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Target-Responsive DNA-Switches for Analytical and Biomedical Applications

Permanent Link: http://ufdc.ufl.edu/UFE0042789/00001

Material Information

Title: Target-Responsive DNA-Switches for Analytical and Biomedical Applications
Physical Description: 1 online resource (160 p.)
Language: english
Creator: ZHU,ZHI
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ANALYTICAL -- APTAMER -- BIOMEDICAL -- COCAINE -- COLORIMETRIC -- DNA -- DNAZYME -- ENGINEERING -- GRAPHENE -- HYDROGEL -- LOGIC -- PB -- PHOTODYMAIC -- SCAFFORLD -- SWNT -- THERAPY
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Detection of molecular recognition events has always been an area of primary focus in analytical science, biomedical research and biotechnological application, with a highly selective molecular recognition element as the key factor. Out of many potential molecular probes, nucleic acid based functional probes hold great promise in a variety of unique application, due to their remarkable molecular recognition properties and structural features. As an ideal building block, DNA is renowned for its predictability and specificity of Watson-Crick base pairing. Upon certain molecular engineering process, some sequence-specific DNA, well-known as aptamer, can effectively recognize other molecules such as ions and proteins. Combining with other significant features such as simplicity of synthesis, diversity of modification, and compatibility with many detection techniques, DNA provides a new opportunity and design medium for molecular engineering, which will fundamentally change the field related to molecular probes. The goal of my research is combining the selectivity of DNA with other functional materials for the development and application of novel probes, named DNA-switches, for target-responsive reactions in different systems. The first part of my research is constructing functional-DNA cross-linked hydrogel for visual detection. Visual detection is an increasingly attractive method, especially for rapid diagnostics in disaster situation, home healthcare settings, and in poorly equipped rural areas. We grafted the DNA recognition into hydrogel and built such a colorimetric detection platform. The hydrogel was made of polyacrylamide and cross-linked into gel by DNA hybridization. AuNPs and enzyme were trapped inside the 3D network of the hydrogel. The dissociation of hydrogel in response to its target released caged AuNPs and enzyme to take part in their role for visual detection and signal amplification. We built up the system with cocaine aptamer for cocaine visual detection, and further extent the system with DNAzyme for Pb2+ detection. The second part of my research is establishing a safer and more efficient photodynamic therapy based on DNA and carbon nanomaterials. Photodynamic therapy (PDT) is gaining wide acceptance as an alternative noninvasive treatment of cancers. To design controllable PDT agent, the photosensitizer was covalently linked with a ssDNA and wrapped onto the surface of carbon materials, such that the photosensitizer can only be activated by light upon target binding. The carbon materials showed great quenching efficiency to singlet oxygen generation (SOG). In the presence of targets, the binding events changed the ssDNA conformation, disturbed its interaction with carbon materials, and caused ssDNA to fall off carbon surface, resulting in the restoration of SOG. SWNTs and thrombin aptamer were used for thrombin mediated SOG. Further, ssDNA/GO as another PDT was design for mRNA imaging and therapy. Similar effect has been achieved with the mRNA triggered singlet oxygen generation. These studies validated the potential of our design as a novel PDT agent with regulation by target molecules, enhanced specificity, and efficacy of therapeutic function, which directs the development of PDT to be safer and more selective. The last research subject of my research is molecule engineering of DNA nanostructure as logic switchboard for medical diagnostics. Logic diagnosis becomes increasingly valuable with the development of modern medical technology, since more complicated and abnormal results from a medical examination need to be interpreted. Using highly developed DNA nanotechnology, DNA nanoscaffold as a logic switchboard was constructed. By smartly designing the DNA sequences to control the communication between sensor tile and reporter based on strand-displacement principle, implementation of OR and AND logic gates have been successfully realized. These results demonstrated the feasibility and capability of DNA nanotiles as scaffold to construct logic operation. In summary, my research mainly focuses on the molecular engineering of DNA along or with other functional materials for more sensitive and selective detection and therapy, which we envision will be useful in analytical and biomedical applications.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 ZHI ZHU.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042789:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042789/00001

Material Information

Title: Target-Responsive DNA-Switches for Analytical and Biomedical Applications
Physical Description: 1 online resource (160 p.)
Language: english
Creator: ZHU,ZHI
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ANALYTICAL -- APTAMER -- BIOMEDICAL -- COCAINE -- COLORIMETRIC -- DNA -- DNAZYME -- ENGINEERING -- GRAPHENE -- HYDROGEL -- LOGIC -- PB -- PHOTODYMAIC -- SCAFFORLD -- SWNT -- THERAPY
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Detection of molecular recognition events has always been an area of primary focus in analytical science, biomedical research and biotechnological application, with a highly selective molecular recognition element as the key factor. Out of many potential molecular probes, nucleic acid based functional probes hold great promise in a variety of unique application, due to their remarkable molecular recognition properties and structural features. As an ideal building block, DNA is renowned for its predictability and specificity of Watson-Crick base pairing. Upon certain molecular engineering process, some sequence-specific DNA, well-known as aptamer, can effectively recognize other molecules such as ions and proteins. Combining with other significant features such as simplicity of synthesis, diversity of modification, and compatibility with many detection techniques, DNA provides a new opportunity and design medium for molecular engineering, which will fundamentally change the field related to molecular probes. The goal of my research is combining the selectivity of DNA with other functional materials for the development and application of novel probes, named DNA-switches, for target-responsive reactions in different systems. The first part of my research is constructing functional-DNA cross-linked hydrogel for visual detection. Visual detection is an increasingly attractive method, especially for rapid diagnostics in disaster situation, home healthcare settings, and in poorly equipped rural areas. We grafted the DNA recognition into hydrogel and built such a colorimetric detection platform. The hydrogel was made of polyacrylamide and cross-linked into gel by DNA hybridization. AuNPs and enzyme were trapped inside the 3D network of the hydrogel. The dissociation of hydrogel in response to its target released caged AuNPs and enzyme to take part in their role for visual detection and signal amplification. We built up the system with cocaine aptamer for cocaine visual detection, and further extent the system with DNAzyme for Pb2+ detection. The second part of my research is establishing a safer and more efficient photodynamic therapy based on DNA and carbon nanomaterials. Photodynamic therapy (PDT) is gaining wide acceptance as an alternative noninvasive treatment of cancers. To design controllable PDT agent, the photosensitizer was covalently linked with a ssDNA and wrapped onto the surface of carbon materials, such that the photosensitizer can only be activated by light upon target binding. The carbon materials showed great quenching efficiency to singlet oxygen generation (SOG). In the presence of targets, the binding events changed the ssDNA conformation, disturbed its interaction with carbon materials, and caused ssDNA to fall off carbon surface, resulting in the restoration of SOG. SWNTs and thrombin aptamer were used for thrombin mediated SOG. Further, ssDNA/GO as another PDT was design for mRNA imaging and therapy. Similar effect has been achieved with the mRNA triggered singlet oxygen generation. These studies validated the potential of our design as a novel PDT agent with regulation by target molecules, enhanced specificity, and efficacy of therapeutic function, which directs the development of PDT to be safer and more selective. The last research subject of my research is molecule engineering of DNA nanostructure as logic switchboard for medical diagnostics. Logic diagnosis becomes increasingly valuable with the development of modern medical technology, since more complicated and abnormal results from a medical examination need to be interpreted. Using highly developed DNA nanotechnology, DNA nanoscaffold as a logic switchboard was constructed. By smartly designing the DNA sequences to control the communication between sensor tile and reporter based on strand-displacement principle, implementation of OR and AND logic gates have been successfully realized. These results demonstrated the feasibility and capability of DNA nanotiles as scaffold to construct logic operation. In summary, my research mainly focuses on the molecular engineering of DNA along or with other functional materials for more sensitive and selective detection and therapy, which we envision will be useful in analytical and biomedical applications.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 ZHI ZHU.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042789:00001


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1 TARGET RESPONSIVE DNA SWITCHES FOR ANALYTICAL AND BIOMEDICAL APPLICATIONS By ZHI ZHU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE O F DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Zhi Zhu

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3 To my parents

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4 ACKNOWLEDGMENTS I am deeply indebted to a long list of people, without whom this dissertation would not be possible. First, I wish to express my gratit ude to my exceptional research advisor, Dr Weihong Tan for providing the tremendous support, guidance and help for my research and life during the five year PhD studies at University of Florida. His advice and suggestions made my projects go more smoothly. The e ncouragements and inspirations he constantly delivered kept me studying. I also appreciate all the valuable opportunities that he provided for m e to train me to be a more mature person and a better scientist. Also, I thank Dr. Charles R. Martin, Dr. Richa rd A. Yost Dr. Gail E. Fanucci and Dr. Donn M. Dennis for being par t of my graduate committee I greatly appreciate their advice, assistance, and encouragement during the past years. The dissertation is a result of successful collaborations with scienti sts in different areas. I would like to thank Dr. Lin Wang and Dr. Hui Chen for the guidance and treme ndous help to lead me into research during my first year. I thank Dr. Zhiwen Tang and Dr. Ronghua Yang for initiating the single walled carbon nanotube pr oject. I greatly appreciate Dr. Chaoyong Yang, Dr. Xiaoling Zhang, and Dr. Huaizhi Kang for their critical comments and help in hydrogel visual detection project. I am grateful to Dr. Haipeng Liu for all the knowledge about nucleic acids he taught me and m any helpful discussions on different projects as well as the valuable friendship. I would like to thank Dr. Kathryn R. Williams, Dr. Yan Chen, Dr. Joseph Phillips, Dr. Youngmi Kim, Dr. Jilin Yan, Dr. Yufen Huang, Dr. Ruowen Wang, Dr. Mohammed Ibrahim Shuko or, Dr. Quan Yuan, Jin Huang, for all the experimental help and research discussions. I really

