Name: Qin Weijie Degree: Ph.D. Department: Chemical and Biomolecular Engineering Thesis title: Fabrication of gold nanoparticle-DNA conjugates bearing specific number of DNA for quantitative detection and well-defined nanoassembly Year of submission: 2007 I FABRICATION OF GOLD NANOPARTICLE-DNA CONJUGATES BEARING SPECIFIC NUMBER OF DNA FOR QUANTITATIVE DETECTION AND WELLDEFINED NANOASSEMBLY QIN WEIJIE NATIONAL UNIVERSITY OF SINGAPORE 2007 II Acknowledgements I would like to sincerely express my greatest gratitude to my supervisor, Dr. Yung Lin Yue Lanry, for his unreserved support and guidance throughout the course of this research project. His continues guidance, constructive criticisms and insightful comments have helped me in getting my thesis in the present form. He has shown enormous patience during the course of my Ph.D. study and constantly gives me encouragements to think positively. More importantly, his rigorous research methodology, objectivity and enthusiasm in scientific discovery will be a model for my life and career. I wish to express my heartfelt thanks to all my friends and colleagues in the research group, Mr. Zhong Shaoping, Miss Zhao Haizheng, Mr. Jia Haidong, Miss Tan Weiling, Mr. Deny Hartono, and Miss Duong Hoang Hanh Phuoc and other staffs of the Department of Chemical and Biomolecular Engineering, especially Miss Li Xiang, Miss Li Fengmei, Mr. Han Guangjun, and Mr. Boey Kok Hong. Without their assistance, this work could not have been completed on time. Special acknowledgements are also given to National University of Singapore for its financial support. I deeply appreciate my girl friend, Miss Liu Ying. Her love and encouragement light up many lonely moments in my life as a graduate student away from home. Last, but not least, I would like to dedicate this thesis to my parents. Without their love, support and understanding, I would not have completed my doctoral study. III Table of contents Acknowledgements III Table of contents IV Summary . VII List of tables .XI List of figures . XII Chapter Introduction 1.1 Background . 1.2 Aims and scope of this project . Chapter Literature review 2.1 Synthesis, stabilization and characterization of metallic nanoparticles . 2.1.1 Reaction mechanism and kinetics . 2.1.2 Mechanism of particle formation 2.1.3 Synthesis of metallic nanoparticles . 2.1.4 Stabilization of nanoparticles 2.1.5 Characterization of nanoparticles 12 2.2 Formation of gold nanoparticle-DNA conjugates (nAu-DNA) 13 2.2.1 Introduction to synthetic DNA 13 2.2.2 Formation of gold nanoparticle-DNA conjugates (nAu-DNA) . 14 2.3 Applications of nAu in nanoassembly and ultrasensitive DNA detection . 15 2.4 Study on the plasmon coupling of metallic nanoparticle dimers . 25 2.5 Gel electrophoresis study on nanoparticle-DNA conjugates 27 2.5.1 Electrophoretic isolation of discrete nAu-DNA conjugates 27 2.5.2 Conformation study of nanoparticle-bound DNA . 29 2.6 Formation of discrete nanoparticle-DNA conjugate groupings . 31 2.7 Enzyme manipulation of the nanoparticle-bound DNA . 32 2.7.1 Introduction to restriction endonuclease . 32 2.7.2 Effect of steric hindrance on DNA hybridization and enzymatic reaction efficiency 33 2.7.3 Enzyme manipulation of nanoparticle bound DNA 34 Chapter Synthesis of gold nanoparticles (nAu) . 39 3.1 Materials and methods 39 3.2 Results and discussion 40 3.2.1 Synthesis and characterization of nAu 40 3.3 Conclusions 42 Chapter Efficient manipulation of nanoparticle-bound DNA via restriction endonuclease . 46 4.1 Introduction 46 4.2 Materials and methods 48 4.3 Results and discussion 53 IV 4.3.1 Importance of short ssDNA modification on the surface of nAu for achieving high enzyme digestion efficiency 53 4.3.2 Effect of ssDNA surface coverage on enzyme digestion nanoparticle-bound DNA . 55 4.3.3 Enzyme digestion efficiency of nanoparticle-bound DNA . 57 4.3.4 Effect of ionic strength on dT-ssDNA surface coverage on nAu and enzyme digestion efficiency 61 4.4 Conclusions 63 Chapter Fabrication of nanoparticle-DNA conjugates bearing specific number of short DNA strands by enzymatic manipulation of nanoparticle-bound DNA . 65 5.1 Introduction 65 5.2 Materials and methods 67 5.3 Results and discussion 71 5.3.1 Enzyme digestion efficiency of nanoparticle-bound dsDNA by polyacrylamide gel electrophoresis 71 5.3.2 Agarose gel electrophoresis of the nanoparticle-DNA conjugates 73 5.3.3 Restriction endonuclease digestion/cleavage of nanoparticle-dsDNA conjugates bearing definite number of DNA strands . 