Incorporating Stimuli-Responsive DNA Strands with Functional Nanomaterial for Biophysical and Analytical Applications

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Incorporating Stimuli-Responsive DNA Strands with Functional Nanomaterial for Biophysical and Analytical Applications
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1 online resource (99 p.)
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english
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Zhang,Yunfei
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University of Florida
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Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
Tan, Weihong
Committee Members:
Powell, David H
Omenetto, Nicolo

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Subjects / Keywords:
azobenzene -- dna -- mercuric -- mesoporous -- nanomaterial -- nanomotor -- plasmonic -- resonance -- surface
Chemistry -- Dissertations, Academic -- UF
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Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Due to their functionality, stability, versatility, and programmability, nucleic acids have received extensive attention in the scientific community as outstanding nanoscale macromolecule materials 1. Several external stimuli such as light irradiation fluctuation, ion concentration variation, temperature change, electric field oscillation, and pH change can cause the geometry or formation change. My research projects focus on combining both the advantages of stimuli-responsive DNA and the optical and structural properties of nanoparticles, in order to realize the development of different applications. The first project was to use silver nanoparticle for developing highly efficient light-driven azobenzene incorporated DNA nanomachine. Light-driven DNA motor with good reversibility has shown its great potential in converting photonic energy into of molecular scale movement; however, the challenge of nanomotor is set by its low conversion efficiency. We developed here a silver nanoparticle enhanced DNA nanomotor system, which significantly improved working efficiency of DNA motors. The enhancement mechanism was proposed through surface-plasmon resonance effect, which was simulated and explained by the theoretical calculation of surface plasmonic localized electric field distribution. The second project employed the light-driven azo-DNA to devise a light-controlled release system based on mesoporous materials. Through capping the pores of mesoporous nanoparticle with azo-DNA strands, cargo release could be controlled by different light irradiations. By switching the irradiation wavelength, a fast-response, highly-reversible controlled release system has been designed and fabricated. The third project was the conjugation of Hg2+ sensitive T-rich DNA to mesoporous silica nanoparticles for sensitively detecting Hg2+. In this project, mesoporous silica nanoparticles acted as a dye carrier and were capped by T-rich DNA. In the presence of Hg2+,T-rich DNA with a stronger binding with Hg2+ detached from the nanoparticle, followed by the release of the dye cargo, and thus the detection of Hg2+ was realized by measuring the fluorescence intensity of the released dye. This approach was approved to have the lowest detection limit down to ppb level in a short time without any interference from other metal ions. In summary, my research has mainly focused on the molecular design of DNA strands functionalized nanoparticles for guest molecule delivery, ion detection, and nanomotor development.
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Yunfei Zhang.
Thesis:
Thesis (M.S.)--University of Florida, 2011.
Local:
Adviser: Tan, Weihong.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-08-31

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1 INCOR PORATING STIM ULI RESPONSIVE DNA STRANDS WITH FUNCTIONAL NANOMATERIAL FOR BIOPHYSICAL AND ANALYTICAL APPLICATIONS By YUNFEI ZHANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Yunfei Zhang

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3 To m y b eloved p arents and f ianc

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4 ACKNOWLEDGMENTS First, I wish to express my sincerest gratitude to my exceptional research advisor, Dr. Weihong Tan for offering me such a precious opportunity to study and research under his guidance during the two year studies at University of Florida. His precious advice, inspiration and encouragement strongly encourage me to be a more confident person and a better scientist. I also appreciate his unselfish supporting to me fighting against all the difficulties I faced in this two years, which will definitely have a great impact throughout my life and career. I would also like to express my sincere thanks to my committee members, Dr. David Powell, and Dr. Nicolo Omenetto for the helpful discussion, advice and assistance I really want to thank Dr Nicolo Omenetto for his guidance in course study and thank Dr Powell for his kind est help in my first year MS study I also want to thank Dr. Ben Smith for all the help and support in this two years. In particular, I would also like to thank Dr. Kathryn R. Williams for her kindness and help with the experiments and manuscript s. This th esis is a result of successful collaboration with many great scientists in different areas. I especially would like to thank Dr. Quan Yuan for the guidance, tremendous help and friendship during me first two years of research. I want to greatly thank Dr Y an Chen for all her guidance in my research and help in my personal help. I want to thank Meghan B. for her patient guidance to teach me and help me in all the cell experiment I am grateful to Mingxu You for the many helpful discussions on dif ferent projects as a knowledgeable friend. I am thankful to Dr. Zhi Zhu for being there whenever I need any help or advice. I thank Dr. Huaizhi Kang for her

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5 help in Azobenzene DNA synthesis and purification Also I would like to thank Tao Chen for all the helpful discussions It has been a great time and experience in the Tan research group, and it is my great pleasure to meet so many kind and nice friends here I would like to thank Dr. Kwame Sefah, Dr. Kelong Wang, Dr. Ling Meng, Dalia Lopez Colon, Hui Wa ng, Suwussa Bamrungsap, Xiangling Xiong, Basri Gulbakan Lu Peng, Da Han, Guizhi Zhu Emir Yasun, Ismail soy and others for their friendship and help. Each of them has made the time very enjoyable and pleasant. Also I am deeply grateful to my fianc H e Wei for his selfless supporting to all my important choice in my life I appreciate all the things he has dedicated for our love, and for our future His smile is the strongest motivation in my life. Finally, I owe a huge debt of gratitude to my parents for their sincerest love, encouragement, and support to me Their endless love and constant guidance make me who I am. I really appreciate the ir unselfish dedication to me all this time. I also want to thank my four years old younger sister; she is the most beautiful sunshine in my life, forever and ever.

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6 TABLE OF CONTENTS page ACKN OWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 BACKGROUND ................................ ................................ ................................ ...... 16 Review of Nucleric Acid ................................ ................................ .......................... 16 Discover y of Nucleic Acids ................................ ................................ ............... 16 Composition and Structure of Nucleic Acids ................................ ..................... 16 The Principle of Complementary Base Pairing ................................ ................. 17 Stimuli response DNA Switches ................................ ................................ ....... 17 PH responsible DNA switches ................................ ................................ ... 18 Light responsible DNA switches ................................ ................................ 18 Ion responsible DNA switches ................................ ................................ ... 19 The Surface Plasma Resonance of Metal nanoparticles ................................ ........ 20 Bio functionalized Mesoporous Silica Nanoparticles and their Applications ........... 21 2 USING SILVER NANOPARTICLE FOR THE ENHANCEMENT OF LIGHT DRIVEN DNA NANOMACHINE ................................ ................................ .............. 25 Experimental Section ................................ ................................ .............................. 26 Chemicals ................................ ................................ ................................ ......... 26 Instrumentation ................................ ................................ ................................ 26 Synthesis of Sil ver and Gold Nanoparticle ................................ ....................... 26 Ag nanosphere ................................ ................................ ........................... 26 Ag nanowire ................................ ................................ ............................... 27 Ag nanoprism ................................ ................................ ............................. 27 Au nanosphere ................................ ................................ ........................... 28 Au nanorod ................................ ................................ ................................ 28 Characterization of Silver and Gold Nanoparticle ................................ ............. 28 Irradiation Source ................................ ................................ ............................. 30 Theoretical Calculation of Electric Field Distribution around Nanoparticle ....... 30 Results and Discussion ................................ ................................ ........................... 31 Design of Light driven Nanomotor ................................ ................................ .... 31 Calculation of Conversion Efficiency ................................ ................................ 31 Enhanced Conversion Efficiency with Ag Nanoparticle ................................ .... 32 Reversibility of Ag Nanowire Enhanced DNA Nanomotor ................................ 33 Fluorescence Micrographs of DNA Nanomotor Conversion ............................. 33

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7 Localized Surface Plasmon of Ag Nanoparticle ................................ ................ 34 Absorbance Spectral of Ag Nanoparticle and Azobenzene Molecule ............... 34 Au nanoparticle and Effects on DNA Nanomotor ................................ ............. 35 Conclusion ................................ ................................ ................................ .............. 35 3 USING LIGHT DRIVEN DNA STRANDS FOR THE PHOTO CONTROLLED REVERSIBLE RELEASE SYSTEM OF MESOPOROUS NANOCONTAINER ....... 51 Experimental Section ................................ ................................ .............................. 51 Chemicals ................................ ................................ ................................ ......... 51 Instrumentations ................................ ................................ ............................... 52 Synthesis of Azobenzene Phosphoramidite ................................ ..................... 52 Synthesis and Purification of DNA Sequences ................................ ................. 53 Synthesis of Mesoporous Silica Nanoparticle ................................ ................... 54 Surface Functionalize of Mesoporous Silica Nanoparticle with Short DNA ...... 54 Dye Loading and DNA Capping of Mesoporous Silica Nanoparticle ................ 54 Irradiation Sources ................................ ................................ ........................... 55 Characterization of the DNA modified Mesoporous Silica Nanoparticle (MSN DNA) ................................ ................................ ................................ ... 55 Dye Release Experiment of DNA Capped MSN R6G ................................ ...... 56 Results and Discussion ................................ ................................ ........................... 56 Dye Releas e Test under Different Irradiation Source ................................ ....... 57 Optimization of A zo DNA Sequences ................................ ............................... 58 Reversibility of Dye Release System ................................ ................................ 58 Conclusion ................................ ................................ ................................ .............. 59 4 USING ION SENSITIVE DNA STRANDS FOR THE DETECTION OF MERCURIC ION ON A MESOPOROUS STIMULI RELEASE SYSTEM ................ 69 Experimental Section ................................ ................................ .............................. 70 Chemicals ................................ ................................ ................................ ......... 70 Buffer Solutions ................................ ................................ ................................ 70 Synthesis of Mesoporous Silica Nanoparticle ................................ ................... 71 Surface Modification and Dye L oading of Mesoporous Silica Nanoparticle ...... 71 Characterization of Mesoporous Silica Nanoparticle ................................ ........ 72 Synthesis and Purification of DNA Sequences ................................ ................. 72 Adjustment of Short DNA on Particle Surface ................................ .................. 73 DNA Capping of the Mesoporous Silica Nanoparticle ................................ ...... 74 Quenching effect of Mercuric Ion on Different Organic Dyes ............................ 75 Dye Release Experiment of DNA Capped MSN R6G ................................ ...... 75 Results and Discussions ................................ ................................ ......................... 75 Fluorescence Test of Free DNA ................................ ................................ ....... 76 Detection of Hg 2+ using Stimuli Release Mesoporous System ......................... 78 Selectivity Test ................................ ................................ ................................ 78 Conclusion ................................ ................................ ................................ .............. 79

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8 5 SUMMARY AND FUTURE DIRECTIONS ................................ .............................. 92 Incorporating Stimuli responsive DNA Strands with Functional Nanomaterial for Biophysical and Analytical Applications ................................ ............................... 92 Future Directions ................................ ................................ ................................ .... 93 LIST OF REFERENCES ................................ ................................ ............................... 94 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 99

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9 LIST OF TABLES Table page 2 1 Sequence of azobenzene nanomotor, normal nanomotor, and complimentary DNA to nanomotor. ................................ ................................ ............................. 50 3 1 Sequence of a zobenze ne DNA, normal DNA, and arm DNA ............................. 68 3 2 S urface area and pore volume of m esoporous silica nanoparticle before surface modification, after surface modification and DNA conjugation, and after dye loading. ................................ ................................ ................................ 68 4 1 Sequence of Arm DNA, Linker DNA, and Fluorefore Linker DNA and Complimentary Linker DNA ................................ ................................ ................ 91