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5 appreciate Hui Wang an d Suwussa Bamrungsap as being supportive friends and colle a g u e s for all five year PhD life we spent together. Tan group is a big family. The help and friendship from former and current group members make my memory of Gainesville enjoyable and unforgettable. I would like to thank Dr. Josh E. Smith, Dr. Colin Medley, Dr. Prabodhika Mallikaratchy, Dr. Marie Carmen Estevez, Dr Kwame Sefah, Dr. Liu Yang, Dr. Karen Martinez, Dr. Kelong Wang, Dr. Parag A. Parekh, Dr. Ling Meng, Dr. Jennifer Martin, Pinpin Sheng, Dalia Lopez Colon, Xiangling Xiong, Ying Pu, Basri Gulbakan, Dimitri Simaeys Van, Elizbeth Jimenez, Tao Chen, Lu Pen g, Guizhi Zhu, D a Han, Cuichen Wu, Yunfei Z hang and others for their friendship encouragement, and help. Each of them has made this journey very enjoyable and pleasant. Finally, I am deeply indebted to my parents for their unconditional love, encouragem ent, support and companion to make me who I am right now. Their hard working, strong spirit and unselfish dedication inspire me to always keep going on.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................... 16 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 19 Review of Nucleic Acids ................................ ................................ .......................... 19 Composition and Structure of Nucleic Acids ................................ ..................... 19 Chemical Synthesis of Nucleic Acids ................................ ............................... 21 Molecular Engineering of Nucleic Acids ................................ ........................... 24 DNA based nanostructures ................................ ................................ ........ 26 DNA based nanomachines ................................ ................................ ........ 27 Nucleic Acid Aptamers ................................ ................................ ..................... 28 DNAzymes ................................ ................................ ................................ ....... 30 Hydrogels and Their Applications ................................ ................................ ........... 32 Hydrogels for Biosensing ................................ ................................ .................. 33 DNA Inspired Hydrogels ................................ ................................ ................... 35 Carbon Nanomaterials and Their Applications ................................ ........................ 37 Single Walled Car bon Nanotubes ................................ ................................ .... 37 Optical properties of SWNTs ................................ ................................ ...... 38 The specific interaction between DNA and SWNTs ................................ ... 38 Graphene Oxides ................................ ................................ ............................. 40 Photodynamic Therapy ................................ ................................ ........................... 41 2 APTAMER CROSS LINKED HYDROGEL AS A COLORIMETRIC P LATFORM FOR VISUAL DETECTION ................................ ................................ ..................... 53 Introduction ................................ ................................ ................................ ............. 53 Experimental Section ................................ ................................ .............................. 54 Chemicals and Instrumentation ................................ ................................ ........ 54 Synthesis of Acrylic Phosphoramidite ................................ .............................. 54 Synthesis of DNA Sequences ................................ ................................ .......... 55 Hydrogel Formation ................................ ................................ .......................... 56 Synthesis and Modification of Colloidal Gold Nanoparticles ............................. 56 Absorbance Measurement ................................ ................................ ............... 57 Results and Discussion ................................ ................................ ........................... 57 AuNPs for Principle Demonstration ................................ ................................ .. 59 Leaking study ................................ ................................ ............................. 60

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7 Target responsive kinetics study ................................ ................................ 61 Competition assay for AuNP hydrogel ................................ ....................... 62 Enzymatic Reaction for Signal Amplification ................................ .................... 63 Target response in enzyme assay ................................ ............................. 63 Specificity test ................................ ................................ ............................ 65 Conclusion s ................................ ................................ ................................ ............ 65 3 DNAZYME CROSS LINKED HYDROGEL FOR VISUAL DETECTION OF PB (II) ................................ ................................ ................................ ................................ 73 Introduction ................................ ................................ ................................ ............. 73 Experimental Section ................................ ................................ .............................. 74 Chemicals and Instrumentation ................................ ................................ ........ 74 Synthesis of DNAzyme ................................ ................................ ..................... 75 Hydrogel Formation ................................ ................................ .......................... 76 PAGE for Probe Validation ................................ ................................ ............... 76 Results and Discussion ................................ ................................ ........................... 77 Sequence Design Strategy ................................ ................................ ............... 78 The Per formance of DS 1 Related Probes ................................ ....................... 79 PAGE for system study ................................ ................................ .............. 79 Leakage test ................................ ................................ .............................. 80 Hydrogel response to Pb 2+ ................................ ................................ ......... 81 The Performance of DS 2/E14 7 Probes ................................ .......................... 82 PAGE for validation ................................ ................................ .................... 82 Hydrogel response to Pb 2+ ................................ ................................ ......... 82 Conclusion s ................................ ................................ ................................ ............ 83 4 REGULATION OF SINGLET OXYGEN GENERATION USI NG SINGLE WALLED CARBON NANOTUBES ................................ ................................ ......... 89 Introduction ................................ ................................ ................................ ............. 89 Experimental Section ................................ ................................ .............................. 90 Chemicals and Instrumentation ................................ ................................ ........ 90 Synthesis of Aptamer Photosensitizer (AP) ................................ ...................... 91 Preparation of Single Walled Carbon N anotube ................................ ............... 92 Fluorescence E xperiment ................................ ................................ ................. 92 AP SWNT Response towards Thrombin ................................ .......................... 93 Gel E lectrophoresis for Demonstration ................................ ............................. 93 The P hototoxicity of AP SWNT toward L iving C ells ................................ ......... 94 DP SWNT Response towards cDNA ................................ ................................ 94 Results and Discussion ................................ ................................ ........................... 95 Fluorescence and SOG Quenching by SWNTs ................................ ................ 96 Fluorescence Enhancement and SOG upon Target Binding ............................ 96 Quantitative Response to Target ................................ ................................ ...... 97 Gel Electrophoresis for Proving the Different States of AP SWNT ................... 97 Specificity of AP SWNTs again Proteins ................................ .......................... 98 Phototoxicity of AP SWNT toward Living Cel ls ................................ ................. 99

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8 F luorescence R esponse and SOG of DP SWNT towards cDNA ..................... 99 Conclusion s ................................ ................................ ................................ .......... 100 5 SSDNA/GRAPHENE OXIDE FOR MOLECULAR MEDIATION OF MRNA IMAGING AND POTENTIAL PHOTODYNAMIC THERAPY ................................ 108 Introduction ................................ ................................ ................................ ........... 108 Experimental Section ................................ ................................ ............................ 110 Chemicals and Instrumentation ................................ ................................ ...... 110 Preparation of Graphene Oxide ................................ ................................ ...... 111 Fluorescence E xperiment ................................ ................................ ............... 111 Gel E lectrophoresis for Demonstration ................................ ........................... 111 Results and Discussion ................................ ................................ ......................... 112 Quenching Efficiency of GO ................................ ................................ ........... 112 Restoration of Fluorescence and SOG by cDNA ................................ ............ 113 Quantitative Response to cDNA ................................ ................................ ..... 113 Detection Selectivity of m Ce6/GO ................................ ................................ 114 Gel Electrophoresis for System Demonstr ation ................................ .............. 114 Confocal Image for Internalization Demonstration ................................ .......... 115 Conclusion s ................................ ................................ ................................ .......... 116 6 DNA SCAFFOLD AS LOGIC SWITCHBOARD FOR SMART DETECTION ......... 122 Introduction ................................ ................................ ................................ ........... 122 Experimental Section ................................ ................................ ............................ 124 Chemicals and Instrumentation ................................ ................................ ...... 124 Self assembly of Nanotiles ................................ ................................ ............. 124 Gel E lectro phoresis for Structure Validation ................................ ................... 125 Fluorescence E xperiment ................................ ................................ ............... 125 Results and Discussion ................................ ................................ ......................... 125 OR Gate Construction and Validation ................................ ............................ 126 PAGE for structure validation ................................ ................................ ... 126 OR operation proved by fl uorescent measurement ................................ .. 127 AND Gate Construction and Validation ................................ .......................... 128 AND operation proved by fluorescent measurement ............................... 128 Conclusion s ................................ ................................ ................................ .......... 129 7 SUMMARY AND FUTURE DIRECTIONS ................................ ............................ 140 Target Responsive DNA Switches for Analytical and Biomedical Applications .... 140 Functional DNA Cross linked Hydrogel for Visual Detection .......................... 141 Regulation of Singlet Oxygen Generation Using Carbon Nanomaterials ....... 141 DNA Scaffold as a Logic Switchboard for Smart Detection ............................ 142 Future Directions ................................ ................................ ................................ .. 143 Hydrogel Lab on chip Device on Paper for Visual Detection .......................... 143 DNA Scaffold as a Logic Switchboard for Smart Detec tion ............................ 144

