金/銀奈米結構合成與光學性質探討應用於生物螢光鑑定與標記

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研究生：	茱蒂 Judy M. Obliosca
論文名稱：	金/銀奈米結構合成與光學性質探討應用於生物螢光鑑定與標記 Synthesis and Optical Properties of Gold and Silver Nanostructures for Biological Fluorescence Assessment and Labeling Applications
指導教授：	曾繁根 Tseng, Fan-Gang
口試委員:	Tseng, Fan-Gang Wang, Pen-Cheng Yang, Chung-Shi Chang, Feng-Chih Li, Lain-Jong
學位類別：	博士 Doctor
系所名稱：	原子科學院 - 工程與系統科學系 Department of Engineering and System Science
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	96
中文關鍵詞：	Fluorescence quenching 、gold nanoparticles 、surface functionalization 、microarray 、DNA 、galvanic replacement reaction 、fluorescence 、core-shell 、synthesis
相關次數：	點閱：2 下載：0
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The development of nanomaterials for biological and optical detections, imaging, bioengineering, DNA sequencing, gene therapy and electrochemical sensing applications has been an active and interesting field of research in recent years. Among the metals, gold (Au) and silver (Ag) nanoparticles (NPs) have been of great interest because of their ease of preparation, better homogeneity, high compatibility with biomolecules, intense absorption bands in the visible region and significant contributions to surface-enhanced Raman spectroscopy (SERS) as active substrates. Many studies utilize metal nanoparticles to tune the luminescence intensity of fluorophores. The enhancement or quenching of a dye’s fluorescence intensity is strongly dependent on the spatial separation of the dye from the nanoparticle surface.
In the first part of the study, the distance-dependent fluorescence behavior of Au–DNA–Cy3 conjugates in solution and array platforms is presented. This was carried out by equilibrating the phosphine-stabilized AuNPs of 10-nm size with the designed rigid spacer ds-DNA consisting of thiol-modified target and Cy3-labelled complementary probe of different lengths (5-20 nm). The Cy3-labelled products were immobilized onto MPTMS (3-mercaptopropyltrimethoxysilane)-modified glass substrates and then excited with a 532-nm laser source. Quenching efficiency of AuNPs with increasing Au-to-dye distance was assessed using ligand exchange of the thiolated oligonucleotide by 2-mercaptoethanol (ME) to obtain free DNA–Cy3 probe, thus eliminating nanoparticle effect on the dye’s luminescence intensity. Effective exchange, revealed by UV-vis absorption and fluorescence profiles, was achieved in a few minutes. Quenching effect on Au–DNA–Cy3 array, consistent with the result in solution phase for the conjugates with up to 10-nm Au-to-Cy3 separation distance, was observed.
This study also aims on tuning the electronic and optical properties of Au and Ag nanostructures by varying their sizes and shapes and by combining them with another nanomaterial from which significant cooperative effects can develop. In the second part of this work, a simple route for synthesizing small-sized Ag/Au core-shell (or simply Ag/Au) on multi-walled carbon nanotube (MWCNT) surfaces via galvanic replacement of AgNPs is presented. The Raman response of MWCNT decorated with Ag/Au was investigated under surface-enhanced Raman scattering (SERS). A relatively weak Raman signal enhancement of the tube was observed due to the large interparticle distance between neighboring small-sized nanostructures. Ag/Au give better enhancing capability than the starting Ag because of the synergistic effect between the localized electric field of the Ag core and the Au shell separated with a hollow space formed during the galvanic replacement reaction. Furthermore, the Ag/Au were removed from the CNT surfaces via sonication with 1-octanethiol (OT), releasing unreplaced AgNPs and Au nanobowls (AuNBs) of 1.3 and 7.6 nm in mean diameter sizes, respectively. The luminescent property of these fine-sized nanocomposites (Au/Ag NCs) was investigated. Interestingly, the separated Au/Ag NCs (i.e., the mixture of AuNBs and unreplaced AgNPs) exhibit significant fluorescence behavior that may be useful for single-molecule probing and detection. The synthetic approach of this study provides the preparation of smallest dimension of AuNBs so far simply achieved by wet chemical process using MWCNTs as templates.