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10 LIST OF FIGURES Figure page 1 1 The g eneral structure of nucleobase ................................ ................................ .. 23 1 2 The b ackbone s tructure of DNA and RNA ................................ .......................... 23 1 3 The p rinciple of complementary base pairing ................................ ..................... 24 1 4 The a bnormal nucleobase pairing ................................ ................................ ...... 24 2 1 Ag nanoparticle with different morphology and size. ................................ .......... 37 2 2. Au nanoparticle with different morphology and size. ................................ ........ 38 2 3 Synth esis procedure of azobenzene p hosphor amidite. ................................ ...... 39 2 4 Open close conversion of DNA nanomotor with three azobenzene moieties.. ... 40 2 5 Scheme of the enhanced DNA nanomotor. ................................ ...................... 41 2 6 Fluorescence spectra of DNA nan omotor with Ag nanoparticle .......................... 42 2 7 Conversion efficiency of DNA nanomotors in the existence of Ag nanomaterials with different morphologies ................................ ......................... 43 2 8 Reversible conversion of DNA nanomotor with Ag nanowire. ............................ 44 2 9 Fluorescence micrographs of DNA nanomotor solution. ................................ .... 45 2 10 Localized electric field intensity distributions IEI 2 calculated for Ag nanoparticle with different morphology ................................ ............................... 46 2 11 Extinction spectrum of Ag nanostructures and absorption spectrum of azobenzene molecule. ................................ ................................ ........................ 47 2 12 Image of Au nanoparticle and fluorescence spectrum of DNA motor.. ............... 48 2 13 The experimental extinction spectrum of Au nanoparticle with absorption spectral of azobenzene. and theoretical calculate d electric field distributions IEI 2 ................................ ................................ ................................ ...................... 49 3 1 TEM image of DNA modified MSN. ................................ ................................ .... 60 3 2 TEM image of DNA modified MSN. ................................ ................................ .... 61 3 3 Small angle XRD pattern of MSN, MSN NCO, and MSN DNA. ......................... 62

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11 3 4 BET nitrogen adsorption/desorption i sotherms of DNA functionalized m esoporo us silica nanoparticle ................................ ................................ .......... 62 3 5 BJH pore size distr ibutions of DNA functionalized m esoporo us s ilica n anoparticle ................................ ................................ ................................ ........ 63 3 6 The s etup of the mesoporous dye release device. ................................ ............. 63 3 7 The s cheme of azobezene modified DNA based light driven reversible release system. ................................ ................................ ................................ .. 64 3 8 Time dependent rhodamine 6G release curves from DNA capped mesoporous silica nanoparticle under different irradiation.. ................................ 65 3 9 Dye release under UV irradiation from mesoporous particle with azo DNA and normal DNA for 1500 min ................................ ................................ ............ 66 3 10 Dye release under UV irradiation from me soporous particle with different azo DNA for 1500 min ................................ ................................ ........................ 66 3 11 Dye release curve from MSN as a function of time while switching ir radiation source every 120 mins ................................ ................................ ....................... 67 4 1 TEM image of mesoporous silica nanoparticle. ................................ .................. 81 4 2 SEM image of mosoporous silica nanoparticle. ................................ .................. 82 4 3 P owder X ray patterns of the solid m esoporous silica nanoparticle. ................... 83 4 4 The titration curve of Arm DNA bin ding on per gram of dye loaded m esoporous silica nanoparticle. ................................ ................................ ......... 84 4 5 Quenching effect of mercuric ion on different organic dyes ................................ 84 4 6 Setup of the mesoporous dye release device ................................ ..................... 85 4 7 The s chematic representation of the synthesis surface modification, dye loading, DNA binding of the MSN and the dye release of the MSN in the existence of the Hg 2+ ................................ ................................ ........................ 86 4 8 H ybridization of the Arm DNA and Linker DNA in the existence of the Hg 2+ ...... 86 4 9 Scheme of the hybridization of the Arm DNA, Fluorophore Linker DNA and Complimentary Linker DNA. ................................ ................................ ............... 87 4 10 Fluorescence intensity of the 100nm Fluorophore Linker DNA solution after the addition of certain amount of Arm DNA, Hg 2+ and Complimentary Linker DNA. ................................ ................................ ................................ ................... 87

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12 4 11 Release curve of r hodamine 6G from Linker DNA capped MSN R6G in mercuric acetate solu tion of different concentration s ................................ ......... 88 4 12 different concentration s ................................ ................................ ...................... 89 4 13 Fluorescence intensities of the released dye from Linker DNA capped MSN R6G in the presence of 1ppm different cation s ................................ .................. 90

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13 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INCORPORATING STIMULI RESPONSIVE DNA STRANDS WITH FUNCTIONAL NANOMATERIAL FOR BIOPHYSICAL AND ANALYTICAL APPLICATIONS By Yunfei Zhang August 2011 Chair: Weihong Tan Major: Chemistry Due to their functionality, stability, versatility, and programmability nucleic acids ha ve received extensive attention in the scientific community as outstanding nanoscale m acromolecule materials 1 Several external stimuli such as light irradiation fluctuation, ion conc entration variation, temperature change, electric field oscillation, and pH change can cause the geometry or formation change. Through the specific and rapid response to external stimuli, it is promising to design nucleic acids that serve into molecular s ensors and nanoscale molecular motors. On the other side, the nanoscale sizes endow particles with interesting properties, including the surface plasmon resonances of noble metal nanoparticles, and the porous structure of mesoporous silica nanoparticles. M y research projects focus on combining both the advantages of stimuli responsive DNA and the optical and structural properties of nanoparticles, in order to realize the development of high efficiency DNA nanomotor, DNA controlled release system, and DNA ba sed ion detection in different systems. The first project was to use silver nanoparticle for developing highly efficient light driven azobenzene incorporated DNA nanomachi ne. Various DNA based nanomotor

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14 have been investigated due to the fast development of DNA technology. For instance, Light driven DNA motor with good reversibility has shown its great potential in converting photonic energy into kinetic energy of molecular scale movement ; however the challenge of nanomotor is set by its low conversion effi ciency. We developed here a silver nanoparticle enhanced DNA nanomotor system which significantly improved working efficiency of DNA motors than previous works. The enhancement mechanism was proposed through surface plasmon resonance effect, which was sim ulated and explained by the theoretical calculation of surface plasmonic localized electric field distribution. The early success makes us believe this work could potentially benefit the future development of more efficient light driven DNA nanomotor. The second project employed the light driven azo DNA to devise a light controlled release system based on mesoporous materials. Light control has the advantages of simple operation, rapid response, and high reversibility. With large inner space and homogeneous porous structure, mesoporous silica nanoparticles have functioned as good cargo carriers. Through capping the pores of mesoporous nanoparticle with azo DNA strands, cargo release could be controlled by different light irradiations. By switching the irradi ation wavelength ( 365nm for releasing, 450nm for capping), a fast response, highly reversible controlled release system has been designed and fabricated, possibly contributing to the controllable drug release in therapeutic medicine and pharmacology The third project was the conjugation of Hg 2+ sensitive T rich DNA to mesoporous silica nanoparticles for sensitively detecting Hg 2+ through a signal amplification process. As is well known, mercury pollution is a serious problem posing severe health risks to

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15 humans even at very low concentrations. Traditional detection methods usually require complicated sample preparation and expensive instruments and it is therefore imperative to develop an easy and simple detection approach for Hg 2+ detection. In this proj ect, mesoporous silica nanoparticles acted as a dye carrier and were capped by T rich DNA. In the presence of Hg 2+ T rich DNA with a stronger binding with Hg 2+ detached from the nanoparticle, followed by the release of the dye cargo, and thus the detecti on of Hg 2+ was realized by measuring the fluorescence intensity of the released dye. This approach was approved to have the lowest detection limit down to ppb level. The detection could be achieved in a short time and without any interference from other me tal ions. The great potential s of this Hg 2+ sensing system would be further tested for detecting aqueous contamination in real sample In summary my research has mainly focused on the molecular design of DNA strands functionalized nanoparticles for guest molecule delivery, ion detection, and nanomotor development. I expect to explore more in the biomedical area in the future.

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16 CHAPTER 1 BACKGROUND Review of Nucle r ic Acid Discovery of Nucle ic Acids Nucleic acids are biological molecules that contain information They were first discovered by Friedrich Miescher from the nuclei of white blood cells in 1869 in Felix Hoppe Seyler's laboratory at the University of Tbingen, Germany 2 The double helix structure of nucleic acid was pioneering elucidated in 1953 by Watson and Crick 3 Composition and Structure of Nucle ic Acids Generally nucleic acids can be divided into two main groups: deoxyribonucleic acid(DNA) and ribonucleic acid(RNA) according to their difference in the formation of nucleotides. Nucleotides are building blocks of nucleic acids macromolecule, and each nucleotide can be divided into three components: a nuc leobase a pentose sugar and a phosphate group DNA and RNA are just distinguished by a hydroxyl group in suga r. DNA ha s 2' deoxyribose while RNA ha s ribose The nucleobase can be also labeled into two groups: the purines group, labeled as adenine(A) and g uanine(G), the pyrimidines group labeled as cytosine(C), uracil(U) and thymine(T) (s tructures shown in Figure 1 1, thymine only exists in DNA an d uracil only exists in RNA). The nucleotides are connected by phosphodiester linkages between sugars and phosphates to form a sugar phosphate backbone(Shown in Figure1 2) and the ends of each nucle ic acid The nucleobases have covalent binding to the sugar phosphate backbone via an N glycosidic linkage between a nuc leobase ring nitrogen and a carbon on the pentose sugar ring.

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17 The Principle of Complementary Base Pairing A significant important discovery was reported by Chargaff et al 4 A double stranded DNA mol ecule has percentage base pair equality between adenine(A) and thymine(T) guanine(G) and cytosine(C), although base compositions are valid for each of the two DNA strands. Afterwards the famous Watson Crick double helix DNA structure was proposed in which the double strands DNA are formed by hydrogen bonding between purine and pyrimidine. In the typical Watson Crick DNA base pairing, adenine (A) forms two hydrogen bonding with thymin e (T) and guanine (G) forms three hydrogen bonding with cytosine (C) (as shown in Figure 1 3) Additionally, thymine is replaced by uracil (U) in RNA In this helix structure of DNA duplex the nucleotides are paired in the middle while the ribose sugar and phosphate groups that form the backbone are exposed at outer edge. Generally DNA molecules are mostly double stranded in contrast RNA molecules are mostly single stranded, but the exceptions also exist. Stimuli response DNA Switches A single strand DNA mol ecule has a strong potential of recognizing and hybri dizing with its complementary sequence through this highly specific complimentary base pairing interactio ns. This remarkable specificity enable s DNA strands coupled other functional moieties to form the specific nanostructures or nanomachine which undergo structural transitions in response to external stimuli. Such stimuli include complimentary strand, pH change, ions, light irradiation, electrical field and temperature change.