75 5.4 Conclusions 81 Chapter Nanoparticle based quantitative DNA detection with single nucleotide polymorphism (SNP) discrimination selectivity 82 6.1 Introduction 82 6.2 Materials and methods 85 6.3 Results and discussion 90 6.3.1 Formation of nAu-DNA conjugate dimers using linker DNA of different lengths . 90 6.3.2 Quantification of target DNA through the formation of nAu-DNA conjugate dimers . 93 6.3.3 Hybridization efficiency of strand A, strand revA with target DNA without nAu . 97 6.3.4 Single nucleotide polymorphism (SNP) discrimination using nAu-DNA conjugate groupings 100 6.4 Conclusions 104 Chapter Fabrication of gold nanoparticle based nano-groupings with well-defined structure 106 7.1 Introduction 106 7.2 Materials and methods 108 7.3 Results and discussion 114 7.3.1 Grouping percentage of nAu-DNA conjugates using linker DNA of different lengths 114 7.3.2 Effect of hybridization conditions on final grouping percentage 120 7.3.3 Electrophoretic mobility of conjugate groupings linked by various length of linker DNA . 123 V 7.4 Conclusions 125 Chapter Conclusions 127 Chapter Suggestions for future work . 130 9.1 Further study on the hybridization of nAu-DNA conjugates . 130 9.2 FRET based quantitative DNA detection using nAu and quantum dot as efficient fluorescent acceptor and donor . 132 9.3 Application of nAu-DNA conjugates in chip-based DNA detection . 133 9.4 Fabrication of multiple functionalized nAu-DNA conjugates bearing different DNA sequences and its application in SNP discrimination . 134 Reference 136 Appendix I Complete sequences of DNA used in Chapters and 155 Appendix II List of Publications 157 VI Summary Self-assembling of gold nanoparticles to form well-defined nano-structures is a field that has been receiving considerable research interests in recent years. In this field, DNA is a commonly used linker molecule to direct the assembly of nanoparticles because of its unique recognition capabilities, mechanical rigidity, enzyme processibility as well as physicochemical stability and has shown great potential in fabrication and construction of nanometer-scale assemblies and devices. This Ph.D. work aims to fabricate gold nanoparticles bearing definite number and length of DNA strands using gel electrophoresis isolation and restriction endonuclease manipulation of the nanoparticle-bound DNA. These specially designed nanoparticles are then applied for quantitative DNA detection and construction of well tailored nanogroupings. Topically this thesis is divided into chapters. Chapter is the introduction and outlines, the specific aims and scope of this thesis. Chapter reviews the current development in the literature. The main results and findings are discussed through Chapter to Chapter 7. The conclusions and suggestions for further work are covered in Chapter and Chapter respectively. VII In Chapter 3, we describe the synthesis of mono-dispersed gold nanoparticles of various sizes by the reduction of hydrogen tetrachloroaurate (III) tetrahydrate by trisodium citrate dihydrate and tannic acid. The size and size distribution of the gold nanoparticles were analyzed by UV-Vis spectroscopy and transmission electron microscopy (TEM). In Chapter 4, we demonstrate a strategy for efficient manipulation of gold nanoparticle-bound DNA using restriction endonuclease. The digestion efficiency of this restriction enzyme was studied by varying the surface coverage of stabilizer, the size of nanoparticles as well as the distance between the nanoparticle surface and the enzyme cutting site of nanoparticle-bound DNA. We found that the surface coverage of stabilizer is crucial for achieving high digestion efficiency. In addition, the surface coverage of this stabilizer can be tailored by varying the ion strength of the system. Based on the results of polyacrylamide gel electrophoresis and fluorescent study, a high digestion efficiency of 90+% for nanoparticle-bound DNA was achieved for the first time. This restriction enzyme manipulation can be considered as an additional level of control on the nanoparticle-bound DNA and is expected to be applied to manipulate more complicated nanostructures assembled by DNA. In Chapter 5, we report our novel approach to generate gold nanoparticle-DNA conjugates bearing specially designed DNA linker molecules that can be used as nanoprobes for quantitative DNA sequence detection analysis or as building blocks to VIII construct nano-groupings with precisely controlled structure. In our approach, gold nanoparticle-DNA conjugates bearing definite number of long dsDNA strands were prepared by gel electrophoresis. A restriction endonuclease enzyme was then used to manipulate the length of the nanoparticle-bound DNA. This enzymatic cleavage was confirmed