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9 LIST OF REFERENCES ................................ ................................ ............................. 146 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 160

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10 LIST OF TABLES Table page 2 1 Sequences of oligonucleotides for making hydrogel. ................................ .......... 67 3 1 Sequences of DNAzymes and substrates. ................................ ......................... 84 4 1 Sequences of oligonucleotides synthesized. ................................ .................... 102 5 1 Sequences of oligonucleotides synthesized. ................................ .................... 117 6 1 Sequences of the sensor and reporter part for OR gate. ................................ .. 136 6 2 Sequences of the sensor and reporter part for AND gate. ................................ 139

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11 LIST OF FIGURES Figur e page 1 2 DNA and RNA structures via phosphodiester linkage. ................................ ....... 44 1 3 Structure of DNA double helix and base pairing. ................................ ................ 45 1 4 Phosphoramidite chemistry. A) Structure of phosphoramidite. B) Four monomers of nucleic acid phosphoramidite. ................................ ...................... 45 1 5 Aut omated oligonucleotide synthesis through phosphoramidite chemistry. ........ 46 1 6 1D DNA self assembly. A) DNA double helix. B) Stick end cohesion. 5 ............ 47 1 7 2D DNA nanostructures. A) Sticky end assembly of branched DNA Holiday motif. 4 B) DX DNA tile and TX DNA tile. 8 C) 4X4 tile resulting in 2D square lattice. 13 D) DNA origami with 2D shapes. 14 ................................ ...................... 47 1 8 3D DNA nanostructures. A) Cube and truncated octahedron. 25, 26 B) Octahedron. 27 C) Tetrahedron and bipyramid. 28, 29 D) Polyhedra. 30 E) Box with controlled lid. 31 ................................ ................................ ............................ 48 1 9 DNA nanomachines. A) B Z DNA transistor. 32 B) DNA tweezer. 33 C) DNA walker. 34 ................................ ................................ ................................ ............. 49 1 10 Systematic Evolution of Ligands by EXponential enrichment (SELEX). ............. 50 1 11 8 17 DNAzyme. A) The Secondary structure of 8 17. 52 B) The two step mechanism of substrate cleavage catalyzed by 8 17 DNAzyme and Pb 2+ 54 ..... 51 1 12 Mechanism of action of photodynamic therapy (PDT). ................................ ....... 52 2 1 Synthesis of acrylic phosphoramidite. ................................ ................................ 67 2 2 Working principle o f DNA cross linked hydrogel for signal amplification and visual detection. Enzyme is trapped inside hydrogel cross linked by sandwiched hybridization of linker Apt sequence to DNA Strand A and Strand B grafted on linear polyacrylamide chains (PS A and PS B ). Physical separation of substrate from enzyme by a 3 D network of hydrogel prevents enzymatic reaction from taking place. However, addition of a target to competitively bind Linker Apt dissolves the hydrogel, which releases the caged enzyme for enzymatic reaction, resulting in a color change. .................... 68 2 3 Leaking test of hydrogels with different crosslinking DNA concentrations over a 96 hour observation period. The absorbance was monitored at 520 n m for AuNPs. ................................ ................................ ................................ ............... 69

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12 2 4 Release of AuNPs from the hydrogel upon introduction of cocaine. A) Photograph of hydrogel before (left) and 30 min after (right) addition of 1 mM cocaine. Four hydrogels with different DNA cross linker concentration (0.1 mM to 0.7 mM) were prepared to study cargo release kinetics. AuNPs were trapped in DNA hydrogel with a cover layer of buffer. B) Release kinetics of AuNPs from four types of hydrogels upon introduction of 1 mM cocaine at 30 min. The absorbance was normalized by the maximum AuNP signal. ............... 70 2 5 Photograph of gels with entrapped AuNPs for competition assay to demonstrate that the aptamer target b inding is the dominant reaction. .............. 71 2 6 Photograph of gel response to different concentrations of cocaine. I 2 solution was always introduced 10 minutes after cocaine addition as the last step to evaluate the results. Ez = amylase. ................................ ................................ .... 71 2 7 Photograph of c ontrol tests for two cocaine analogs, benzoylecgonine (BE) and ecgonine methyl ester (EME) I 2 solution was always introduced 10 mi nutes after cocaine addition. ................................ ................................ ........... 72 3 1 Working Principle of DNAzyme cross linked hydrogel for Pb 2+ visual detection. AuNPs are trapped inside hydrogel cross linked by hydridization of substrate and enzyme strands grafted onto linear polyacrylamide chains (PS DS and PS E). Addition of target Pb 2+ catalyzes the cleavage reaction and dissolves the hydrogel, which releases the caged AuNPs for visual detection. .. 84 3 2 Sequence design strategy. A) Schematic illustration of the reaction. B) The actual sequences of S DS and S E. ................................ ................................ ... 85 3 3 Polyacrylamide gel electrophoresis (PAGE) results of DS 1 related probes when treated with different concentrations of Pb 2+ ................................ ............ 86 3 4 Photograph of DS 1 hydrogel with entrapped AuNPs. A) The AuNP leaking study of hydrogels made of DS 1 with E10 9, E10 7 and E10 5. B) The hydrogels with and without the addition of 100 M Pb 2+ for 30 minutes. ............. 87 3 5 Polyacrylamide gel electrophoresis (PAGE) results of DS 2/E14 7 treated with different concentrations of Pb 2+ ................................ ................................ .. 88 3 6 Photograph of DS 2/E14 7 gel response to different concentrations of Pb 2+ ..... 88 4 1 Synt hesis of Chlorin e6 conjugated DNA. ................................ ......................... 102 4 2 Schematic of aptamer photosensitizer SWNT complex and the regulation of SOG upon target binding: (I) AP and SWNTs were mixed together to form AP SWN T complex. The ssDNA aptamer is wrapped on the surface of SWNTs, which brings the photosensitizer close to the SWNT to quench SOG. (II) Target binding with am aptamer can disturb the interaction between AP and SWNTs, resulting in the restoration of SOG. ................................ ....... 103

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13 4 3 Fluorescence and SOG quenching by SWNTs. A) The Ce6 fluorescence spectra of buffer, AP and AP SWNT. B) The SOSG signal readout after 10.5 minutes of irradiation with light at 404 nm. ................................ ....................... 103 4 4 Fluorescence and SOG regulation by thrombin. A) The Ce6 fluorescence spectra of the buffer, AP SWNT, and AP SWNT + Tmb. B) The SOSG signal readout after 10.5 minutes of irradiation wi th excitation at 404 nm. ....... 104 4 5 Ce6 and SOSG response to thrombin concentration. A) With increasing of thrombin concentration, the Ce6 fluorescence gradually increased. B) The SOSG signal p lotted as the function of thrombin concentration. The purple ................................ ........................... 104 4 6 Polyacrylamide gel electrophoresis results. A) Fluorescence image of the PAGE gel recorded by Ce6 fluorescence to show the DNA position. B) The photo of the PAGE gel after staining by Coomassie blue to indicate the position of proteins as blue bands. ................................ ................................ ... 105 4 7 SOG specifici ty. SOG selectivity of AP SWNT towards different proteins: thrombin, bovine serum albumin (BSA), protein A (PA), protein L (PL), NeutrAvidin (NA) and IgG. The SOSG fluorescence signals were normalized to that of AP SWNT. ................................ ................................ ......................... 105 4 8 The cell viability affected by the phototoxicity of AP SWNT, determined by CellTiter 96 AQueous One Solution Reagent and plate reader. .................... 106 4 9 Fluorescenc e and SOG regulation by cDNA. A) Ce6 fluorescence spectra of the buffer, DP SWNT, DP SWNT + cDNA and DP SWNT + rDNA. The DP the Ce6 fluorescence increased significantly. B) The SOSG signals were normalized to that of DP SWNT. The same trend as Ce6 fluorescence was observed for SOG. ................................ ................................ ............................ 106 4 10 Ce6 and SOSG response to cDNA concentration. A) The Ce6 fluorescence spectra of b uffer and DP SWNT with different concentrations of cDNA. B) The SOSG signal plotted as the function of cDNA concentration. The purple ................................ ........................... 107 5 1 TEM imag e of soluble GO. ................................ ................................ ............... 117 5 2 Schematic representation of photosensitizer ssDNA/GO complex and the regulation of SOG upon target binding. The photosensitizer ssDNA attaches onto GO to get photosens itizer quenched. Upon target mRNA binding, the duplex leaves away from GO and restores the SOG. ................................ ....... 118 5 3 Optimization of GO quenching conditions. A) Investigation of GO quenching in different buffer conditions. B) With the increasing of GO concentration, the