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18 PH responsible DNA s witches The famous pH responsive DNA structure i motif, discovered by Gueron et al. in 1993, is the C by base pairing between short stretches of protonated and unprotonated cytosines 5 The protonated cytosine can form a self hybridized structure with three hydrogen bonding (shown in Figure 1 4 A) d(TCCCCC) and quadruplex i n the acidic surrounding while in basic surrounding this structure would be destroy and return back to the initially linear structure. This pH responsive DNA s witching has been widely used as a pH sensor or pH driven nanomotor. Liu and Balasubramanian et al have developed an i motif based pH DNA sensor by labeling the ends of i motif with fluorophore and quencher 6 At acidic pH i motif strand folded into the closed i motif structure then fluorophore was quenched while at basic pH i motif strands returned to linear state and the fluorescence expressed again. Shu et al. developed an i motif based DNA motor based motivated by pH switching 7 which converted biochemical reactions into mechanical work through the conformational ch ange of i motif strands. Chen et al. developed a pH controlled release system by capping pore outlets of mesoporous silica nanoparticles with i motif DNA strand 8 The open close of the pore was controlled by the geometry change of i motif at different pH and this process was highly reversible. And the pH sensitive DNA can even be utilized to map pH changes in living Caenorhabditis elegans worm 9 which suggested a great poten tial of i motif DNA strands in in vivo pH mon itoring. Light responsible DNA s witches The light driven DNA strands are usually coupled with photo sensitive molecules such as azobenzene 10 and 2 nitrobenzyl linker 11 The synthesis protocol of 2 nitrobenzyl

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19 linker bridging photocleavable DNA was reported by Bai et al. in 2002 11 By introducing 1 nitrobenzyl linker into DNA strands, the DNA molecule can be efficiently and rapidly cleaved under UV irradiation. In 2007 Asanuma et al. reported a detailed protocol for the synthesis of azobenzene phorsphoramid ite tet hered to DNA to make it photoresponsive 10 Azobenzene is a photo responsive molecule that isomerizes between its planar trans form and non planar cis form under different light irradiation. Tethered with azobenzene molecule, the hybridization of DNA duplex can be controlled by switching the irradiation source. Light driven DNA nanomotor structure were under this mechanism 12 The nanomotor was completely reversible, and did not decompose or induce undesirable side reactions after repeated more than 10 cycles Also a light controlled DNA 3 dimensional structure was discovered wher e the geometry of a DNA tetrahedron was controlled by switching light irradiations of different wavelengths 13 Ion responsible DNA switches Besides the principle of complementary base A T AND C G p airing roles, DNA nucleobases have some abnormal pairings with the existence of metal ions such as Hg 2+ and Ag + Hg 2+ can selectively bind to two thymine (T) bases and forms stable T hymine Hg 2+ T hymine complexes, which are even more stable than the T A Watson Crick matching (shown in Figure 1 4 B ). And additionally, this interaction is highly s pecific as only Hg 2+ can form the T hymine T hymine mismatching base pair. Ag + has a similar property but associate with two cytosine form ing C ytosine Ag + C ytosine complexes ( shown in Figure 1 4 C) This specific interaction between metal cation and DNA has been thoroughly used in the design of molecular b eacon for the ion detection. Lin et al. developed a colorimetric approach using DNA functionalized gold nanoparticles for the detection of mercuric ion 14 This approach took advantage of both

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20 catalytic properties of DNA functionalized gold nanoparticles and the specific binding of a C ytosine Ag + C ytosine mismatch, and consequently it realized selectively detection of Ag + from 19 other metal ions. Lin et al. reported an inexpensive method, where DNA functionalized hydrogel system for ultrasensitive detection and removal of Hg 2+ in w ater 15 In these works DNA strands always give a rapid and sensitive response to the target metal ions, suggest ing a brightly future of using ion stimuli responsive DNA for the ultrasensitive, instrument free, rapid and visual detection of metal ions. The Surface Plasma R esonanc e of M etal nanoparticles Nanoscale metal particle has the unique optical property of surface plasmon resonances, which a re quantized electron oscillations that confined to nanoscale volumes The first theory of light scattering an d absorption by gold nanosphere was reported by Gustav Mie more than a hundred years ago 16 and several modern theories have been developed after that. While metals or semiconductors is irradiated by visible electromagnetic waves, the collective oscillation of con duction band electrons will occur and the electron excitations is defined as Plasmons 17 On bulk metal or extended metal surface plasmons are free to propagate, while on a nano scale metal particle plasmons are imposed with a boundary condition to a finite volume From Mie theor y, the extinction cross section C ext for the scattering of a metal nanosphere can be calculated by equation: 17 In this equation R is the radius of nanosphere m is the frequency independent )

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21 1 2 while 2 t he material dielectric constant Plasmons spherical particles are dipolar which results in only a single plasmon resonance peak, while the anisotropic nanoparticles such as nanorod and nanoprism can support many plasmon modes 18 Several method s are applied to get a deeper understand ing of the relationship between geometry and optical properties on nanoparticle. D iscrete dipole approximation ( DDA ) is the most widely numerical tool for investigating shape ef fects on nanoparticle plasmonic i n which particle is discretized into elementary subunits that are modeled as dipoles. From all this equation and principle t he optical properties of plasmonic atoms of metal nanoparticle are decided by size and morphology of the particle material composition and surrounding dielectric environment 19 21 while the size and morphology are the dominating facts. So controlling the size and morphology of metal nano particles in synthesis step is the best way to engineer the position and strength of plasmonic resonance bands Bio functionalized Mesoporous Silica Nanoparticles and their A pplications M esoporous material is the homogeneous material defined by pores diameters between 2 and 50 nm ( by IUPAC) Th e most commonly used mesoporous materials are silica and alumina while other mesoporous oxides as niobium, tantalum, titanium, zirconium, cerium and tin have also been reported In 1992 a homogeneous mesoporous silica nanosphere was synthesized in Mobil Re search and Development Corporation Central Research Laboratory and named as Mobil Crystalline Materials(known as the famous MCM 41) 22 In 1998 another fiber shaped meso porous silica nanoparticle was synthesized by Zhao et al. in University of California, Santa Barbara and named as Santa Barbara Amorphous type material(noted as SBA 15) 23

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22 With the advantages of good biocompatibility, large loading capacity, highly ordered pore structu re and adjustable pore size, mesoporous silica nanoparticle has already been widely used as carrier vehicles 8 24 And more because of the numerous si lanol group that on the surface mesoporous silica nanoparticle c an be easily be immobilized with various organic or biological molecules as linkers then link to some large molecule to cap the pore, while those linkers can also be cle aved by some specific stimuli which lead to the uncapping of pore and the release of th e cargo 24 The stimuli are like capping is mesoporous silica nanoparticle can be capped by various of materials such as DNA 25 protein 26 27 nanoparticle 28 29 polymers 30 33 and can also be uncapped by various of stimulates such as hea t 26 magnetic field 30 pH 8 24 29 target molecules 28 34 enzyme 31 33 35 or even light 32

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23 Figure1 1 The g eneral structure of nucleobase Figure1 2 The b ackbone s tructure of DNA and RNA

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24 Figure1 3 The p rinciple of complementary base p airing Figure 1 4 The a bnormal nucleobase pairing s A) Protonated Cytosine C ytosine pairing. B) Thymine Hg 2+ Thymine pairing C) Cytosine Ag + Cytosine pairing

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25 CHAPTER 2 USING SILVER NANOPAR TICLE FOR THE ENHANCEMENT OF LIGHT DRIVEN DNA NANOMACHINE In the recently years, stimuli responsive DNA has received much attention as nanomachines to make nanoscale movements in response to external stimuli 6 36 39 In 2009 Kang et al. reported a photo responsive DNA nanomachine with incorporated azobenzene moieties 12 using the photoisomerization of azobenzene to regulate DNA hybridization then control the open and close of DNA Nanomachine. In theoretical calculation the contraction force of a single DNA nanomotor could be in pN level. With the good reversibility of DNA nanomotor, this kind of photo driven nanomotor promised us a approach to generate photonic energy into mechanical movement. However, the disadvantage of azobenzene DNA na nomotor is the low close open conversion due to the low photoisomerization efficiency of azobenzene moieties, which is the bottle neck problem to the further development of light driven nanodevice applications. Here, we reported our discovery that silver nano particle could enhance the open close conversion efficiency of DNA nanomotor by plasmonic near field coupling and spectral overlap with incorporated azobenzene moieties. The open close conversion efficiency of light driven DNA nanomotor was enhanced fr om initial 20% to 85% with the existence of silver nanowire. The experimental results were confirmed by theoretical modeling calculations of the localized electric field distribution and absorbance spectral overlap of silver nanoparticle with different mor phology The DNA nanomotor with enhanced conversion efficiency shows a g reat potential in light driven n anomachine design and solar energy conversion

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26 Experimental Section Chemicals All the chemicals to synthesis a zobenzene p hosphoramidite Silver and Gol d nanoparticle we re purchased from Sigma Aldrich chemical, Inc. The materials for DNA synthesis, including CPG columns and reagents for DNA modification and coupling, were purchased from Glen Research Co. Instrumentation The synthesis and coupling of all t he DNA sequences were taken out on ABI3400 DNA/RNA s ynthesizer ( Applied Biosystems) .Purifications were carried out on a ProStar HPLC system with a gradient unit(Varian) and a C 18 column(Econosil, 5U, 250X4.6mm) The concentration of DNA sequences were cal culated by measuring the absorbance of DNA solution at 260nm on a Cary Bio 300UV spectrometer(Varian). The absorbance spectrum of the entire silver and gold nanoparticle was measured on the same spectrometer. Fluorescence measurements of the DNA motor solu tion were performed on a Fluorolog Tau 3 spectrofluorometer (Jobin Yvon).Transmission electron microscopy (TEM) images of all the gold and silver nanoparticle were obtained on a Hitachi H 7000 NAR transmission electron microscope under a working voltage of 100 kV. The fluorescence images were taken by CLSM (IX81, Olympus). Synthesis of Silver and Gold Nanoparticle Ag n anosphere Firstly 2.50 g p olyvinylpyrrolidone ( PVP M W = 10,000) was dissolved in 10 mL double distilled water (Millipore, Bioscience Research Reagents), then 3 mL of AgNO 3 solution (188 mM) was added. Then the solution was heated at 60 C for certain time

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27 under stirring (15 min for 6 nm Ag nanosphere and 60 min for 50 nm ones), and then the product was obtained. Ag n anowire Ag nanowires were synthesized following a reporte d method 40 Firstly 5 mL ethylene glycol was heated to 160 C and then 0.5 mL 0.12 mM PtCl 4 solution (ethylene glycol as solvent) was injected into the solution with magnetic stirring for 4 min. Afterward, 2.5 mL ethylene glycol solution of AgNO 3 (20 mg/mL) and 5 mL ethylene glycol solution of poly(vinyl pyrrolid one) (PVP, 40 mg/mL, M W = 55 000) were simultaneously injected to the ethylene glycol containing platinum seeds. This reaction mixture was stirred at 160 C 10 min for 1 m nanowires and 1h for 40 m ones, respectively. After cooled down to room temperature, the reaction mixture was diluted with acetone (approximate 10 times by volume), and centrifuged at 20 00 rpm for 20 min. The nanowire settled down to the bottom of the container under centrifugation while the nanoparticles that still remained i n the liquid phase was removed using a pipet. This separation procedure was repeated several times until nanowire samples essentially free of particles were obtained. Ag n anoprism 25 mL AgNO 3 aqueous solution (0.1 m M ) was prepared, and then 300 L of sodiu m citrate (30 m M ), 1.5 mL of PVP (M w = 29000 g/mol, 3.5 m M ), and 60 L of aqueous H 2 O 2 (30 wt % ) were added under vigorously stirring at room temperature. Fresh prepared NaBH 4 (100 m M 250 m L) was then rapidly injected into this mixture. Ag triangular nanoplates were obtained after approximately 1 h reaction.