TABLE OF CONTENTS

ABSTRACT………………………………………………………………………………..    ii 
ACKNOWLEDGMENTS…………………………………………………………………    iv
LIST OF FIGURES………………………………………………………………………..    ix
LIST OF TABLES…………………………………………………………………………    xii 
NOMENCLATURE………………………………………………………………………..xiii
CHAPTER 1: INTRODUCTION ……………………………………………………….    1 
1.1    Motivations of the Study ……………………………………………………….    4
1.2    Objectives of the Study…………………………………………………………    5
CHAPTER 2: REVIEW OF RELATED LITERATURE    ……………………………...    6 
2.1 Dye Fluorescence Quenching Assessment…………………………………….    6
2.1.1 Principle of Fluorescence…………………………………………………….    6    
2.1.2 Fluorescent Dye Cyanine……………………………………………………    7
2.1.3 Quantum Yield and Lifetime of a Fluorophore    ……………………………...    8
2.1.4 Optical Properties of Metal Colloids………………………………………..    10
2.1.4.1 Rayleigh and Mie Scattering……………………………………………    10
2.1.4.2 Raman Scattering………………………………………………………….    11
2.1.5 Quantum Yield and Lifetime of a Fluorophore Near a Metal………………..    12
2.1.6 Factors Affecting the Dye’s Fluorescence Due to Metal…………………….    13
2.1.6.1 Distance from the Particle Surface to the Dye……………………………..    13
2.1.6.2 Size of the Colloids………………………………………………………...    14
2.1.6.3 Orientation of Fluorophore to the Particle Surface…………………………    15
2.1.7 Properties, Surface Functionalization and Applications
 of Au Nanostructures    ……………………………………………………………...    16
2.1.8 Typical Dye Quenching Assessment…………………………………………    20
2.2 Synthesis of Au Nanobowl/Ag Nanoparticle Nanocomposites………………..    22
2.2.1 Sizes, Shapes, Properties and Applications of Au and
 Ag Nanomaterials………………………………………………………………….    22
2.2.2 Preparation Techniques of Core-Shell Ag and Au Nanostructures…………………..    26
2.2.3 Preparation Techniques of Au Nanocrescent or Nanobowl………………….    30
2.2.4 Optical Property of Au Nanocrescent or Nanobowl …………………………    32
CHAPTER 3: METHODOLOGY………………………………………………………    36 
3.1 Dye Fluorescence Quenching Assessment…………………………………….    36
3.1.1 Materials and Instrumentation……………………………………………….    36
3.1.2 Synthesis of Phosphine-stabilized AuNPs…………………………………..    37
3.1.3 Target and Probe DNA Sequences…………………………………………...    37
3.1.4 Synthesis of Au–DNA–Cy3 Conjugates……………………………………...    40
3.1.5 Gel Electrophoresis of Au–DNA–Cy3 Conjugates………………………......    40
3.1.6 Fluorescence Quenching Assessment………………………………………..    41
3.1.6.1 Solution Phase……………………………………………………………...    41
3.1.6.2 Array Format……………………………………………………………….    41
3.1.7 Surface Modification of a Glass Substrate with MPTMS……..……………..    42
3.2 Synthesis of Au Nanobowl/Ag Nanoparticle Nanocomposites……………….……….    43
3.2.1 Materials and Instrumentation………………………………………………..    43
3.2.2 Synthesis of Multi-walled Carbon Nanotube (MWCNT)…………………....    43
3.2.3 Synthesis of Silver Nanoparticles on MWCNT (Ag-CNT)…………………..    44
3.2.4 Synthesis of Ag/Au core-shell on MWCNT (Au-Ag-CNT)………………….    45
3.2.5 Preparation of MWCNT, Ag-CNT and Au-Ag-CNT Samples
 for Raman Analysis………………………………………………………………...    45
3.2.6 Separation of Au Nanobowls (AuNBs) from MWCNT Surfaces……………    45
CHAPTER 4: RESULTS AND DISCUSSION ………………………………………...    46 
        4.1 Dye Fluorescence Quenching Assessment……………………………………..    46
        4.1.1 Preparation of Au–DNA–Cy3 Array…………………………………………    46
4.1.2 Synthesis of Phosphine-stabilized AuNPs…………………………………..    48
4.1.3 Synthesis of Au–DNA–Cy3 Conjugates…………………..…………………    49
4.1.4 Binding Efficiency of DNA to Phosphine-stabilized AuNPs………………..    50
4.1.5 Fluorescence Quenching Assessment………………………………………..    53
4.1.5.1 Solution Phase    ……………………………………………………………...    53
4.1.5.2 Array Format……………………………………………………………….    57
4.2 Synthesis of Au Nanobowl/Ag Nanoparticle Nanocomposites………………..    65    
        4.2.1 Synthesis of Au Nanobowls………………………………………………….    65
4.2.2 Optimization of the Au-Ag-CNT Products…………………………………...    71
4.2.3 Raman Response of Au-Ag CNT Products…………………………………..    76
4.2.4 Release of Fluorescent Au/Ag Nanocomposites……………………………...    78
CHAPTER 5: CONCLUSION…………………………………………………………..    82
CHAPTER 6: FUTURE WORK AND RECOMMENDATION……………………….    84
Appendix 1: Estimation on the Number of AuNPs Used in 
Solution and Array Platforms…………………………………………………………….86
Appendix 2: Publications Produced from This Study…………………………………    87 
Journal Papers……………………………………………………………………...    87
Conference Proceedings……………………………………………………………    87
International Conference Papers……………………………………………………    88
Appendix 3: Awards Received from This Study………………………………………..    90
References…………………………………………………………………………………    91

                                

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