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14 fluorescence intensity of mj Ce6 gradually decreased. C) The fluorescence intensity of m Ce6 versus the concentration of GO. ................................ ......... 118 5 4 Fluorescence and SOG regulation by target cDNA. A) The Ce6 fluorescence spectra of the buffer GO, m Ce6/GO, and m Ce6/GO + cDNA. B) The SOSG signal readout after 10.5 minutes of irradiation with excitation at 404 nm. ................................ ................................ ................................ ................... 119 5 5 Ce6 fluorescence and SOSG response to cDNA concentration. A) With the increase of cDNA concentration, the Ce6 fluorescence gradually increased. B) The SOSG signal plotted as the function of cDNA concentration. ............... 119 5 6 Polyacrylamide gel electrophoresis results. Gel was stained with stains all to show DNA position. ................................ ................................ .......................... 120 5 7 Intracelluar imaging of ssDNA/GO complex. Confocal fluorescence microcopy of MDA MB 231 cells treated with m Ce6/GO (A and B) and control r Ce6/GO (C and D). Ce6 fluorescence field (left) and bright filed Ce6 fluorescence overlay (right) are shown. ................................ ............................ 121 6 1 The symbols and truth tables of basic logic gates. ................................ ........... 131 6 2 Sequence directed DNA strand displacement. A) Full length complement strand AB l eads to the irreversible displacement of strand A via a three way brand migration mechanism. B) For shorter length complement strand two situations might happen. (1) Strand A keeps hybridize with strand and form three strand structure. (2) Strand A releases from the because of the unstable duplex formation. ................................ ................................ ............... 132 6 3 Strand structure and DNA sequences used in nanotiles. A) OR gate nanotile. B) AND gate nanotile. ................................ ................................ ....... 133 6 4 The design strategy of DNA scaffold based logic switchboard. ........................ 134 6 5 The principle of OR gate based on DNA sensor nanotiles and duplex repor ters. ................................ ................................ ................................ .......... 135 6 6 The PAGE gel experiment results for nanotile structure validation. .................. 136 6 7 Fluorescence measurement results of OR nanotile logic gate. A C) The time based measurement with input 1, input 2 or input 1&2. D) Symbol and truth table of OR logic gate. E) Fluorescence intensities are summarized as a truth table. ................................ ................................ ................................ ...... 137 6 8 The principle of AND gate based on DNA sensor nanotiles and duplex reporters. ................................ ................................ ................................ .......... 138

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15 6 9 Fluorescence measurement results of AND nanotile logic gate. A) and B) Time based measuremen t with addition of input 1&2 and input 2&1. C) Symbol and truth table of OR logic gate. D) Fluorescence intensities are summarized as a truth table. ................................ ................................ ............ 139 7 1 Illustration of paper based lab o n chip device with different color patterns for buffer and target solution. A) Construction of hydrogel paper strip. B) Dipping the strip in buffer solution, the sensing molecules will move up until gel region. C) Dipping the strip in target solution, t arget dissolves the gel, and sensing molecules can move up cross the gel region. .............................. 145

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16 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Req uirements for the Degree of Doctor of Philosophy TARGET RESPONSIVE DNA SWITCHES FOR ANALYTICAL AND BIOMEDICAL APPLICATIONS By Zhi Zhu May 2011 Chair: Weihong Tan Major: Chemistry Detection of molecular recognition events has always been an area of pri mary focus in analytical science, biomedical research and biotechnological application, with a highly selective molecular recognition element as the key factor. Out of many potential molecular probes, nucleic acid based functional probes hold great promise in a variety of unique application, due to their remarkable molecular recognition properties and structural features. As an ideal building block, DNA is renowned for its predictability and specificity of Watson Crick base pairing U pon certain molecular e ngineering process, some sequence specific DNA, well known as aptamer, can effectively recognize other molecules such as ions and proteins. Combining with other significant features such as simplicity of synthesis, diversity of modification, and compatibil ity with many detection tech niques, DNA provides a new opportunity and design medium for molecular engineering, which will fundamentally change the field related to molecular probes. The goal of my research is combining the selectivity of DNA with other fu nctional materials for the development and application of novel probes, named DNA switches, for target responsive reactions in different systems.

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17 The first part of my research is constructing functional DNA cross linked hydrogel for visual detection. Visua l detection is an increasingly attractive method, especially for rapid diagnostics in disaster situation, home healthcare settings, and in poorly equipped rural areas W e grafted the DNA recognition into hydro gel and built such a colorimetri c detection pla tform. The hydrogel was made of polyacrylamide and cross linked into gel by DNA hybridi zation. AuNPs and enzyme were trapped inside the 3D network of the hydrogel. The dissociation of hydrogel in response to its target released caged AuNPs and enzyme to ta ke part in their role for visual detection and signal amplification. We built up the system with cocaine aptamer for cocaine visual detection, and further extent the system with DNAzyme for Pb 2+ detection. The second part of my research is establishing a safer and more efficient photodynamic therapy based on DNA and carbon nanomaterials. Photodynamic therapy (PDT) is gaining wide acceptance as an alternative noninvasive treatment of cancers. To design controllable PDT agent, the photosensitizer was covalen tly linked with a ssDNA and wrapped onto the surface of carbon materials, such that the photosensitizer can only be activated by light upon target binding. The carbon materials showed great quenching efficiency to singlet oxygen generatio n (SOG). In the pr esence of target s the binding events changed the ssDNA conformation, disturbed its interaction with carbon materials, and cause d ssDNA to fall off carbon surface, resulting in the restoration of SOG. SWNTs and thrombin aptamer were used for thrombin media ted SOG. Further, ssDNA/GO as another PDT was design for mRNA imaging and therapy. Similar effect has been achieved with the mRNA triggered singlet oxygen generation. These studies validated the potential of our design as a novel PDT agent with regulation by target

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18 molecules, enhanced specificity, and efficacy of therapeutic function, which directs the development of PDT to be safer and more selective. The last rese arch subject of my research is molecule engineering of DNA nanostructure as logic switc hboard for medical diagnostics. Logic diagnosis becomes increasingly valuable with the development of modern medical technology, since more complicated and abnormal results from a medical examination need to be interpreted. Using highly developed DNA nanotechnol ogy, DNA nanoscaffold as a logic switchboard was constructed. By smartly designing the DNA sequences to control the communication between sensor tile and reporter based on strand displacement principle, implementation of OR and AND logic gates have been su ccessfully realized. These results demonstrated the feasibility and capability of DNA nanotiles as scaffold to construct logic operation. In summary, my research mainly focuses on the molecular engineering of DNA along or with other functional materials f or more sensitive and selective detection and therapy, which we envision will be useful in analytical and biomedical applications.

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19 CHAPTER 1 INTRODUCTION Review of Nucleic Acid s Nucleic acids are biological macromolecules that are essential to life. Ove r the past decade, increasing evidence reveals that nucleic acid molecules are responsible for a wide range of cellular functions, such as regulation/ silencing of gene expression, structural support for molecular machines, and precise control of cell behav ior Nucleic acid s which are polymers of nucleotides, can be largely divided into two groups: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) depending on the nucleotides involved. Since the elucidation of nucleic acid structure in the middle 1950 s, 1 nucleic acids have been transformed and diversified into many fields, from biology to material science engineering. He re a brief overview of nucleic acid structure and function is given. Composition and Structure of Nucleic Acids Chemical degradation studies in the early twenty century on material extracted from cell nuclei found that the high molecular weight nucleic ac id polymer was composed of repeating units, termed nucleotides, each of which comprises three units linked together a phosphate group, a sugar ring, and a nucleotide base (Figure 1 1). The bases are planar aromatic heterocyclic molecules and are divided into two groups: the purine s, adenine (A) and guanine (G) and the pyrimidine s, cytosine (C) thymine (T) and uracil (U) As presented in Figure 1 2, individual nucleotide units are joined between phosphate group and sugar ring.

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20 The important early experimental data of Chargaff 2 on DNA reveal ed a universal 1:1 ratio of A with T and G with C, although the base compositions of DNA betwee n species varied The base equivalency in conjunction with the X ray data by Rosalind Frank l in and Maurice Wilkins, led James Watson and Francis Crick to propose the famous double helix structure in 1953 in which pairs of purine and pyrimidine bases on each strand are linked by specific hydrogen bonds. The A T base pair has two hydrogen bonds and G C base pair has three hydrogen bonds, often referred as Watson Cr ick base pairing. In the double helix structure shown in Figure 1 3, the outer edges of the helix is formed of alternating ribose sugar rings and phosphate groups and the inner layer contains the nucleotide bases paired and preserved inside the helix. Bec ause of the phosphate groups, the nucleic acids normally are highly negative charged. Regarding the base pairing, the NH groups of the bases are good hydrogen bond donors, while the carboxyl oxygens and ring nitrogens serve as hydrogen bond receptors. Alth ough only four types of different nu cleotide bases occur in a nucleic acid, the numbers of nucleotides can vary enormously from around 80 nucleotides in transfer RNA to over 10 8 base pairs in a eukaryotic chromosome. The mutual recognition of A by T and G by C established the fidelity of DNA transcription and translation. DNA codes for protein synthesis by first transcribing to messenger RNA which leaves the nucleus for translation into protein with three nucleotide sequences corresponding to one amino a cid. This process involves ribosomes messenger RNA s (mRNA s ) which carry the transcribed code to ribosome, and transfer RNA s (tRNAs) which match the three nucleotide sequence with the