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28 Au n anosphere Firstly 20 mL HAuCl 4 (0.1 mM) solution was heated to boiling under vigorous stirring. Then certain amount of fresh prepared 1 wt% sodium citrate solution (0.40 mL for the 13 nm Au nan ospheres and 0.5 0.175 mL for the 50 nm Au nanospheres ) was added. The solution was kept boiling until the color turned from dark blue to red. Keep stirring until the solution cool down to the room temperature, then the products were obtained. Au n anorod Cetyltrimethylammonium bromide (CTAB) aqueous solution (0.1 M, 10 mL) was mixed with 0.25 mM HAuCl 4 (0.25 mL). A freshly prepared, ice cold 0.01 M NaBH 4 (0.6 mL) was added to this solution all at once, followed by rapid inversion mixing for 2 min. The re sulting CTAB stabilized gold nanoparticle seed solution was kept at room temperature for 2 hrs. For seed mediated growth, HAuCl 4 (2 mL, 0.01 M) and AgNO 3 (0.4 mL, 0.01 M) were added into CTAB (40 mL, 0.1 M). After gentle mixing of the solution, a freshly p repared ascorbic acid (0.32 mL, 0.1M) was added as a mild reducing agent. Then a portion of the seed solution (0.096 mL) was added. The reaction mixture was mixed by gentle inversion for 10 s and left undisturbed at least overnight to form Au nanorod Characterization of Silver and Gold Nanoparticle The size and morphology of silver and g old n anop article were characterized by Transmission electron microscopy (TEM) on a Hitachi H 7000 NAR transmission electron microscope under a working voltage of 100 kV The high definition image were shown from Figure 2 1 (Ag nanoparticle) and Figure 2 2(Au nanoparticle)

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29 Synthesis of Azobenzene P hosphoramidite Azobenzene phosphoramidite was synthesized according to the prot ocol reported by Asanuma et al 10 (Figure 2 3 ). Compound 1. 1H NMR (CDCl 3 7.96 2. 1H NMR (CDCl 3 (CDCl 3 6.79 149. Synthesis and P urification of DNA S equences All t he DNA sequences used in this project are listed in Table 2 1 Three DNA sequences were all synthesized on ABI 3400 DNA synthesizer. The DNA sequences were loaded and syhthesis protocol was set up according to the operation handbook manufacturer. Debcyl CPG was used for the synthesis of Azobenzene DNA nan omotor and Normal DNA nanomotor, and the Azobenzene Phosphoramidite was used for the synthesis of Azobenzene DNA nanomotor. All the DNA products were deprotected and cleaved from CPG by incubating with 2mL 1:1 Ammonium hydroxide/ Methylamine for 30min in a 65 o C water bath. Afterwhile the supernatant was transferred to a 15mL eppendorf tube and added 250 L 3.0M NaCl and 6.25mL ethanol. The mixture were kept in 20 o C until the DNA preci pitated. Afterward the mixture was centrifuged at 400 rpm for 30 min. The supernatant solution was removed while the precipitated DNA was redissolved in 400 L trithylamine acetate ( TEAA) for the next purification step.

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30 The DNA/TEAA solution was purified in a C18 column on Varian Prostar HPLC machine. The collected DNA solution was dried in a vacuum dryer and then detritylated in 80% acetic acid for 30mins. Then the detritylated DNA was precipitated in 500 L ethanol with 20 L 3M NaCl again. The ethanol super natant was removed and then the DNA was dissolves in 400 L de ionized water. The concentration of DNA was calculated by measuring t he absorbance of DNA solution, while the extinction coefficient of each sequence was calculated on Integrated DNA Technologie s website. The extinction coeffi cient of the azobenzene moiety was calculated as 4,100 M 1 / cm 1 12 All the DNA products were kept in 20 o C freezer, while the a zobenzene DNA should avoid all kind of direct light irradiation. Irradiation S ource In order to test the conversion efficiency of a zobenzene DNA nanomotor under irradiation s with specific wavelength two kinds of light sources have been used: a 60W table lamp with a 450 nm filter was used as a visible light source to turn the azobenzene DNA motor transfer from Trans to Cis which opened the DNA hairpin structure, and a 23W 60Hz UV lamp with the wavelength at 365 nm was used as a UV light source to turn the azobenzene DNA motor transfer from Cis to Trans which closed the DNA hairpin structure Theoretical Calculation of Electric F ield Distribution around Nanoparticle The electric field (|E| 2 ) intensity distributions around the different nanostructures were calculated by finite element method (FEM) using the 3D RF model of COMSOL Femlab. The incident light wavelength was settled as 350 nm while the optical constant of the silver and gold was settled as data reported by Johnson et al 41 perfectly matched

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31 layers were adopted t o avoid spurious reflection effects at the simulation zone boundaries Results and Discussion Design of Light driven Nanomotor The design of light driven DNA nanomotor is shown in Figure 2 4. The DNA motor we designed having a stable hairpin structure with a six base pair stem and a 19 base loop And additionally, three azobenzenes were incorporated into the pair stem part so the hybridization of the stem part would be controlled by the irradiation sources. A fluorophore (fluorescein isothiocyanate, noted as FAM) and a quencher (4 dimethylaminoazobenzene 4 carboxyl ic acid, noted as Dabcyl) were labeled on each ends of the DNA strand so the final sequence design of the azobenzene DNA nanomotor was: 5 FAM CCT AGC TCT AAA TCA CTA TGG TCG C Azo GC Azo TA Azo GG Dabcyl 3 As shown in Figure 2 4, when the DNA nanomotor was under the irradiation with wavelength larger than 400nm, such as the visible light, the DNA motor and Debcyl would quench the fluorescence. On the other hand, with the irradiation source in the UV range( 300nm< <400nm ), the photoisomerization of azobenzene would lead to the open of DNA motor, no energy transfer between FAM and Debcyl so the fluorescence could be detected. In this project fluorescent intensity of FAM ex cited at 488 nm wa s used to monitor the light driven close open movement of the DNA nanomotor Calculation of Conversion Efficiency T he fluorescence intensity of DNA nanomotor under visible light irradiation ( I Blank ) was settled as baseline and t he fluorescence intensity of fully open DNA nanomotor

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32 after addition of excess amount of complementary DNA ( 5 GGA TCG AGA TTT AGT GAT ACC AGC GCG ATC C 3 noted as cDNA ) was settled as 100% ( I cDNA ). T he average close open conversion efficiency ( ) of the DNA nanomotor for each photon regulation cycle can be evaluated from the following equation while I UV is the fluorescence intensity of DNA motor after UV irradiation Enhanced Conversion Efficiency with Ag Nanoparticle T he azobenzene DNA nanomotor without any Ag nanoparticle only has 20% conversion efficiency after 5 min UV irradiation (wavelength= 350 ). Only with the existence of Ag nanoparticle, the conversion efficiency could be enhanced due to the localized electric field created by the Ag nanoparticle under light irradiance(Scheme shown in Figure 2 5). Enhancement of efficiency also depended on the morphology and size of Ag nanoparticle. To find the Ag nanostructure with the maximum enhancement on DNA nanomotor, Ag nanosp here Ag nanoprisms and Ag nanowires with different sizes were all investigated. 1 mL of Ag nanoparticle (2 nM) was washed and centrifuged at 4,000 rpm fo r 20 min. The precipitated nanoparticle was dispersed in 1 mL of deionized water. the azo DNA nanomotor and incubated overnight to get the maximum absorbance on the surface of Ag nanoparticle. Then the mixture was centrifuged and discarded unadsorbed DNA in supernatant, and the precipitate was dispersed in 1 mL of 8 mM Tris buffer. I Blank was the fluorescence intensity of DNA nanomotor solution that measured under visible light,

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33 while I UV was measured after 5mins UV irradiation ( operate in dark room ) I cDNA was measured after excess amount of cD NA was added to the solution. Conversion efficiency was calculated using the equation described before. From the results Ag nanowire offered the largest enhancement on DNA nanomotor conversion efficiency, while Ag nanoprism almost had no effect (Results sh own in Figure 2 6, Figure 2 7). Reversibility of Ag Nanowire Enhanced DNA N anomotor Although Ag nanowire significantly enhanced the conversion efficiency of DNA nanomotor the reversibility of this high efficiency should be confirmed. The revers ibility o f DNA nanomotor with Ag nanowire was tested by alternating 5 min of visible irradiation and 5 min of UV irradiation for ten cycles. As shown in Figure 2 8 even after ten cycles there was no obvious decrease in conversion efficiency These results confirmed that the enhancement effect was constant in this system. Fluorescence Micrographs of DNA Nanomotor C onversion The f luorescent images of DNA motor switching were taken on a confocal laser scanning microscope DNA na nomotor solutions with and without Ag nanowire were both exposed under the same UV irradiation Fluorescence micrographs of the DNA nanomotor solution were taken every 30 s to monitor the fluorescence change (Figure2 9) From the fluorescence image It is obvious that the DNA nanomotor solution with Ag nanowire showed higher fluorescence intensity than that without Ag nanowire after UV illumination, confirming the results from spectrafluorometer. From the image DNA nanomotor not only had higher conversion efficiency but also had a faster response with Ag nanowire.

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34 Localized Surface Plasmon of Ag N anoparticle In the recently research of light driven molecules, the interaction between azobenzene m olecules and metal nanoparticle has been reported 42 44 For the further study of the origin of this Ag nanoparticle enhancement effect, the near fi eld behavior of Ag nanoparticle was mimicked using finite element method based commercial software. When excited by electromagnetic field the conduction elec trons of metallic nanostructure would create nonpropagating l ocalized surface plasmon The silver nanoparticle used in this project (Ag nanosphere, Ag nanowire, Ag nanoprism)all been tested. The calculation results shows that (Figure 2 10) the electric fields around the Ag nanowires, Ag nanoparticles, and Ag nanoprisms are all strongly localized. Th e Ag nanowire ga ve the highest enhanced electric field distribution especially on both ends, while Ag nanosphere shown a homogeneously e nhanced electric field around it Ag nanoprism also gave a localized enhan ced electric field Absorbance Spectral of Ag Nanoparticle and Azobenzene M olecule Only with the calculation results of Ag nanoparticle could not give a complete explanation of Ag For further study, the extinction spectral of Ag nanoparticle and absorbance spectral of azobenzene were measured .(Figure 2 11) The azobenzene molecule shown two major absorbance peaks The low intensity peak at 440 nm was due to the symmetry forbidden n transition while the high intensity at 320 nm was due to the symmetry transition*. Ag nanos phere ( 50 nm ) and Ag nanowire both show n a narrow ex tinction peak centered at 430 nm, with the Ag nanoprism did not show any peak here. T heir extinction peaks overlap the absorption peak of the azobenzene

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35 molecul e. The coupling strength was reported strongly dependent on the spectral overlap between the mol ecular and plasmonic resonances 45 So coupling betwee n azobenzene and the plasmonic resonance of both Ag nanosphere and nanowire were confirmed, while the Ag nanoprism which only ga ve a peak located at 820 nm did not overlap with the absorption peak of azobenzene. This calculated result strongly supported experimental results, the localized electric field created from surface plasmonic resonance and spectral overlap were both the main reason for Ag nanowire to give the highest enhanced convers ion efficiency of DNA n anomotor while Ag nanoprism did not. Au nanoparticle and Ef fect s on DNA N anomotor In order to further confirm the mechanism that concluded f or the Ag nanoparticle enhancement effect on DNA nanomotor conversion efficiency, Au nanosphere and nanorod were a lso tested with DNA nanomotor but none of them shown an efficiency ( shown in Figure 2 12 ) The calculation results shown that Au nanosphere also ga ve localized electric field under the same incidence wavele ngth but the extin ction peak of Au nanoparticles ( around 520 nm ) had no overlap with azobenzene absorption peak ( shown in Figure2 1 3 ). Although the surface plasmonic resonance of Au nanoparticle could create a localized electric field, the conversion efficiency cannot be enhanced due to no coupling between Au nanoparticles and DNA nanomotor azobenzene moiety Conclusion In conclusion, we reported the enhancement effect of Ag nanoparticle for the conversion effi ciency of light driven DNA nanomotor. For original mechanism, we their surface

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36 plasmon resonance induced localized electric field With the largest localized electric field and extinction s pectral overlap with azobenzene Ag nanowire increased open close conversion efficiency of DNA nanomotor from 20% to 85% For Au nanoparticle with similar localized electric field did not have any enhancement effect as the extinction spectral had no coupli This discovery promise d a bright future for future development of high efficiency light driven nanomotor for multiple purposes

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37 Figure 2 1 Ag nanoparticle with different morphology and size. A) Ag nanosphere (6 nm diameter). B) Ag nanosphere (50 nm diameter). C) Ag nanoprism(50 nm length and 3 nm thickness). D) E) Ag nanowire 46

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38 Figure 2 2 Au nanoparticle with different morph ology and size. A) Au nanosphere (13 nm diameter). B) Au nanosphere (50 n m diameter). C) Au nanorod ( average length of 60 nm and width of 13 nm ) 46

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39 Figure 2 3 Synthesis procedure of azobenzene p hosphoramidite 46

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40 Figure 2 4 Open close conversion of DNA nanomotor with three azobenzene moieties FAM.