by gel electrophoresis, and digestion efficiency of 90% or more was achieved. With this approach, nanoparticle conjugates bearing definite number of strand of short DNA with less than 20-base can be achieved. Sequence-specific DNA detection is important in various biomedical applications such as gene expression profiling, disease diagnosis and treatment, drug discovery and forensic analysis. In Chapter 6, we develop a gold nanoparticle-based method that allows DNA detection and quantification and is capable of single nucleotide polymorphism (SNP) discrimination. The precise quantification of single stranded DNA is due to the formation of defined nanoparticle-DNA conjugate groupings in the presence of target/linker DNA. Conjugate groupings were characterized and quantified by gel electrophoresis. A linear correlation between the amount of target DNA and conjugate groupings was obtained at lower target DNA concentration and can further be exploited for target DNA quantification. For SNP detection, single base mismatch discrimination was achieved for both the end-and center-base mismatch. The method holds promise for creating a quantitative and highly specific DNA detection method for biomedical applications. IX Many interesting properties of nanoparticle-based materials are highly dependent upon their structural parameters. In Chapter 7, we describe the fabrication of DNA induced gold nanoparticle nano-groupings with well-defined structures (dimers, trimers and other higher order multimers) using gold nanoparticles bearing definite number and length of DNA. These nano-conjugate groupings were analyzed using gel electrophoresis and discrete gel bands corresponding to groupings with defined structures were obtained. Various factors that affect the formation of nano-groupings were explored as well. The results show that direct linkage of two nanoparticle-DNA conjugates without linker DNA, longer hybridization time and higher ion strength buffer lead to higher degree of grouping. For nano-grouping formation, a minimum length of linker DNA of 24-base is needed for our nanoparticle-DNA conjugate system. Further increase in the linker length results in little improvement in the grouping percentage. Furthermore, it was found that the number of nanoparticles involved in the grouping structure is more effective in deciding its electrophoretic mobility than the length of linker DNA. TEM characterization further demonstrated that conjugate groupings extracted from each gel band consist of the expected grouping structure. This confirms that gel electrophoresis is an efficient tool for isolation of small grouping structures of nanoparticle-DNA conjugates. 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Edit. 2002, 41, 1277-1289. 154 Appendix I____________________________________________________________ Appendix I Complete sequences of DNA used in Chapters and Figure A1 Complete sequences of Strand A’ and Strand revA’. 155 Appendix I____________________________________________________________ Figure A2 Complete sequences of Strand C’, Strand revC1’ and Strand revC2’. 156 Appendix II____________________________________________________________ Appendix II List of Publications Journal publication: (1) Qin, W. J.; Yung, L. Y. L. Fabrication of gold nanoparticle based nanogroupings with well-defined structure. Manuscript in preparation. (2) Qin, W. J.; Yung, L. Y. L. Nanoparticle based quantitative DNA detection with single nucleotide polymorphism (SNP) discrimination selectivity. Manuscript in preparation. (3) Tan, W. L.; Qin W. J.; Yung, L. Y. L. Difference in "Base Pair to Termini" affects the enzymatic digestion of nanoparticle-bonded DNA. Biomacromolecules 2007, 8, 750-752. (4) Qin, W. J.; Yung, L. Y. L. Efficient manipulation of nanoparticle-bound DNA via restriction endonuclease. Biomacromolecules 2006, 7, 3047-3051. (5) Qin, W. J.; Yung, L. Y. L. Nanoparticle-DNA conjugates bearing a specific number of short DNA strands by enzymatic manipulation of nanoparticle bound DNA. Langmuir 2005, 21, 11330-11334. Conference publication: (1) Qin, W. J.; Yung, L. Y. L. AIChE Annual Meeting, San Francisco, CA, USA, November 2006. (2) Yung, L. Y. L.; Qin, W. J. Abstracts of Papers of The American Chemical Society 2006, 231, Atlanta, GA, USA, March 2006. (3) Qin, W. J.; Yung, L. Y. L. Abstracts of Papers of The American Chemical Society, 230, Washington, DC, USA, August 2005. (4) Qin, W. J.; Yung, L. Y. L. International Conference on Materials for Advanced Technologies (ICMAT 2005) Suntec City, Singapore, July, 2005. 157 [...]