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21 corresponding amino acid and bring it to the growing polypeptide chain As a result, the ribosome works with mRNA and tRNA to build a sequence of amino acid s into a protein. Chemical Synthesis of Nucleic Acids Since the pioneer work in 1950s, chemical synthesis of nucleic acids has been highly developed and optimized with th e process fully automated in the late 1970s. 3 Currently the chemical synthesis of relatively short nucleic acids ( fewer than 20 0 bases) with defined sequence is a remarkably simple, rapid and inexpensive task on an automated instrument, called a DNA synthesizer D evelopmen ts in nucleic acid research have spurred a dramatic expansion of the range of available modifications, either on the end s or in the middle which, in turn, greatly broadens the nucleic acid applications. Fairly speaking, Synthetic oligonucleotide can be re garded as the fuel that drives the engine of molecular biology, evidenced by their wide applications in the poly merase chain reaction (PCR), DNA sequencing, mutagenesis, molecular beacons, microarrays, single nucleotide polymorphism (SNP) assays, and rapid expanding world of small RNAs. In th is s ection the basics of the solid support synthesis of o li g o nucleotide via phosphoramidite chemistry will be reviewed. First t he building block, phosphramidite (Figure 1 4A), is composed of different functional grou ps : a nucleoside base plus sugar ring, a phosphate, and protecti on groups A di iso hydroxyl of a nucleoside, hydroxyl of deoxyribose is capped with an acid labile dimetho xytriyl (DMT) group, which can be selective ly activated under acidic conditions during the synthesis. To prevent undesired side reactions, the phosphite is protected by a 2 cyanoethyl group, and all primary amines of nucleosides have to be rendered unreact ive by attaching specific protecting groups(Figure 1 4B), all

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22 of which are basic sensitive so that can be effectively removed by strong bases after the synthesis. Other modifier s such as fluorophore s amine s biotin, and spacers, although different in str ucture and protecting group, share the same strategy to design functional phosphoramidites. synthesis of oligonucl eotide is carried out a column containing the solid support (controlled pore glass bead (CPG)) with holes and channels in it, where first nucleot terminal hydroxyl group with a long space arm. The synthesis column has filters on both sides, to hold the beads while the reagents are passed through to react with beads and excess reagents can be easily remove d During the synthesis, the phosphoramidite building blocks are sequentially coupled to the growing oligonucleotide chain on the be ads. One synthesis cycle involves four chemical reactions: detritylation, cou pling, capping and oxid ation, shown in Figure 1 5. The first step, detritylation, is removal of the trityl group protecting hydroxyl at the end of the gr owing oligonucleot ide chain attached to the CPG by flushing the column with dilute acidic solution, either 3% dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in dichloromethane (DCM). hydroxyl group becomes the only group on column to react with the subs equent nucleotide The intensity of the orange color from DMT liberated at this step is measured by a UV Vis detector inside the instrument to determine the coupling efficiency of the previous step. The second step, coupling, is achieved by adding a ph o sp horamidite derivative of the next nucleotide with tetrazole, a weak acid, together through the column. The

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23 tetrazole protonates the diisopropylamine leading to the formation of the tetrazole phsphoramidite intermediate, which is susceptible to nucle ophilic hydroxyl group on CPG to form an unstable phosphate triester linkage. The reaction column is then washed to remove any extra tetrazole, unbound bases and by products. Since the coupling yield is not always 100%, some of the solid su pport bound hydroxyl site remains unreacted, which could react in later cycles resulting in an oligonucleotide with a deletion. To prevent this from happening, the third step, capping, is needed to terminate or cap the u hydroxyl groups by ac etylation to This is achieved by co delivering acetic anhydride and 1 methylimidazole through the column. The DMT group of the successful coupling step hydroxyl group from being capped. This step can minimize the length s of failure products and facilitates post synthesis HPLC purification. The last step, oxidation, convert s the internucleotide link age from the less stable phosphi te to the much more stable phosphate linkage. It is achieved by treating the support b ound materia ls with iodine and water in presence of weak base, such as pyridine. Iodine is used as the oxidizing agent and water as the oxygen donor. Other modifications, such as fluorophores, quenchers, biotin etc, can be introduced into any desired posi tion of an olignucleotide, if they can be converted into phosph or amidite derivatives and are compatible with DNA synthesis process. Otherwise, the post synthesis coupling or off machine coupling can also be applied for further modification of nucleic acids After t he synthesis and modification are complete, the full protected products are cleaved from the solid support and deprotected. The method varies according to the

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24 functional groups on the oligonucleotides. Normally, t his is done by incubating the CPG in concentrated ammonia at high temperature for an extended amount of time. All the protecting groups on the nucleosides and the 2 cyanoethyl group on phosphate are hydroxyl remains. Then during incubation in ethanol at 2 0 o C for 30 minutes, the oligonucleotide and some salts precipitate from the solution. The precipitate is collected by centrifugation at high speed The solid is re dissolved in 0.1 M triethylammonium acetate (TEAA, pH 7) for reverse d phase ion pairing HP LC with C 18 column as stationary phase and acetonitrile (ACN) and 0.1M TEAA water as the mobile phase. The TEAA, an organic cation, interact s with the negatively charged oligonucleotide to make a nonp o lar ion pair which can have better interaction with t he stationary phase. In revered phase HPLC, the alkyl chains interact with analytes and reverse the elution order. The polar compounds (failed products) are eluted first while non polar compounds (successful products with DMT group attached ) are retained. After the purified oligonucleotide is dried, the DMT group is removed by incubation with 80% acetic acid. The reaction is terminated with ethanol followed by vacuum drying. The pure oligonucleotide can be quantified by UV absorbance measurement at 260nm Molecular Engineering of Nucleic Acids Although n ucleic acids have long been known to define our genetic makeup, it w a s not until 30 years ago, 4 that structural DNA nanotechnology led DNA beyond just biology. Today, DNA molecular engineering and DNA nanotechnology have evolved into a unique interdisciplinary field, crossing chemistry, physics, biology, computer science, and material science. The biological function molecular recognition property

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25 biocompatibility and biodegradability of nucle ic acids have made DNA one of the most appealing and versatile bu ilding blocks for nanotechnology Scientists have used DNA to construct various nanostructures, nanomachines, and smart materials as well as functional ligands for a broad range of applicati ons from new materials to devices. DNA is an excellent structural building block for nanotechnology with several important features 5 7 The first and most important property is the molecular recognition with predict able hybridization energy offered by Watson Crick base pairing, which makes the DNA highly programmable and designable. Secondly, the DNA molecule has well known minuscule structural geometry, with a 3.4 nm helical repeat with about 2 nm in diameter Third it has combined stiffness and flexibility, and the mechanical properties can be easily tailored by simply adjusting the number of base pair s (bp). T he persistence length of ds DNA is about 50 nm, corresponding to 150 bp and ssDNA is much more flexible, s o the combination of dsDNA and ssDNA can form stable motifs with a desired topology. Fourth, molecular biology provides rich tools for DNA synthesis, manipulation and modification, such as automated phosph o ramidite chemistry mentioned above well establish ed methods for DNA purification and characterization, and various enzymes commercially available for DNA ligation, cleavage, labeling, amplification, etc. Fifth DNA is chemically and physically stable, nontoxic and biocompatible, making it suitable for i n vitro and in vivo applications. Last but not the least, through the evolutionary mechanisms, some functional DNA s such as apt amers and DNAzymes, provide a direct molecular bridge linking DNA with proteins and catalytic reactions, thus endow ing the DNA w ith enhanced capability. All the

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26 aforementioned advantages make DNA an attractive, versatile, and programmable building block for DNA nanotechnology. DNA based nanostructures The simplest and most basic DNA self asse mbly method is the spontaneous hybridiz ation between two complementary ssDNA strands to form a dsDNA double helix according to Watson Crick base pairing rules (Figure 1 6A) Two DNA molecules t e with each other preferentially ( Figure 1 6B ) However, these linear DNA chains alone cannot yield larger and more complicated nanostructures. As a result, branched DNA motifs have been created to extend the DNA self assembly into two dimension s (2D) an d even three dimension s (3D) For example, more than twenty years ago, Seeman first combined the four arm Holiday junction with sticky end cohesion to make periodic 2D and 3D lattices 4 a s illustrated in Figure 1 7A By joining two double helixes together through strand exchange, Seeman and coworkers constructed a gro up of branched complexes called crossover tiles (Figure 1 7B) such as double crossover (DX) tiles, 8 and triple crossover (TX) tiles 9 With p roper sticky ends design, these tiles were successf ully self assemble d into linear arrays, 10 2D lattices, 9, 11 and DNA tubes. 12 As shown in Figure 1 7C H ao and coworkers designed 4X4 tiles containing four four arm DNA branched junctions oriented with a square aspect ratio, resulting in nanoribbons and 2D nanogrids. 13 Another milestone in DNA structure de sign was the concept of DNA origami proposed by Rothemund in 2006 to increase the complexity and versatilit y in DNA self assembly (Figure 1 7D) 14 The long strand is folded into the desired shape by a number of short helper strands Almost any arbitrary patterns can be