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41 Figure 2 5. Scheme of the enhanced DNA nanomotor. A) DNA nanomotor shown low c onversion efficiency without Ag nanoparticle B ) field.

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42 Figure 2 6 Fluorescence spectra of DNA nanomotor ex = 488 nm) incubated with A) DNA only B) Ag nanowires (40 m) C) Ag nanospheres (6 nm) D) Ag nanospheres (50 nm) E) Ag nanoprisms ( side length of 50 nm ) 46

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43 Figure 2 7 Conversion efficiency of DNA nanomotors in the existence of Ag nanomateri als with different morphologies 46

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44 Figure 2 8 Reversible conversion of DNA nanomotor with Ag nanowire. Each cycle: UV (350 nm), 5 min; Vis (450 nm),5 min 46

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45 Figure 2 9 Fluorescence microgr aphs of DNA nanomotor solution. A) DNA nanomotor only. B) DNA nanomotor with Ag nanowire under UV irradiation Image taken from confocal microscopy 46 0 s 3 0 s 9 0 s 6 0 s 15 0 s 12 0 s 30 0 s 24 0 s 27 0 s 18 0 s 21 0 s A 0 s 3 0 s 6 0 s 9 0 s 12 0 s 18 0 s 24 0 s 21 0 s 15 0 s 27 0 s 30 0 s B

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46 Figure 2 10 Localized e lectric field intensity distributions (IEI 2 ) calculated for A) Ag nanowire C) Ag nanoprism (50 nm)(calculated at irradiation wavelength of 350 nm) 46

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47 Figure 2 11 Extinction spectrum of Ag nanostructures and absorption sp ectrum of azobenzene molecule 46

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48 Figure 2 12 Image of Au nanoparticle and fluorescence spectrum of DNA motor A) Image of Au nanosphere (1 3 nm). B) Fluorescence spectrum of DNA motor incubat ed with Au nanosphere (13 nm). C) Fluorescence spectrum of DNA motor incubated with Au nanosphere (13 nm) after 5 min UV irradiation. D) Fluorescence sp ectrum of DNA motor incubated with Au nanos phere (13 nm)with excess cDNA. E) Image of Au nanosphere (5 0 nm). F) Fluorescence spectrum of DNA motor incub ated with Au nanosphere (50 nm). G) Fluorescence spectrum of DNA motor incubated with Au nanosphere (50 nm) after 5 min UV irradiation. H) Fluorescence spectrum of DNA motor incubated with Au nano sphere (50 nm)with excess cDNA. I)Image of Au nanorod (60 n m x1 3 nm). J) Fluorescence spectrum of DNA motor incubated with Au nanorod (60 nmx13 nm). K) Fluore scence spectrum of DNA motor incubated with Au nanorods (60 nmx13 nm) after 5 min UV irradiation. L) Fluorescence spectrum of DNA motor incubated with Au nanorods (60 nmx13 nm)with excess cDNA 46

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49 Figure2 13 The exper imental extinction spectrum of Au nanoparticle with absorption spectral of azobenzene. A) Au nanosphere (13 nm). B) Au nanosphere (50 nm). C) Au nanorod(13 nm 60 nm) T heoretical calculated electric field distributions (IEI 2 ) was calculated at wavelength of 350 nm. D) Au nanosphere (13 nm). E) Au nanosphere (50 nm). F) Au nanorod (13 nm 60 nm) 46

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50 Table2 1 Sequence of azobenzene nanomotor, normal nanomotor, and comp limentary DNA to nanomotor. Sequence Name Sequence(Azo for Azobenzene Phosphoramidite) Azobenzene DNA Nanomotor 5 FAM CCT AGC TCT AAA TCA CTA TGG TCG C Azo GC Azo TA Azo GG Dabcyl N ormal DNA 5 FAM CCT AGC TCT AAA TCA CTA TGG TCG CGC TAG G Dabcyl 3 cDNA 5 GGA TCG AGA TTT AGT GAT ACC AGC GCG ATC C 3

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51 CHAPTER 3 USING LIGHT DRIV EN DNA ST R A NDS FOR THE PHOTO CONTROLLED REVERSIBLE RELEASE S YSTEM OF MESOPOROUS NANOCONTAINER Stimuli responsive n ucleic acids with specific geometry change respond to external stimuli such as pH 8 metal ion 47 48 light sourc e 12 have already shown their great potential in molecular sensing, logic gat e operation 49 and nanomachine design 12 In previous research we have developed a high conversion efficiency light driven DNA nanomotor. In this project, we combine the technique of light driven DNA motor and Mesoporous silica nanoparticle to investigate a new light control reversible release system. Based on stable structure, lack of cytotoxicity, bio compatible, large inner space ease of surface modification and homogeneous pore size distribution mesoporous silica have recently bee n rapidly developed as a controlled release system for guest molecules 24 29 34 35 In m ost cases guest molecules will be released with the external stimuli to mesoporous silica nanoparticle and this process is not reversible. C ompared to target molecule stimuli, photo control release ha s significant ad vantages of fast response and ease of control without loss of efficiency. In addition, by switching the irradiance source a reversible controlled release process can be realized In this chapter we report a new pho to controlled responsive DNA/mesoporous silica hybrid release system by introducing light sensitive azobenzene DNA nanomotor to the surface of mesoporous material. Experimental Section Chemicals The chemicals tetraethylorthosilicate(TEOS), n cetyltrimethy lammonium bromide (CTABr), 3 (Triethoxysilyl)propyl isocyanate(TSPI),sodium hydroxide(NaOH), Sodium

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52 Nitrate(NaNO3), 3 (N morpholino)propanesulfonic acid (MOPS), Triton X 100, Rhodamine 6G, anhydrous toluene, ethanol, methanol were provided by Sigma Aldrich Fluorescein was provided by Invitrogen. De ionized water was obtained from Milli Q pump. Phosphate buffered saline (PBS) and tris(hydroxymethyl)aminomethane (Tris) were purchased from Fisher Scientific. Instrument ations Fluorescence measurements were ca rried out on a FluoroLog 3 spectrofluorometer (Jobin Yvon). The concentrations of all DNAs were calculated by measuring the absorbance of DNA at 260 nm on a Cary Bio 300UV spectrometer (Varian), as the extinction coefficient was calculated on Integrated DN A Technologies website. The extinction coeffi cient of the azobenzene moiety was calculated as 4,100 M 1 / cm 1 12 Scanning electron microscopy (SEM) images were taken from a JEOL JSM 6700 scanning electron microscope. Transmission electron microscopy (TEM) images were obtained on an H 7000 NA R transmission electron microscope (Hitachi) with a working voltage of 100 kV. XRD patterns were recorded on a D/MAX 2000 diffractometer (Rigaku), using Cu K radiation ( = 1.5406 ).The nitrogen adsorption and desorption isotherms at 78.3 K were measured using an ASAP 2010 analyzer (Micromeritics). The BET model was applied to evaluate the specific surface areas. Pore size and pore volume were determined from the adsorption data by the BJH method. Synthesis of Azobenzene P hosphoramidite Azobenzene phosph oramidite was synthesized according to the prot ocol reported by Asanuma et al 10 The detailed synthesis procedure was described in chapter 2.

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53 Synthesis and Purification of DNA S equences The azobenzene modified DNA sequences were synthesized using an ABI3400 DNA/RNA synthesizer (Applied Biosystems). The synthesis started with a controlled The a zobenzene phosphoramidite coupling reagent was prepared by dissolving in acetonit incorporation. A 15 min coupling time was used to improve the synthesis yield. DNA products were cleaved from CPG by incubating with 2mL 1:1 Ammonium hydroxide/ Methylamine ( AMA) for 30min in a 65 o C water bath. Then the cleaved DNA was precipitated by adding 250 L 3.0M NaCl and 6.25mL ethanol and kept in 20 o C freezer overnight. Then the precipitated DNA was dissolved in 400 L trithylamine acetate (TEAA) for the next purification step. The DNA was purified in a C18 column on Varian Prostar HPLC machine. The collected DN A solution was dried in a vacuum dryer and then detritylated in 80% acetic acid for 30mins. Then 500 L ethanol with 20 L 3M Na Cl was added to detritylated DNA again. The ethanol supernatant was removed and then the DNA was dissolves in 400 L de ionized wat er. The concentration of DNA was calculated by measuring the absorbance of DNA solution, while the extinction coefficient of each sequence was calculated on Integrated DNA Technologies website. The molar extinction coeffi cient of the azobenzene moiety was calculated as 4,100 M 1 / cm 1 12 All the DNA products were kept in 20 o C freezer Note that all the a zobenzene product s should avoid all kind s of direct light irradiation. All t he DNA sequences used in this project are listed in Table 3 1.

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54 Synthesis of Mesoporous Silica Nanoparticle CTAB (1.00 g, 2.74 mmol) was dissolv ed in 480 mL of nanopure water with 3.50 mL 2.00 M s odium hydroxide aqueous solution in a 1L flask. The solution was stirred by a magnetic stir bar and the temperature of the mixture was adjusted to 80 o C. 5.00 mL of the TEOS soluti on (22.4 mmol) was added drop by drop to the surfactant solution under vigorous stirring. The react ion was kept at 80 o C for 2 h to give a white precipitate. This solid product was filtered, washed with deionized water and methanol for more than 3 times and dried in air to yield the as synthesized mesoporous silica nanoparticles (denoted as MSN). To remove the surfactant template (CTAB), 1.50 g of the as synthesized MSN was refluxed for 24 h in a methanolic solution of 9.00 mL of HCl (37.4%) in 160.00 mL methanol. The resulting material was filtered and extensively washed with deionized water and methanol again Surface Functionalize of Mesoporous S ilica Nanoparticle with S hort DNA 1.00g MSN was refluxed in 80.00 mL of anhydrous toluene with 0.25 mL of 3 i socyanatopropyltriethoxysilane for 20 h to get surface functionalized with 3 isocyanat opropyl group (noted as MSN NCO). After discarded the toluene by centrifuge the purified MSN NCO (100 mg) was dispersed in 2 mL of de ionized water, certain amount of 20 amine modified Arm DNA T A C C T A NH2 Then the mixture was shake in a incubate overnight at room temperature to allow t he amino groups of DNA react with the NCO functional groups on the surface of MSN (noted as MSN DNA) Dye Loading and DNA Capping of Mesoporous Silica Nanoparticle 50 mg MSN DNA was added to 5 mL 5.00 mM Rhodamine 6G solution in 1X PBS buffer (pH = 7.4). After the mixture was shaking overnight in a dark room the final

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55 mixture was the n centrifuged and washed with PBS buffer solution three times. The azobenzene modified ssDNA ( noted as azo DNA 1 mL, 120 M) was added to hybridize with arm DNA on MSN and cap the pores on the mesoporous particles (noted as MSN R6G) Then the capped MSN was washed again to remove the dye absorbed on the surface The maximum amount of Rh odamine 6G loaded on the MSN was determined by analyzing the supernatant solution spectrophotometrically. All the washing solutions were collected, and the loa ding amount was calculated. Compared to the initial concentration of rhodamine 6G solution, the maximum loading amount of rhodamine 6G was approximately g ram MSN. Irradiation S ources A 6 W UV light source was chosen as the UV light source to convert the azoben zene from cis to trans transition ( DNA dehybridize, MSN uncap) As the visible light source, a 60 W table lamp with a 450 nm filter was selected to cause the azoben z ene converse from cis to trans (DNA hybridize, MSN cap). For all the experiments, the light sources were carefully positioned to avoid the heating effect on the samples. Characterization of the DNA modified Mesoporous Silica Nanoparticle ( MSN DNA) The size and morphology of Mesoporous silica nanoparticle nanoparticle were characterized by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy ( SEM). The TEM image was taken on a Hitachi H 7000 NAR transmission electron microscope under a working voltage of 100 kV. The high definition image were shown from Figure 3 1 Scanning electron microscopy (SEM) images were taken from a JEOL JSM 6700 scanning electron microscope ( Figure 3 2 ).

