITH LAB-ON-VALVE SYSTEM FOR TRACE ANALYSIS OF MULTIPLE HEAVY METALS…………………………………………………………….….    2-1
 2.1  INTRODUCTION………………………………………………………...    2-1
 2.2  EXPERIMENTAL SECTION……………………………………………    2-4
  2.2.1  Reagents…………………………………………………………….................    2-4
  2.2.2  Preparation of MNPs-PAA……………………………………………………    2-5
  2.2.3  Characterization of MNPs-PAA……………………………………………...    2-5
  2.2.4  The adsorption capacity of MNPs-PAA……………………………….........    2-6
  2.2.5  Analytical procedure…………………………………………………………..    2-7
  2.2.6  Performance evaluation………………………………………………………    2-8
 2.3  RESULTS AND DISCUSSION…………………………………………..    2-9
  2.3.1  Physical characteristics of MNPs-PAA……………………………………..    2-9
  2.3.2  Chemical characteristics of MNPs-PAA…………………………………….    2-11
  2.3.3  On-line pre-treatment…………………………………………………...........    2-13
  2.3.4  The adsorption capacity of MNPs-PAA……………………………….........    2-14
  2.3.5  Analytical performance……………………………………………………….    2-14
 2.4  CONCLUSIONS………………………………………………………….    2-17
2.5  REFERENCES……………………………………………………………    2-18
 2.6  TABLES...…………………………………………………………............    2-21
 2.7  FIGURES………………...………………………………………………..    2-25
CHAPTER 3  SYNTHESIS OF NANOCOMPOSITE CONSISTED OF MAGNETIC IRON-OXIDE NANOPARTICLES AND CARBON NANOTUBES AS GAS ADSORBENT………………………............................    3-1
 3.1  INTRODUCTION………………………………………………………...    3-1
 3.2  EXPERIMENTAL SECTION……………………………………………    3-2
  3.2.1  Reagents………………………………………………………………..............    3-2
  3.2.2  Synthesis of MCNT……………………..………………………………..........    3-2
  3.2.3  Characterization of MCNT……….…………………………………………..    3-3
  3.2.4  Application of MCNT as gas adsorbent………………………………….…    3-4
        (1) MCNT adsorbents…………………………………………………………    3-4
        (2) Blank analysis of adsorbent……………………………………………...    3-5
        (3) Efficiency of adsorbent……………………………………………………    3-5
 3.3  RESULTS AND DISCUSSION…………………………………………..    3-5
  3.3.1  TEM measurement of MCNT………………...………………………………    3-5
  3.3.2  Mechanism and yield………………………………………………………….    3-6
  3.3.3  TGA measurement of MCNT…………………………………………………    3-7
  3.3.4  FT-IR measurement of MCNT………………………………………………..    3-7
  3.3.5  The composition of MCNT……………………………………………………    3-8
  3.3.6  Magnetic properties of MCNT……..………………………………………...    3-8
  3.3.7  Raman measurement of MCNT……….………………………………..........    3-9
  3.3.8  Application of MCNT as gas adsorbent ……………………………………    3-10
        (1) Blank analysis of adsorbent……….……………………………….........    3-10
        (2) Efficiency of adsorbent……………………………………………………    3-11
 3.4  CONCLUSIONS………………………………………………………….    3-12
3.5  REFERENCES……………………………………………………………    3-13
 3.6  TABLES…………………………………………………………………...    3-17
 3.7  FIGURES…………………………………………………………………    3-20
CHAPTER 4  IN VITRO CYTOTOXICITY OF ZINC OXIDE (AND 68ZINC OXIDE) NANOPARTICLES IN HUMAN KERATINOCYTE CELL……………………………………………………………………………..    4-1
 4.1  INTRODUCTION………………………………………………………...    4-1
 4.2  EXPERIMENTAL SECTION……………………………………………    4-5
  4.2.1  Reagents………………………………………………………………..............    4-5
  4.2.2  Synthesis of ZnO (and 68ZnO) NPs………….………………......................    4-5
        (1) Synthesis of ZnO NPs……………………………………………………..    4-5
        (2) Synthesis of 68ZnO NPs………………………………………...…………    4-5
  4.2.3  Characterization………………………………………………………............    4-6
  4.2.4  Cell culture and treatment with ZnO (and 68ZnO) NPs………….………..    4-6
  4.2.5  Assessment of cytotoxicity…….………………………………………………    4-7
        (1) Mitochondrial activity………………………………………………    4-7
        (2) LDH release………………………………………………………………..    4-7
  4.2.6  Dissolving behavior of ZnO (and 68ZnO) NPs………….……….…………    4-7
  4.2.7  Sample preparation for CSLM and ToF-SIMS analysis.………………….    4-8
        (1) CSLM imaging analysis…………………………………………………..    4-8
        (2) ToF-SIMS surface mass spectral and chemical imaging analysis…..    4-9
 4.3  RESULTS AND DISCUSSION…………………………………………..    4-9
  4.3.1  Characterization of ZnO (and 68ZnO) NPs………….……………………..    4-9
  4.3.2  Assessment of cytotoxicity………………………….…………………………    4-10
        (1) Mitochondrial activity…………………………………………………….    4-10
          - The dosage-response…………………………………………………….    4-10
          - The time-response……………………………………………………….    4-11
        (2) LDH release………………………………………………………………..    4-11
          - The dosage-response…………………………………………………….    4-11
          - The time-response……………………………………………………….    4-12
  4.3.3  Dissolving behavior…………………………….……………………………..    4-13
  4.3.4  CSLM and ToF-SIMS analysis……………………………………...............    4-15
        (1) CSLM imaging analysis…………………………………………………..    4-15
        (2) ToF-SIMS surface mass spectral and chemical imaging analysis…..    4-16
          - ZnO NPs treatment……………………………………………………...    4-16
          - 68ZnO NPs treatment……………………………………………………    4-18
 4.4  CONCLUSIONS……………………………………………….........……    4-20
 4.5  REFERENCES….……………………………………………………...…    4-21
 4.6  TABLES…..…………………………………………………………....….    4-25
 4.7  FIGURES…….……………………………………………………………    4-26
CHAPTER 5  SUMMARY AND PERSPECTIVE......………………………..    5-1
 5.1  REFERENCES……………………………………………………………    5-3
 5.2  FIGURES………………………………………………………………….    5-4

                                

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