... nanoparticledsDNA conjugates bearing Strand A or strand B Lane P1 and Q1 correspond to the conjugates bearing 5 strands of digested Strand A and Stand B respectively; Lane P2 and Q2 correspond to conjugates bearing 5 strands of intact Strand A and B respectively; Lane N corresponds to mixed conjugates bearing different strands of Strand A and Lane O correspond to the conjugate bearing only 5-base ssDNA without... growth of nanocircuitry11, 12, and in developing DNA sequence detection3 6, 37 1.2 Aims and scope of this project This Ph.D work aims to fabricate gold nanoparticles bearing definite number and length of DNA strands The scope of this work includes studying the enzyme manipulation efficiency of gold nanoparticle- bound DNA, preparing well defined 3 Chapter 1 _ nanoparticle- DNA conjugates. .. including (i) stabilizer surface coverage on nanoparticle, (ii) distance between nanoparticle surface and enzyme-cutting site of particle-bound DNA, and (iii) size of nanoparticles 2 To fabricate gold nanoparticles bearing definite number of DNA strands with predetermined length Nanoparticle- DNA conjugates bearing definite number of long double-stranded DNA strands are prepared by gel electrophoresis A... 4, 3 and 2 strands of Strand B respectively; Lanes I2, J2 and K2 correspond to the conjugates bearing 4, 3 and 2 strands of Strand B without EcoR V digestion respectively; Lane L corresponds to mixed conjugates bearing different strands of Strand B and Lane M correspond to the conjugate bearing only 5-base ssDNA without Strand B 78 Figure 5.7 Comparison of electrophoretic mobility of the digested nanoparticledsDNA... Strand A Lanes A1, B1 and C1 correspond to EcoR V digested conjugates bearing 4, 3 and 2 strands of Strand A respectively; Lanes A2, B2, C2 correspond to the conjugates bearing 4, 3 and 2 strands of Strand A without EcoR V digestion respectively; Lane D corresponds to mixed conjugates bearing different strands of Strand A and Lane E correspond to the conjugate bearing only 5-base ssDNA without Strand... picture of the formation of nanoparticle- DNA conjugate groupings 89 Figure 6.3 PAGE characterization of enzyme digested 9.7 nm nAu-bound DNA Lanes 1-8 correspond to: 10bp DNA ladder, strand A' (free), strand A' (bound), strand A' (no enzyme), strand revA' (free), strand revA' (bound), strand revA' (no enzyme) and 10bp DNA ladder 91 XV Figure 6.4 Formation of nAu -DNA conjugate dimers with various length of. .. manipulate the length of the nanoparticle- bound DNA 3 To conduct quantitative DNA detection and SNP discrimination study using the well defined gold nanoparticle- DNA conjugates fabricated in the previous experiments Establish a reliable correlation between the amount of target DNA and conjugate groupings formed and study the selectivity of SNP discrimination using single base mismatched DNA 4 Chapter 1 ... Comparison of the electrophoretic mobility of the enzyme treated and nonenzyme treated 5-base ssDNA modified nanoparticles Lanes F-H correspond to enzyme treated nanoparticles, non-enzyme treated nanoparticles, and nanoparticledsDNA mixture respectively 76 Figure 5.6 Agarose gel electrophoresis of nanoparticle- dsDNA conjugates bearing Strand B Lanes I1, J1 and K1 correspond to EcoR V digested conjugates bearing. .. efficiency of free DNA and nanoparticle- bound DNA by endonuclease EcoR V 73 Table 6.1 Hybridization efficiency of strand & strand revA with various ratios of target DNA 98 Table 7.1 Relative mobility of conjugate groupings linked by different length of linker DNA 124 XI List of figures Figure 2.1 Electrostatic stabilization of nanoparticles 10 Figure 2.2 Steric hindrance stabilization of nanoparticles... These specially designed nanoparticles are then applied for quantitative DNA detection and construction of well tailored nano-groupings The specific objectives of this thesis include: 1 To investigate the feasibility of enzymatic manipulation of the gold nanoparticle- bound DNA A systematic study on various factors that affect the enzyme manipulation efficiency of nanoparticle- bound DNA is conducted, including . Chemical and Biomolecular Engineering Thesis title: Fabrication of gold nanoparticle -DNA conjugates bearing specific number of DNA for quantitative detection and well-defined nanoassembly Year of. Year of submission: 2007 II FABRICATION OF GOLD NANOPARTICLE -DNA CONJUGATES BEARING SPECIFIC NUMBER OF DNA FOR QUANTITATIVE DETECTION AND WELL- DEFINED NANOASSEMBLY QIN. nanoparticle- dsDNA conjugates bearing Strand A or strand B. Lane P1 and Q1 correspond to the conjugates bearing 5 strands of digested Strand A and Stand B respectively; Lane P2 and Q2 correspond to conjugates