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56 The porosity of the Mesoporous silica nanoparticle was characterized by XRD. XRD patterns were recorded on a D /MAX 2000 diffractometer (Rigaku), using Cu K radiation ( = 1.5406 ). The XRD pattern was shown in Figure3 3. The nitrogen adsorption and desorption isotherms at 78.3 K were measured using an ASA P 2010 analyzer (Micromeritics) (Figure3 4). The BET model was applied to evaluate the specific surface areas Pore size and pore volume were determined from the adsorption data by the BJH method. (Pore size distribution see Figure 3 5, Surface area and pore volume calculation results see Table 3 2) Dye Release Experiment of DNA C apped MSN R6G Dye loaded DNA modified MSN (MSN R6G) was dispersed in Tris buffer (20 mM Tris HCl, 20 mM NaCl, 2 mM MgCl 2 pH = 8). In each release experiment, 0.5mg MSN R6G was kept in a mini dialysis tube (cut off molecular weight= 7,000),and tube was immersed in a 10ml beaker with 5ml same buffer (shown in Figure3 6) During the dye release process, the released dye could disperse through the dialysis membrane, while the MSN and DNA would be kept inside tube Samples were ta ken from the solution in beaker, and the dye release process was monitored by measuring the fluorescence intensity. Results and Discussion Light driven molecule az obenzene and its derivatives isomerizes from the trans to cis forms under irradiation at 300 380 nm and isomerizes from cis to trans under irradiation at wa velengths > 400 nm 42 For the double strand azobenzene DNA, the azobenzene phosphoramidite in cis form(under UV irradiation)would cause the dehybridization of the duplex and it will reverse back with the irradiation of visible l ight 12

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57 It has been already reported that b y the geometry change of the DNA attached on the pore of mesoporous silica nanoparticle, the open close of the pore can be controlled 8 In this project azobenzene DNA was used to cap the mesoporous silica nanoparticle as shown in Figure 3 7 single stranded Arm T A C C T A attached on the pore edge of mesoporous silica particle The linker DNA azo DNA strands with azobenzene moieties (azo DNA) was hybridized to the arms. Under visible nm), the azobenzene moieties were in the trans form, and the linkers kept hybridize d to the arms, then the pores were tightly capped, and the loaded guest molecule was trapped in mesoporous particle Switched the irradiation source to UV converted to the cis form. This would cause the dehybri dization between Arms and azo DNA and the pores were uncapped Because of the reversibility of azobenzene molecule, the process was also reversible as if the irradiation source switched back to visible light, the azo DNA would hybridize with Arm DNA again to cap the pore. Dye Release Test under Different Irradiation S ource T he dye release process under different irradiance was monitored by measuring the fluorescence intensities measured on fluorescence spectroscopy as a function of time The experimental procedure has been described before. As shown in Figure 3 8, u nder the irradiation of visible light (450 nm), the fluorescence intensity increased very slowly that no obvious dye release was observed, indicating that the mesoporous particle was well capped by azo DNA and dye was trapped well without leaking When the irradiation source was switched to UV ( 365 nm ) t he fluorescence intensity of the released dye in solution increased rapidly and reached 91% release after 1500 min. This result showed that the azo DNA capped mesoporous silica nanoparticle was a

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58 good carrier for guest molecule, and rapidly response to the UV irradiation which lead to a large release of guest molecule. For comparison, a normal DNA without azobenzene mod ification was also used to c ap mesoporous silica particle and dye release test was taken under UV and visible irradiation. However, no dye release was detected after UV irradiation ( Figure 3 9) Optimization of azo DNA S equences To optimize the design of azo DNA sequence with a bette r response to UV irradiation DNA strands (azo DNA 1 /azo DNA 2/azo DNA 3/azo DNA 4 ) sequences shown in Table3 1 ) were prepared with different numbers and positions of azobenzene moieties. Dye release from mesoporous silica nanoparticle capped with each se quence s was tested. As shown in Figure 3 10, azo DNA 4 gave the best release results so this sequence was used in the entire project. Reversibility of Dye Release System One significant advantage of this release sy stem was the reversibility of open close cycles of pores via alternating irradiation wavelengths. As shown in Figure 3 11 started with visible irradiation only a very small amount of released dye was detected, while a rapid increase of released dye was detected when the irradiation wa velength was suddenly changed from visible(450nm) to UV light ( 365 nm ) at 120 min. At 240 min s when changing the irradiation source back to visible light at 450 nm, the pore was capped again and release of the trapped dye was sonly restricted and this cyc les could be repeated for many times. The release process shows that the on off switching is reversible and can be repeated several times with a good reversibility.

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59 Conclusion In this project we have developed a photo controlled reversible release system b y capping mesoporous container with light sensitive DNA strand. The photo sensitive capping/uncapping sensitively response to different irradiations, then exhibited a light controlled release process After the optimization of azo DNA stra nds 91% of cargo release can be reached after 1500 mins release time. This capping/uncapping was reversible by switching di fferent irradiation sources with a rapid response and high reversibility after several cycles. This new design already showed a gre at potential toward the development of light controlled guest molecule release systems. Therefore rapid switching between the open and closed states can be used to control drug release, which can be great help in a wide range of desired applications, especially for targeted drug delivery to avoid the side effects

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60 Figure 3 1 TEM image of DNA modified MSN.

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61 Figure 3 2 TEM image of DNA modified MSN.

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62 Figure 3 3 Small angle XRD pattern of MSN, MSN NCO and MSN DNA Figure 3 4 BET nitrogen adsorption/desorption isotherms of DNA functionalized m esoporous silica nanoparticle(MSN DNA)

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63 Figure 3 5 BJH pore size distrib utions of DNA functionalized mesoporous silica n anoparticle (MSN DNA) Figure 3 6 The s etup of the mesoporous dye release device

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64 Figure 3 7 The s cheme of a zobe zene modified DNA based light driven reversible r elease s ystem.

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65 Figure 3 8 Time dependent rhodamine 6G release curves from DNA capped mesoporous silica nanoparticle under different irr adiation. Fluorescence intensities have been normalized to the maximum level of dye released.

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66 Figure 3 9 Dye release under UV irradiation from mesoporous particle with azo DNA and normal DNA for 1500 min, data were normalized to the maximum of dye released in the experiment. Figure 3 10 Dye release under UV irradiation from mesoporous particle with azo DNA1/ azo DNA2/ azo DNA3/ azo DNA4 for 1500 min, data was normalized to the maximum of dye released in the exp eriment.

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67 Figure 3 11. Dye release curve from MSN as a function of time while switching irradiation source every 120 mins showing the reversible capping/uncapping control of mesoporous particle by changing the irradiation wavelength. Data was normalized to the maximum of dye released.

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68 T able3 1 Sequence of Azobenzene DNA, normal DNA, and arm DNA. Sequence Name Sequence(Azo for Azobenzene Phosphoramidite) azo DNA 1 T A G G Azo T A CCC CCC CCC CCC T A G G Azo T A azo DNA 2 T A Azo G G Azo T A CCC CCC CCC CCC T A Azo G G Azo T A azo DNA 3 A Azo T A Azo G G Azo T A CCC CCC CCC CCC A Azo T A Azo G G Azo T A azo DNA 4 T Azo A G Azo G T Azo A CCC CCC CCC CCC T Azo A G Azo G T Azo A Normal DNA T A G G T A CCC CCC CCC CCC T A G G T A Arm DNA T A C C T A NH 2 Table 3 2 Surface area and pore volume of Mesoporous silica nanoparticle before surface modification (MSN), after surface modification and DNA conjugation(MSN DNA), and after dye loading(MSN R6G) Sample Surface area (m 2 /g) Pore volume (m 3 /g) MSN 842.27 1.0322 DNA functionalized MSN (MSN DNA) 628.38 0.79572 Rh6G loaded MSN (MSN R6G) 502.45 0.82572

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69 CHAPTER 4 USING ION SENSITIVE DNA STRANDS FOR THE DETECTION OF MERCURI C ION ON A MESOPOROUS STIM ULI RELEASE SYSTEM Mercury pollution is a global contamination problem with varies of nature origins and seriously s health 50 This problem is attracting more and more high toxicity at low concentration, cause severe damage to kidney, nervou s system and other organs 51 The mercury can be accumulated in human body through several processe s, and one of the major source is the water soluble Hg 2+ ion contamination in drinking water. In 2001 The United States Environmental Protection Agency (EPA) has acclaimed the top limit of 2 ppb (10 nM) for Hg(II) in drinking water 52 Therefore, developing a new mercuric ion detection methods which is sensitive, easy to operate low cost and applicable to aqueous system becomes important and urgent 53 There are many t raditional methods for the detect ion of mercuric ion in aqueous systems but almost all of them require complicated sample preparation and expensive instruments such as atomic absorption spectroscopy, cold vapor atomic fluorescence spectrometry, and gas chromatography 53 In order to simplify the sample preparation and reduce the cost on the instrument many mercuric ion detec tion methods have been developed basing on small organic molecules like fluorophores 54 nanoparticle like gold nanosphere 14 55 or biomaterials such as DNAzymes 48 DNA hydrogel 15 and protein 56 The original mechanism of these approaches basicly rely on directly binding or reaction between the probe and Hg 2+ that causes the detectable color or fluorescence change 57

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70 In this project we designed a DNA functionalized mesoporous silica nanoparticle based Hg 2+ responsible dye release system to realize the direct, fast, sensitive detection of Hg 2+ in aqueous system. The detection was realized by capping the mesoporous silica nanoparticle ( MSN) with Hg 2+ sensitive DNA to trap the dye inside and releasing dye molecule inside MSN in the existence of Hg 2+ We made use of the unique structure of MSN as the carrier of the dye and use T base rich DNA as the mercuric ion responsible cap for MSN. In our design we capped MSN by a Hg 2 + sensitive DNA T rich strands to trap the dye molecule inside MSN, and when immersed in solution with Hg 2+ pores on MSN would be opened and dye would be released. By measuring the fluorescence intensity of released dye a detection of Hg 2+ was realized. Experimental Section Chemicals The chemicals tetraethylorthosilicate(TEOS), n cetyltrimethylammonium bromide(CTABr), 3 (Triethoxysilyl)propyl isocyanate(TSP I),sodium hydroxide(NaOH), Sodium Nitrate(NaNO 3 ), 3 (N morpholino)propanesulfonic acid (MOPS), Triton X 100, Rhodamine 6G, Mercury(II) Acetate(HgAc 2 ), Lead(II) Acetate( PbAc 2 ), Barium(II) Acetate(BaAc 2 ), Iron(II) Acetate (FeAc 2 ), Iron(III) Chloride (FeCl 3 ) Zinc(II) Acetate(ZnAc 2 ), Magnesium Acetate(MgAc 2 ), Cadmium(II) Acetate( CdAc 2 ), Copper(II) Acetate(CuAc 2 ), Cobalt(II) Acetate (CoAc 2 ), Nickel(II) Acetate(NiAc 2 ), Calcium(II) Acetate(CaAc 2 ), anhydrous toluene, ethanol, methanol were provided by Sigma Ald rich. Fluorescein was provided by Invitrogen. De ionized water was obtained from Milli Q Buffer Solutions Binding buffer consisting in 10 mM MOPS, 50 mM NaNO 3 0.05%(in volume)Triton X 100 (pH=7.0) was used for DNA binding and dye release experiences