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27 designed by computer and self assemble d such as rectangle, stars, smiley faces, maps and other 2D shapes. These 2D DNA templates provide the opportunity to position materi als with n anoscale precision, such as nanoparticle assembly 15 17 and protein nanoarrays. 13, 18, 19 Distance related reactions have also been studied on these templates, for e xample, distance dependent protein ligand binding, 20 and single molecule reactions 21, 22 Meanwhile, biosensors based on these DNA structures have also be en developed. 23, 24 Compared to 2D nanostructures, 3D DNA nanostructures are significantly more chal lenging and their construction has become a hot topic in recent years. The first example was achieved by Seeman and coworkers (Figure 1 8A) who prepared DNA cubes 25 and truncated octahedrons. 26 Hence then different unique 3D DNA motifs have been designed. Shih et al. 27 built a rigid DNA octa hedron by folding a 1.7 kb ssDNA in the presence of five 40 mer helper strands (Figure 1 8B) Turberfield et al. 28, 29 constructed a series of DNA tetrahedra and bipyramid s (Figure 1 8C) Mao et al. 30 devel oped a robust, one pot strategy for assembling 3D str uctures, such as tetrahedra, dodecahedra and buckyballs (Figure 1 8D) Moreover, DNA origami has also been extended from 2D to 3D. Anderson et al. 31 designed 3D nanoboxs with controllable lids (Figure 1 8E) These 3D DNA nanostructures although still in their infancy, have great potential in drug delivery and tissue engineering. DNA based nanomachines In addition to static DNA nanostructures, a number of DNA based nanomachines have been generated. The first devic e consisted of two double crossover molecules that can be converted from the normal right handed helix (B DNA) to the unusual left handed conformation (Z DNA) triggered by high salt concentrations and low

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28 temperatures (Figure 1 9A) 32 Later, Yurke et al. 33 constructed a pair of DNA tweezers that could be transformed between the close d and open state s by single stranded branch migration (Figure 1 9B) Shi n and Pierce 34 built a walker device with two distinguishable feet that can walk along a track with four different anchorages (Figure 1 9C) Meanwhile, various DNA nanomotors have been developed that can be powered by ions, DNA, protons, light and o ther biomolecules. The development of this field has demonstrated again that DNA is a versatile material in nanotechnology. They have huge potential to be used in nano mechanics, nano electronics, DNA computation, etc. Nucleic Acid Aptamers Aptamers are s ingle stranded (ss) oligonucleotides or peptide molecules that bind to a specific target molecule. Aptamer s are generated by an in vitro method known as SELEX (Systematic Evolution of Ligands by EXponential enrichment) 35, 36 The SELEX process normally starts with a random library of 10 13 ~ 10 16 ssDNA or ssRNA molecules and is followed by an iterative process to specifically amplify sequences hav ing high binding affinity to a target The aptamers can bind to small molecules, including metal ions 37, 38 organic dyes and amino acids 39, 40 proteins 41 as well as biological cells 42 45 viruses 46 and bacteria 47 Folding aptamers into distinct secondary and tertiary structures allows them to bind to their target s with high affinity (dissociation constants o n the order of uM to pM) and to recognize their targets with a specificity that is comparable to antibodies Moreover, as aptamers are structurally and characteristically unique from antibodies, they provide significant advantages including: rapid and repr oducibl e synthesi s, easy and controllable modification to fulfill different diagnostic and therapeutic purpose s long term stability as dry powder or in solution ability to sustain reversible denaturation, non toxicity and lack of immunogenicity and fast tissue

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29 penetration with short blood residence time. These chemical properties make aptamers ideal candidates as molecular probes for use in cancer diagnosis and therapy. To start the SELEX process, first, the target and possibly a negative control are c hosen. Then a DNA library is designed to have a random sequence of 30 40 bases and a region flanked by 18 20 bases primer sequences for polymerase chain reaction amplification (PCR) This leads to 10 13 10 16 different DNA molecules. After everything is pr epared, the typical select in cycle is shown in Figure 1 10 First, the library is incubated with the targets for the desired time and at the desired temperature. Some sequences will bind to the target molecules tightly, but othe rs may bind weakly or not a t all. T he second step is to physically se parate the binders from the undesired oligonucleotides The s uccess of the entire process depends largely on this step because narrowing the pool with high affinity sequences will speed up the enrichment process After the separation, the eluate is amplified by PCR or used for counter selection. In counter selection, the negative controls are incu bated with eluate and unbound pro bes are collected and amplified by PCR. In this way, the nucleic acids with unwanted b i nd ing are successfully removed. The eluted ssDNA is considered the enriched pool for the first round and used as the library for the next round. In gen eral, the concentration of targets DNA, ionic strength, incubation time and temperature can be varied t o provide more stringent condition s to favor the selection of ligands with high affinity. In most cases, after 10 20 rounds of selection cycl es, the pool is highly enriched. Then the enriched pool is cloned and sequenced to identif y sequences Identified s equences are chemically synthesized an d labeled with suitable tags to

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30 evaluate their binding affinities and the K d s of a ptamers towards their target are determined by generating a binding affinity curve. Based on this process, the Tan group has developed a whole cell SELEX strategy to generate panels of new aptamer probes targeting several types of cancer cells including lymphocytic leukemia myeloid leukemia liver cancer small cell lung cancer, and non small cell lung cance r 42 45 Th e utility of cell SELEX lies in selection of cancer cell specific probes without the adv antage of known target molecules on the cell surface These aptamers all showed high affinity and excellent specificity towards their target cells DNAzymes A DNA enzyme, also named a DNA zyme or a deoxyribozyme, is another type of widely investigated functional nucleic acid with the catalytic ability towards various chemical reactio ns, such as RNA or DNA cleavage, ligation, porphyrin metalation, an d DNA self modification. 48, 49 In 1994, the first DNAzyme, 10 23, which catalyzed the cleavage of RNA phosphodiester bonds in presence of Pb 2+ was isolated by an in vitro selection technique. 50 A variety of D NAzymes have emerged since the development of t his techniqu e. Compared to enzymes, DNAzymes are impressively stable even at elevated temperatures, and the production and modification of a DNAzyme is relatively easy. While metal ions are the most important cofactors for enzymes many DNAzymes have also recruited metal ions, especially transition metal ions to increase the reaction diversity and catalytic effic i ency 51 Different transition meta l ions have a board range of properties that can help expand of DNAzyme function. Transition metal ions are better Lewis acids than alkaline earth metals and their metal bound water processes a

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31 sufficiently l ow pK a to catalyze pho sphod iester transfer or cl eavage efficiently. Moreover, transition metal ions have rich spectroscopic feature s that can facilitate the study of reaction mechanisms. Heavy metal contamination oft en poses significant health issues to the general public; therefore, these DNAzymes can work as analytical tools for metal detection. One of the most thoroughly investigated RNA cleaving DNAzyme s is 8 17 DNAzyme first reported in 1997 by Santoro and Joyce 52 Interestingly, because of its small catalytic domain and high variable sequence requirements, the 8 17 motif has been selected from random sequence libr aries at least ten times. 53 The structure of the 8 17 motif i s presented in Figure 1 10 A T he 8 17 DNAzyme binds its subs trate through Watson Crick base pairing upstream and downstream with t he catalytic loop domain and the cleavage site in the middle. nucleotide remains nucleotide engages in nonstandard G(substrate) T(DNAzyme) wobble pairing for optimal activity. In the DNAzyme catalytic core, o nly four residues are absolutely conserved as circled in Figure 1 11 A For the substrate, there are no stringent sequence requirements, except for the identity of two nucleotides at the scissile phosphodiester linkage. The cleavage mechanism as demonstrat ed involves two steps 54 (Figure 1 11 B) : the hydroxyl group at the cleavage site undergoes cyclic phosphate hydroxyl group; t he n cylic p hosphate subsequently hydro lyzes phosphate The catalytic activity of 8 17 can be supported by a wide variety of divalent metal ions w ith the activity order as reported 55 : Pb 2+ > > Zn 2+ >> Mn 2+ Co 2+ > Cd 2+ > Ni 2+ > Mg 2+ Ca 2+ > Sr 2+

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32 Ba 2+ The se divalent metal cofactors act as Lewis acids to assist in the coordination or hydroxyl group. The Pb 2+ with the highest catalytic effect can further catalyze the second step of hy cyclic phosphate This capability is not observed in the presence of other divalent cations, such as Zn 2+ and Mg 2+ The eff icient RNA cleavage activity, small catalytic core, flexible metal ion and substrate requirements make 8 17DNA zyme a useful platform for various applications, such as model system investigation computational devices, molecular biology studies, and analytical tools for toxic heavy metal ions Hydrogels and Their Applications Hydrogels, cross linked networks of hyd rophilic polymers swollen with water, are general ly soft and wet materials. The hydrophilic polymers without crosslinking are called hydrosol (soluble in water) when they are dissolved in aqueous solution. Hydrosols (sol) display liquid behavior that canno t retain a shape, and hydrogels (gel) display solid behavior that can maintain certain geometry. The properties of a hydrogel are generally determined by the type of polymer, the type of crosslink, the degree of crosslink ing, and the water content. Dependi ng on the type of crosslink, hydrogels can The traditional chemical gels have been synthesized by chemical crosslinking methods via covalent bonds, polymerization of monomers or macromers, or the conjugation reactions between functional groups present in the polymer backbone. Due to their mild processing condition s and reversible sol gel conversion the physical gels have drawn much interest to the scientists in recent years. They are spontaneously formed by weak non covalent forces, such as hydrogen bonding, ionic bonding, Van der Waals force s coordinate interaction, hydrophobic interaction, entanglement, and helix formation.