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71 Hybridization buffer in 10 mM MOPS, 50 mM NaNO 3 20mM MgAc 2 (pH=7.0) was used f or DNA hybridization. Synthesis of Mesoporous Silica Nanoparticle The MCM 41 mesoporous silica nanoparticle was prepared by the following procedure: 500mg n cetyltrimethylammon ium bromide(CTABr) was dissolved and suspended in 250ml DI water. Then 3.5ml NaOH (1M) was added. The CTABr solution was transferred to a 500ml triple neck bottle, then heated to 80 degree with the magnetic stirring. After while 2.5ml tetraethylorthosilica te (TEOS) was added drop by drop to the CTABr solution. Then reaction was taken for 2 hour with magnetic stirring to obtain the write powder precipitate. The mixture was cooled down under room temperature, then centrifuged at 2000rpm for 10 min to get rid of the supernant The solid was washed with ethanol for three times. The final product was reflux in methanol for 20 hours to get rid of CTABr. After washed with ethanol three times the product mesoporous silica nanoparticle ( MSN) was obtained and kept dr ied under room condition. Surface Modification and Dye Loading of Mesoporous Silica N anoparticle 1g extracted MSN was refluxed in 80ml anhydrous toluene with 0.25ml 3 (Triethoxysilyl) propyl isocyanate( TSPI) for 20hours to immobilize the isocyanate group on the surface. The isocyanate functionalized MSN(MSN NCO) was collected and washed with anhydrous toluene three times, then dried in oven under 70 o to evaporate toluene. The 1g functionalized MSN NCO wa s suspended in 20ml 2.5mg/ml Rhodamine 6G water solution inside a 50ml centrif uge tube to load the dye in the pores of MSN NCO scaffolding. The mixture was ultrasonic for 1min then shake 24hours with aim to achieving maximum dye loading. Afterward the dye loaded MSN NCO was washed

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72 quickly with ethanol for three times and dried in oven under 70 o C. The dye loaded MSN NCO(MSN R6G) was kept dried until next step. Characterization of Mesoporous Silica Nanoparticle The size and morphology of Mesoporous silica nanoparticle nanoparticle were characterized by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy ( SEM). The TEM image was taken on a Hitachi H 7000 NAR transmission electron microscope under a working voltage of 100 kV. The high defi nition image were shown from Figure 4 1. Scanning electron microscopy (SEM) images were taken from a JEOL JSM 6700 scanning electron microscope ( Figure 4 2). The porosity of the m esoporous silica nanoparticle (MSN) surface functionalize m esoporous silica nanoparticle (MSN NCO) and dye loaded m esoporous silica nanoparticle (MSN R6G) were characterized by XRD. XRD patterns were recorded on a D/MAX 2000 diffractometer (Rigaku), using Cu K radiation ( = 1.5406 ).The XRD pattern was shown in Figure4 3. Synthesi s and Purification of DNA Sequences All the DNA sequences used in this project are listed in Table 4 1 Three DNA sequences were all synthesized on ABI 3400 DNA synthesizer. The syn thesis process was similar to the process in chapter 3 and 4. Debcyl CPG was used for the synthesis of Linker DNA and Amino CPG was used for the synthesis of Arm DNA All the DNA products were deprotected and cleaved from CPG by incubating with 2mL 1:1 Ammonium hydroxide/ Methylamine for 20 min in a 65 o C water bath. After whil e the supernatant was transferr ed to a 15mL eppendorf tube. 250 L 3.0M NaCl and 6.25mL ethanol were added to help the precipitation of DNA The mixture were kept in 20 o C

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73 until most of the DNA precipitated to bottom After while the mixture was centrifuged to get rid of the supernatant solution and the precipitated DNA was redissolved in 400 L trithylamine acetate ( TEAA) for the next step. The DNA was purified on Varian Prostar HPLC machine through a C18 column The purified DNA should also be detritylated in 80% acetic acid for at least 30mins. Then the detritylated DNA was precipitated in 500 L ethanol with 20 L 3M NaCl The mixture was kept in 20 o C freezer until most of the DNA precipitate again. The ethanol supernatant was remove d and then the DNA was dissolved in 400 L de ionized water. The concentration of DNA was calculated by measuring the absorbance of DNA solution, while the extinction coefficient of each sequence was calculated on Integrated DNA Technologies website. All the DNA products were kep t in 20 o C freezer, the Fluorefore Linker DNA with the FAM coupling should be kept in dark and avoid all kind of irradiations. Adjustment of Short DNA on Particle S urface In order to confirm the maximum amount of Arm DNA to saturate the surface of dye loaded m esoporous silica nanoparticle, the saturation titration of Arm DNA has benn tested on MSN R6G. Certain amount ( 2uL/4uL/8uL/12uL/16uL/20uL/24uL) of 1mM Amino modified A rm DNA solution was added to 1mg MSN R6G in a 1.5ml centrifuge tube. Binding buffer was added to adjust the overall volume to 200uL. The mixture was kept shaking overnight in incubator under room temperature ( with speed 1000rpm). Afterward the mixture was centrifuged and the supernatant was separated from particle

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74 The amount of Arm DNA attached on the MSN was calculated by the absorbance change of the supernatant From the titration curve (see Figure 4 4 ) 12nmol of Arm DNA was the optimal amount to satura te the surface of 1mg MSN R6G, while around 10nmol of Arm DNA would finally bind on the surface of MSN R6G For the preparation of solid MSN Arm DNA, 500 g MSN R6G was suspended in 200 L binding buffer containing 6nmol of the amino modified Arm DNA. The su spension was ultrasonic for 30min then sh ake in an incubator overnight ( 1000rpm, room temperature). The solution was centrifuged and washed wit h binding buffer for three times This step was operated fast to avoid the free release of the Rhodamine 6G from the particle The absorbance of the supernatant was measured to calculate the binding efficiency of Arm DNA which was in the range of 85.71% 90.63%. DNA Capping of the Mesoporous Silica N anoparticle After the binding of the Arm DNA on the surface of the dye loaded Mesoporous silica nanoparticle, t he Linker DNA was introduced to hybridize with the Arm DNA imm obilized on the MSN then cap the pore 500 g MSN Arm DNA solid was suspended in 200 L hybridization buffer with 20nmol of Linker DNA. The solution w as shake in an incubator for 2 hours(1000rpm, room temperature), then centrifuged and the supernatant was kept. The particle was washed with binding buffer for three times. The absorbance of the supernatant was measured to calculate the hybridization effic iency of Linker DNA to the Arm DNA. According to the calculation results 22.37% 23.29% of the Linker DNA was hybridized to the Arm DNA, which means the ratio of Linker DNA to Arm DNA on the MSN is from 2.23:1.00 to 2.14:1.00. From the result one

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75 Linker DNA will bridge link two Ar m DNA on the surface of the MSN, which was exactly the expect result. Quenching effect of Mercuric Ion on Different O rganic D yes In order to confirm the dye used in mercuric ion detection system would not be quenched by the mercuric ion itself, the quenching effect of different organic dyes was tested. Fluorescence intensity of rhodamine 6G and fluorescein ( dye concentration: 100ng/ml) with the existence of different concentrations of mercuric ion (from 1 ppb to 1000ppm) were measured Mercuric ion shows a obvious quenching effect on Fluorescein but only little effect on Rhodamine 6G especially when the concentration of mercuric ion was in ppm range ( See Figure 4 5 ) Dye Release Experiment of DNA C apped MSN R6G In order to monitoring the dye release from the DNA capped MSN R6G with the existence of mercuric Ions, 0.5 mg of DNA c apped MSN R6G was suspended in m ercuric a cetate binding buffer solution of different concentration(0ppb 10ppb 20ppb 50ppb 100ppb 200ppb 500ppb 1ppm or the complimentary Linker DNA). As shown in Figure 4 6 the particle was suspended in 200 l mercuric s olution in a Mini Dialysis Filter( Thermo Scientific, cut off molecular weight 7000) with a small piece of foam f loating on the surface of 5ml mercuric s olution in a 10ml beaker. The solution was kept stirring with a magnetic stir at 400rpm under room temperature and 150 l of solution was taken from the beaker to measure the fluorescence intensity with fluorescence spectrometer( HORIBA Jobi nYvon) every five minutes. Results and Discussions In our design, the Hg 2+ wa released system an d the detection signal was detect ed by measuring the fluorescence

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76 intensity of released dye, which likes sign al amplification process. The detailed paradigm is shown in Figure 4 7. The solid MSN was surface modified with isocyanate functional groups(MSN NCO). After while this functionalization MSN NCO wa s loaded with dye(Rhodamine 6G). We chose Rhodamine 6G as it had a relatively low quenching effect comparing with the other organic dyes (results shown in experimental section) And then dye loaded particle was obtain the dye doped MSN(MSN R6G). Arm DNA with the amino end( NH2 AAA AAC AAC AAG AAG t o the surface of MSN R6G b y the chemical bind of NCO and NH2 group. After wash ed away the suspend Arm DNA, a certain amount of T base rich Linker DNA ( GTT GTT CTT CCT TTG TTT CCC CTT TCT TTG GTT GTT CTT C s introduced to hybridize with the Arm DN A. The single strand DNA wa s flexible and soft with a cross sectional diameter of 0.6nm, which display a poor coverage of the pores on MSN 8 24 After the addition of the Linker DNA, the Linker DNA would sonly hybridized with th e Arm DNA. As shown in Figure 4 8 one Linker DNA had two binding sites with the Arm DNA, we expect it cross link with the Arm DNA on the MSN surface, then form the more straig ht double strand structure that signific antly inhibit dye release by closing the pores. But only in the existence of Hg 2+ T rich linker DNA would prefer to form intermolecular duplex then leave the MSN to release the dye molecule. Fluorescence Test of Free DNA In order to confirm that the Hg 2+ can efficiently break the Linker Arm DNA hybridization to form the new Linker DNA intermolecule T Hg T duplex, we d id the fluorescence test of the free DNA strands without particle in solution first. We design ed a sim ilar fluorophore Linker DNA but coupled fluorophore and quencher on each end( FAM GTT GTT CTT CCT TTG TTT CCC CTT TCT TTG GTT GTT CTT C Debcyl

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77 (Shown in Figure 4 9) While this DNA wa s in the state, cause the distance between each end no fluorescence reso nance energy transfer ( FRET) would happen between the fluorophore and the quencher so the fluorescence signal could be detected Only wh en this DNA wa s in the duplex state, for example, when the Hg 2+ bond with the T bases to form a intermolecular T Hg T duplex, the fluorophore a nd quencher would be brought together then quench the fluorescence signal was quenched Result as shown in Figure 4 10, fluorephore Linker DNA itself shown a strong fluorescence .Firstly we use d excess amount of free Arm DNA to hybridized with fluorephore Linker DNA first then fluorephore Linker DNA without any FRET so fluorescence intensity was still strong The small reduce of intensity was due to the quenching effect of duplex DNA on fluorophore. After while we titrate d th is solution with Hg 2+ and a signi ficant fluorescence reduction wa s been detected, which mean Hg 2+ could efficiently dehybridize the Linker Arm duplex and form the new T Hg T duplex. Then the excess amount of complimentary DNA to the Linker DNA (cLinker DNA 5' GAA GAA CAA CCA AAG AAA GGG GAA ACA AAG GAA GAA CAA C 3') wa s introduced to the solution. We expected to see if this T Hg T duplex wa s strong enough to inhibit the hybridization between the fluorephore Linker DNA and cLinker DNA. If the complimentar y Linker DNA could open this T Hg T duplex then hybridize with it, then the fluorescence intensity should increase. But from the result only a small incre ase of fluorescence intensity wa s detected after the addition of complimentary Linker DNA From this result it was confirmed that thi s intermolecular T Hg T duplex wa s very stable, even the complimentary DNA cannot open it. T his result

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78 confirm ed that Hg 2+ could be efficiently dehybridize d the Linker DNA from Arm DNA then release d the dye to ex press the fluorescence signal. Detection of Hg 2+ using Stimuli Release Mesoporous System In order to test the ability of this stimuli release mesoporous s ystem on the detection of Hg 2+ th e dye release curve of MSN was been monitored under fluorescence spectrometer .We prepared 8 groups in each 0.5mg Linker DNA capped MSN R6G and then dispersed in mercuric acetate solution of different concentrations (0ppb 10ppb 20ppb 50ppb 100ppb 200ppb 500ppb 1ppm) Also we prepared the 9 th group of adding complimentar y DNA (cLinker DNA 5' GAA GAA CAA CCA AAG AAA GGG GAA ACA AAG GAA GAA CAA C 3') to test the maximum opening of these system. The experimental process was described before. As can be seen in Figure 4 11, without Hg 2+ a negligible release of Rhodamine 6G was tested, confirmed MSN R6G wa s tightly capped by Linker DNA (Brown curve in Figure 4 11 ). In the presence of Hg 2+ the DNA capped MSN R6G was uncapped, the release of the Rhodamine 6G was detected and this release increase with the increase of the Hg 2+ concentration This system also gave a fast response to stimuli. Figure 4 12 shows the fluorescence was det ected in the solution with the increase of Hg 2+ concentration also this detectable limitation of the mercuric ion concentration can be down to 10ppb. The good limitation and the fast response suggest ed this method can be a fast and sensitive process for the detection of the Hg 2+ Selectivity T est To avoid the potent ial interference by other metal ions, the selectivity of this approach was tested. The dye release of Linker DNA capped MSN R6G particle was

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79 also tested in the 1ppm solution of Lead(II) Acetate( PbAc 2 ), Barium(II) Acetate(BaAc 2 ), Iron(II) Acetate (FeAc 2 ), Iron(III) Chloride (FeCl 3 ), Zinc(II) Acetate(ZnAc 2 ), Magnesium Acetate(MgAc 2 ), Cadmium(II) Acetate(CdAc 2 ), Copper(II) Acetate(CuAc 2 ), Cobalt(II) Acetate (CoAc 2 ), Nickel(II) Acetate(NiAc 2 ), Calcium(II) Acetate(CaAc 2 ). The dye release process was measured as the same process as described before. The fluorescence intensities ware measured and normalized to the control groups (free dye release in buffer solution ). Figure 4 13 shows that, in this specific stimuli responsive dye release system only the 1ppm Hg 2 + solutions shown a 6.4 times fluorescence intensity to the control group no interference from other metal ions was detected. Con clusion In this project we developed a new DNA functionalized mesoporous silica nan oparticle dye release system to realize the direct, rapid simple and sensitive detection of mercuric ion in aqueous system. This design was a combination of both DNA sensor technique and mesoporous material Mosoporous silica nanoparticle was used as a co ntainer of dye and w ith the unique porous structure DNA sequences with specific length could be used for the capping of the pore. T he long T rich Linker DNA was used to cap the pore while only with the existence of Hg 2+ the Linker DNA will form the more s table T Hg T duplex with Hg 2+ to uncap the pore and leave MSN. Only a amount of cargo molecule through this signal amplification process. Fluorescence test r esults shows that this design can was rapid (less than 20min) and sensitive ( ppb level) response to Hg 2+ in aqueous solution. And due to the specific binding between T rich DNA and Hg 2+ this approach shown good selectivity and no potential interference by

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80 other metal ions was detected. Comparing to other device dependent ion detection techniques 58 this approach has no need for sample preparatio n, low cost in instruments, ease to operate, and the sensor particle is very stable under room condition, w e strongly believe thi s approach would show great potential in developing new metal ion sensing system for the detection of aqueous contamination

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81 Figure 4 1. TEM image of mesoporous silica nanoparticle

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82 Figure 4 2. SEM image of me soporous silica nanoparticle

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83 Figure 4 3. P owder X ra y patterns of the solids. A) mesoporous silica nanoparticle as synthesized and calcined. B ) iso cyanate group functionalized m esoporous silica nanoparticle. C ) r hodamine 6G doped m esoporous silica nanoparticle

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84 Figure 4 4 The titration curve of Arm DNA bin ding on per gram of dye loaded m esoporous silica nanoparticle Figure 4 5 Quenching effect of mercuric ion on different organic dyes

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85 Figure 4 6 Setup of the mesoporous dye release device

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86 Figure 4 7 Schematic representation of the synthesis, surface modification, dye loading, DNA binding of the MSN and the dye release of the MSN in the existence of the Hg 2+ Figure 4 8. H ybridization of the Arm DNA and Linker DNA in the existence of Hg 2+

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87 Figure 4 9. The s cheme of the hybridization of the Arm DNA, Fluorophore Linker DNA and Complimentary Linker DNA. Figure 4 10 Fluorescence intensity of the 100nm Fluorophore Linker DNA solution after the addition of certain amount of Arm DNA, Hg 2+ and Complimentary Linker DNA.

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88 Figure 4 11. Release curve of r hodamine 6G from Linker DNA capped MSN R6G in mercuric acetate solution of different concentration L aser excitation wavelength : 520 nm

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89 Figure 4 12 Fluorescence signal detected solu tion of different concentration s L aser ex citation wavelength : 520 nm

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90 Figure 4 13 Fluorescence intensities of the released dye from Linker DNA capped MSN R6G in the presence of 1ppm different cation. The fluorescence intensities were normalized to the control. Release time: 20mins

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91 Table4 1. Sequence of Arm DNA, Linker DNA, and Fluorefore Linker DNA and Complimentary Linker DNA Sequence Name Sequence Arm DNA GAA GAA CAA CAA AAA NH 2 Linker DNA GTT GTT CTT CCT TTG TTT CCC CTT TCT TTG GTT GTT CTT C F luorefore Linker DNA FAM GTT GTT CTT CCT TTG TTT CCC CTT TCT TTG GTT GTT CTT C Debcyl Complimentary Linker DNA G AAG AAC AAC CAA AGA AAG GGG AAA CAA AGG AAG AAC AAC

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92 CHAPTER 5 SUMMARY AND FUTURE D IRECTIONS Inc o r porating Stimuli responsive DNA Strands with Functional Nanomaterial for Biophysical and Analytical Applications Stimuli responsive DNA strands, with their sensitive and selectively response, have shown great potentials in molecular motor design, sensor design and drug delivery system development. With the help of functionalized nanoparticles DNA technology can be developed to fulfill different applications, in both analytical and biophysical areas. All the three projects have one similarity: the combination of DN A and nanotechnology. Whereas DNA always acted as a sensitive probe, nanoparticles contributed from their driven nanomotor electric field. In the second project, DNA was a light silica nanoparticle was a carrier of guest molecule. In the thir d project, DNA was an ion numerous stimuli responsive DNA strands and functional nanoparticles, it can be envisioned that a huge space of i nterdisciplinary between these t wo techniques has not been explored so far. The successful outcomes from these studies may lead to the advanced DNA nanoparticle hybridization in the analytical, biophysical, or even biomedical areas.

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93 Future Directions One future direction of DNA functionalized nanomaterials will serve as tools for target specific drug delivery and release system. In particular, solving the serious side effect caused by commercial available drugs in the anti cancer study, a specific drug delivery system is highly d emanding. This system should fulfill the requirements of (1) specific delivery of the drug to the tumor (2)selectively release of the drug around tumor biocompatibil ity. Considering these requirement I have designed an aptamer capped target responsive drug delivery and release system. Aptamers are single stranded DNA or RNA oligonucleotides that with the specific recognition to a wide range of target molecules, from simple molecules to peptides, proteins and even living cells 59 The anti cancer drug doped mesoporous nanoparticles can be designed to be encapsulated by aptamer with high binding affinity to specific cancer cells( such as Sgc8 aptamer to CEM cell or TDO5 aptamer to RAMOS cell) After aptamer binding to cancer cell surface pores on mesoporo us silica nanoparticles will be opened and anti cancer drugs will be released accoradingly. Both aptamer and mesoporous silica nanoparticles are stable and biocompatible. We therefore expect this design can achieve the directly and targeted drug delivery t oward tumor cells and reduce side effect in cancer chemotherapy.

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94 LIST OF REFERENCE S 1 Campolongo M. J. et al. Adaptive DNA based materials for switching, sensing, and logic devices. Journal of Materials Chemistry 21 6113 6121 (2011). 2 Dahm, R. Discovering DNA: Friedrich Miescher and the early years of nucleic acid research. Human genetics 122 565 581 (2008). 3 Watson, J. D. & Crick, F. H. C. Molecular Structure of Nucleic Aicds a Structure for Deoxyribose Nucleic Acid. Nature 171 737 738 (1953). 4 Chargaff, E., Zamenhof, S. & Green, C. Composition of human desoxypentose nucleic acid. Nature 165 756 757 (1950). 5 Gehring, K., Leroy, J. & Guron, M. A tetrameric DNA structure with protonated cytosine cytosine base pairs. Nature 3 63 561 565 (1993). 6 Liu D. & Balasubramanian, S. A Proton Fuelled DNA Nanomachine. Angew. Chem. Int. Ed. 42 5734 5736 (2003). 7 Shu, W. et al. DNA Molecular Motor Driven Micromechanical Cantilever Arrays. Angew. Chem. Int. Ed. 127 17054 17020 (2005 ). 8 Chen, C. et al. Stimuli responsive controlled release system using quadruplex DNA capped silica nanocontainers. Nucleic Acids Research 39 1638 1644 (2011). 9 Surana, S., Bhat, J. M., Koushika, S. P. & Krishnan, Y. An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism. Nature Communications 2 340 (2011). 10 Asanuma, H. et al. Synthesis of azobenzene tethered DNA for reversible photoregulation of DNA functions: hybridization and transcription. Nature Protocols 2 203 212 (2007). 11 Bai, X., Li, Z., Jockusch, S., Turro, N. J. & Ju, J. Photocleavage of a 2 nitrobenzyl linker bri dging a fluorophore to the 5' end of DNA. Proceedings of National Academy of Sciences of the United States of America 100 409 413 (2003). 12 Kang, H. et al. Single DNA Molecule Nanomotor Regulated by Photons. Nano Letters 9 2690 2696 (2009). 13 Han, D. et al. Molecular engineering of photoresponsive three dimensional DNA nanostructures. chemical Communications 47 4670 4672 (2011).

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99 BIOGRAPHICAL SKETCH Yunfei Zhang was born in Hefei, Anhui Province, China, on 1990. In 2002 she enter ed the Hefei NO.1 High school scientific genius class as the only full score in Chemistry of Anhui Pr ovince. In 2005 Yunfei entered University of Scienc e and Technology of China with major in Chemical Physics start her individual research o n biomaterial extraction. After two year work, Yunfei wrote a first author paper that published on Chinese Polymer Bulletin, which al so made her be of USTC. In 2009 Yunfei came to US to continue her research under the guidance of Dr Weihong Tan of University of Florida.