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33 The b iophysical similarity of hydrogels to soft biolo gical tis sues makes them widely applicable in 3D cell culture, tissue engineering, and organ regeneration. The enviro intelligent and stimuli sensitive gel systems that can undergo physicochemical change in response to pH, 56 temperature, 57 ionic strength, 58 electric field, 59 or biological trigger 58 are promising candidates for biosensing, drug delivery, and other biological applications. Recently, DNA or DNA inspired hydrogels 60 66 have aro u se d great interest and they rival oth er peptide or pro tein hydrogels due to the intrinsic advantages of DNA, such as reproducible synthesis, easy manipulation, excellent stability, non toxicity, as well as flexibility and sequence programmability. Especially, the gelling processes are achieve d under physiological conditions, and the mechanical properties and pore size can be easily adjusted by the length, ratio and concentration of DNA. In this section, the field of hydrogels for biosensing and DNA based hydrogels will be briefly reviewed. Hy drogels for Biosensing Since T in 1978 67 hydrogels, especially th e stimuli responsive smart hydrogels have attracted particular attention in the novel and most intensively developing field of polymers Hydrogel properties, such as gel volume or shape, optical properties, mechanical q uality, surface properties, etc., exhibit abrupt response to changes in the environmental conditions or external stimuli. Temperature is one of the stimuli that must be precisely controlled and monitored in various a pplications. Therefore, thermo responsive hydrogels that are sensitive to temperature changes have been developed and widely explored. This thermo sensitivity usually relies on water soluble polymers that show a reversible thermal phase transition

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34 in solub ility, characterized by a critical solution temperature. There are two cases either positive or negative temperature sensitive systems, depending on whether they are contracted below or above a critical temperature, respectively. The pH is another very i mportant stimulus for biological systems, and has been taken into account in designed responsive hydrogels Suitable building macromolecules for this purpose are those polymers w ith weakly acidic or basic pendant groups that are capable of accept ing or rel easing protons in response to the environmental pH and ionic composition. Consequently, the charge density of the polymer is changed as well as the swelling state d ue to electrostatic inter and intra chain interactions. For the gels containing weakly aci dic pend a nt groups, the degree of swelling increases with the environmental pH increase while t he gels containing weakly basic pend a nt groups behave oppositely The conventional hydrogels that rely only on the properties of polymers for an environmental response limit the range of stimuli to which they can respond. Promising approaches involve the incorporation of molecular recognit i o n sites into the hydrogel system for specific analyte sensing. For instance, by immobilizing an enzyme within the network, with the presence of specific chemicals, the enzyme triggers a reaction to change the microenvironment of the hydrogel and lead to gel swelling or collapse. Another approach is based on competitive binding. For example, the hydrogels are cross linked by an tigen/antibody interactions. In the presence of a soluble antigen, either the same one or one which binds more strongly to antibody, the competitive binding can reduce the crosslinking density and break the gel. Hydrogels containing

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35 functional biological m olecules as a part of their structure s such as oligopeptides, oligonucleotides, and proteins, have tremendous potential applications in biosensing As a biosensor, at least two elements should be maintained, bio recognition and signal transduction. Curr e n tly, m ost hydrogel based sensors are transduced by the mechanical work performed by gel swelling and shrinking (e.g. the bending of micromechanical bilayer cantilevers ), 68 or by property changes of gels (e.g. chan ges in optical transmission, 69 refractive index, 70 or resonance frequency ) 71 In another sensing modality, fluorescence tags have been integrated into hydrogels to provide a n easy readout of the change i n the hydrogel network density; 72 or photonic crystals can be encapsulated i nside the hydrogel network, making the Bragg diffraction tunable to g el swelling and shrinking. 73 Most of these transduction methods rely on time consuming manipulation and sophisticated instruments. DNA Inspired Hydrogels A DNA hydrogel is a network of chemically cross linked DNA strands swollen in aqueous solution. Such soft materials have a wide range of biomedical applications, including drug delivery, tissue engineering, selective sorbents, and biosensors. Generally, there are two ways to create DNA hydrogel structures: one is assembled from DNA either natural DNA or synthetic DNA and the other is synthesized of polymers cross linked by DNA. N atural DNA hydrogel s can be made by the solution cross linking of DNA using a chemical cross linker such as ethylene glycol diglycidyl ether (EGDE). 74 76 EGDE contains epoxide groups on both ends that can react with nucleophiles, including the amino groups on the nucleosides. Thus, interstrand cross links form during th e amino EGDE cross linking reactions, which lead to the formation of a 3D DNA network. The

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36 swel ling behavior of hydrogels can be adjusted by different cosolutes, such as metal ions, polyamines, proteins and surfactants. 76 Lee, C. K. et al 77 also reported the formation of DNA hydrogel fiber s by injecting aqueous natural DNA solution into a coagulation bath of room temperature hydrophilic ionic liquids wi thout any crosslinki ng agents. Hydrogels can be made not only of large natural DNA, 1000 2000bp, but also of the small syntheti c DNA, less than 100nt As reported, 62, 78 the synthetic DNA first self assembled into branched X or Y shape d DNA motifs, which were constructed into hydrogels by DNA hybridization with or without further enzyme ligation These types of DNA hydrogels are biocompatible and biodegradable and can be applied to drug delivery, 3D cell culture and tissue engineering. 62 The only drawback may be the large amount of DNA which is needed, although DNA is becoming cheaper with the biotechnique development. H ybrid DNA hydrogels with synthetic polymers as backbone and DNA as cross linker are attract ing increasing attention and interest. The most commonly used cross linking agent in polyacryla methylene bis(acrylamide ), called bis for short. Langrana and coworkers 60 took advantage of DNA complementarity and replaced the traditional cross linking agent bis with a three oligonuleotide system. Two acrydite labeled DNA strands are covalently attached to the polymer chains by copolymerization with acryla mide monomers, and cross linker DNA strand base pairs with both side chains, thus forming a 3D network. Incorporation of DNA cross linking provides a number of advantages: the resulting gel is biocompatible and sequence programmable; the gel sol reversibil ity can be easily realized by introducing a removal strand complementary to the cross linker strand; the mechanical properties and the

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37 pore size of the gel can be adjusted by the length and density of the cross linker DNA; and the melting temperature of th e gel can also be engineered through the sequence and length of the DNA. The mechanical properties of these DNA hydrogels have been systematically studied 60 and applied for neurite outgrowth 79 and fibroblast growth. 80 Functional DNA, such as aptamers, have also been introduced into this system to achieve for instance, the capture and release of proteins, 64 aptamer hydrogel based recognit ion and separation, 81 and visual dete ction with enzyme amplification 66 Carbon Nanomaterials and Their Applications Over the past decade, rapid develo pment in nanoscience and nanotechnology has resulted in the successful synthesis and characterization of various inorganic nanomaterials, including nanoparticles, nanocrystals (quantum dots), nanorods, nanowires and carbon nanotubes (CNTs) The unique phy sical properties of these materials induced by their extremely small size, make them highly suitable for a wide range of biological, electronic, optical, environmental, and medical applications. Single Walled Carbon Nanotubes Specifically, because of the ir rich chemical, electrical, optical and mechanical prop erties, CNTs have emerged as some of the most extensively studied nanomaterials since their discovery by Iijima in 1991. 82 CNTs are mosaics o f carbon atoms that form graphen e sheets and curl into seamless tubules. St r ucturally, CNTs are classified as single walled carbon nanotubes (SWNTs) and multi walled carbon nanotubes (MWNTs) based on the number of graphe n e layers. CNTs have been intensively applied in molecular electronics 83 as field effect transistors 84 and in biomedicine a s biosensors 85 and drug delivery carriers. 86

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38 Optical properties of SWNTs SWNTs, as quasi one dimensional qua ntum wires, have sharp densities of electronic states at the van Hove singularities, resulting in unique optical properties. 87 It has been reported that semic onducting SWNTs can be excited at the second van Hove absorption transition (typically between 500 and 900 nm) and detected through first van Hove emission (t ypically between 800 and 1600 nm). 88 Therefore, the NIR absorption and photoluminescence of SWNTs have been exploited in the development of photother mal therapy 89, 90 and biological imaging. 91 93 In addition, recent research has also found that SWNTs can act coll ectively as quenchers for covalently tethered and/o r stacked pyrenes, porphyrins and chromophores, 94 96 as well as nearby quantum dots, 97 99 through energy transfer or electron transfer processes. SWNTs have several advan tages over conventional organic quenchers, making them particularly suitable as biosensing platform s First, as a new class of universal fluorescence quenche rs, SWNTs possess a broad absorption spectrum, setting them apart from organic quenchers by their i mproved quenching efficiency with low background and high signal to noise ratio. Secon d, SWNTs, as nanocarriers, have ultra high surface area for loading, including multiple molecules along the sidewall for multiplexed sensing. Third, the interactions of S WNTs and biological molecules have been intensively studied in recent years. SWNTs, whether covalently or noncovalently attached to nucleic acids or proteins, have shown the ability to protect these biomolecules from enzymatic digestion or degradation in b iological environment s 100, 101 The specific interaction between DNA and SWNTs At the beginnin g of this century, DNA was initially utilized for dispersing CNTs, after it was discovered that single stranded DNA (ssDN A) could interact noncovalently

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39 with SWNTs to make them water soluble and even sort them based on subtle structures. 102, 103 The ssDNA molecules form stable complexes with individual SWNTs, wrapping around them by m stacking interactions between the nucleotide bases and the SWNT sidewalls. The strength of this binding is demonstrated to be strong enough to disrupt the intertube interactions a nd disperse nanotube aggregates into bundles. 102 104 Atomic force microscopy (AFM) has been employed to determine the structure of DNA SWNT complexes, suggesting a model of helically wrapped ssDNA that is closely arranged along the entire SWNT in a single layer, forming peaks where they are bound and leaving valleys whose height reflects the bare nanotube. 105 This phenomenon revealed that SWNTs have nearly complete DNA coverage with nucleotides aligning parallel to the surface of CNTs and electrically charged phosphate groups pointing outward, thereby minimizing the unfavorable interaction s of the SWNT surface with the aqueous solution and making the regular surface pattern sequence independent. The results agreed with the theoretical predictions of molecular dynamic simulations 106, 107 and were further confirmed by sca nning tunneling microscopy 108 Double stranded DNA (dsDNA) has also been proposed to interact with SWNTs, but its affinity is significantly weaker than that of ssDNA, and is highly dependent on the surface charge of SWNTs. 109 111 dsDNA will weakly absorb onto an uncharged SWNT with either end of a DNA molecule interacting with SWNT, as a result of the partially exposed hydrophobic base pairs at the ends. Even for positively charged SW NTs, dsDNA segments absorb in a roughly parallel configuration, as distinguished from the wrapping mode of ssDNA. Under these conditions, the binding is promoted by electrostatic attraction between the charged SWNT and the charged phosphodiester

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40 backbone o f DNA. 110 In either case, the interaction of SWNTs with dsDNA is much weaker than the interaction between S WNTs and ssDNA. Based on the difference in the propensities of the binding interactions between SWNTs and ssDNA/dsDNA a sensing platform has been generated and applied for biosensing 112, 113 Upon binding to its cDNA or targets, ssDNA on the SWN T surface can undergo a conformation al change to dsDNA, thereby weakening its interaction with SWNT or even resulting in dissociation from SWNTs, and inducing a property change in either the DNA or the SWNTs Graphene Oxi des G raphene, a single atom thick, two dimensional nanostructure of sp 2 hybridized carbon atoms has attracted tremendous attention since the first isolation of single layer graphene in 2004 by Geim and coworkers 114 Graphene oxides (GO s ) can be regarded as graphene covalently decorated with oxygen containing functional groups. GOs have carboxylic groups at their edges and epoxy and hydroxyl groups on the basal planes, so that they contain a mixture of sp 2 and sp 3 hybridized carbon atoms Distinguished from the highly conductive property of graphene with unique electronic structure, GO is an insulator which limits its interest to physicists in the material s area. However, chemists have been attracted by its heterogeneous chemical and ele ctronic structures, and have applied it in a variety of field s like materials, field effect transistors, bio sensors, and biomedical applications. 115, 116 The reactive oxygen func tional groups on GO allow GO to interact with a wide range of materials in covalent, non covalent and/ or ionic manner s for various chemical functionalizations Meanwhile, by carefully controlling the sp 2 /sp 3 carbon fraction on GO, it is possible to tailor the electrical, optical and/or chemical properties of GO.

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41 Due to the heterogeneous atomic and electronic structures, f luoresce nce has been observed from GO over a wide range including ultraviolet, visible, and near infrared (NIR) regions 117, 118 This intrinsic an d tunable fluorescence opens exciting optical applications for GO, especially its visible and NIR fluorescence for biological studies. 117, 119 Although GO is itself fluorescent, it can also quench fluorescence of nearby species, such as dyes, conjuga ted polymers, and quantum dots, through fluorescence resonance energy transfer or non radiative dipole dipole coupling. 116 Such effective quenching could cover a long distance up to 20 nm as experimentally demonstrated 120 and 30 nm as theoretically predicted 121 Similar to SWNT, GO s also interacts differently with ssDNA and dsDNA. ssDNA adsorbs stably on the GO surface due to the stacking interaction between the ring structures of the nucleosides a nd the hexagonal cells of GO. According to a molecular dynamics simulation, 122 the distance between atoms of nucleoside and GO is around 3.5 fairly close to the van de Waals distance, which suggests t he direct adsorption of ssDNA on GO. In contrast, dsDNA could not be adsorbed stably on GO, because the nucleosides of dsDNA are effectively shielded inside the n egatively charged phosphate back bone and the interaction of the phosphate group s of dsDNA with the hydroxyl groups on GO is minimal. As a result, combined with fluorescenc e quenching, GO has been used in optical sensors for sensing ssDNA and biomolecules. 122 124 Photodynamic Therapy Photodynamic therapy (PDT) is gaining wide acceptance as an alternative noninvasive treatment of cancers s uch as head and neck, brain, lung, pancreatic breast, prostate, and ski n cancer s 125, 126 Referring to Figure 1 12 PDT involves a two step process whereby a nontoxic photosensitizer is delivered to an organism and then

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42 activated by an appropriate harmless light source. By absorption of a ligh t photon, the ground state photosensitizer is promote d to the short lived singlet excited state. If the singlet excite d state decays back to the ground state, it results in emission of light, known as fluorescence. Different from normal fluorophores, a pho tosensitizer can convert to the more stable triplet excited state by intersystem crossing, where the promoted electron in a higher orbit undergoes a spin conversion. When it returns to the ground states, the triplet state photosensitizer can initiate photo chemical reaction s directly, generating reactive free radicals, or it can have an electron spin exchange with group state triplet diox yg en ( 3 O 2 ), generating a cytotoxic excited singlet state of oxygen (singlet oxygen 1 O 2 ) Singlet oxygen, the most damaging agg ressive chemical species generated during PDT can react rapidly with cellular molecules and mediate cellular toxicity to cause cell damage, ul timately leading to cell death 127 The efficacy of PDT in cancer treatment depends on the type and concentration of photosensitizer, the light dose and do se rate, and the intracellular localization and oxygen availability. 127 There are three main mechanisms by which PDT mediates tumor destruction: direct tumor cell kill; damage to the tumor associated vasculature, leading to tumor infarction; rapid recruitment and activation of an immune response agai nst tumor cells. These three mechanisms can also influence each other and t he outcome of PDT is dependent on all these mechanisms. 125 Compared to typical curative radiotherapy, chemotherapy and surgery, PDT has several advantages. 127 First PDT treatment only requires a single injection of drug followed by a single illumination which have proven to be cost effective with inc reased life expectancy. Second PDT is a local, rather than systematic treatment; therefore, it is

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43 suitabl e for localized disease. Third the technology development, such as laser and fibe r optic technique s can facilitate the delivery of light with the desired wavelength, dose and rate to the local tumor region. Fourth t reatment of PDT can be repeated in case of recurrence or a new tumor in the previously treated area and it can also be given in combination with other therapeutic methods. However, PDT still has some disadvantages and c hallenges which need to be addressed One is its side effects due to the nonspecific localization In the short term, patients who receive PDT treatment are sensitive to light for around six weeks or more. Other symptoms include blistered, red or swollen s kin, malaise and soreness on swallowing. In the long term, it will accumulate oxidative damage in human tissues and cause aging acceleration, even heart disease and can c er. Another challenge is low therapeutic efficiency if the drug is not located at tum or site, because 1 O 2 can diffuse only approximately 10 20 nm in cells during its very short lifetim e ( 10 6 10 9 s). 128 As a result, a controllable PDT that is localized or activated at the target tumor site would lead to more effic ient and reliable therapy as well as fewer side effects. Two strategies have been applied 129 : the utilization of site specific delivery agents that carry photosensitizers to the target region, such as antibodies, aptamer, and peptide ; or the design of targe t activated PDT agents that are photodyn amically inactive until they encounter the molecular targets.

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44 Figure 1 1 General structure of nucleic acids and nucleiosides Figure 1 2. DNA and RNA structure s via phosphodiester linkage.

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45 Figure 1 3. Structure of DNA double helix and base pairing. Figure 1 4. Phosphoramidite chemistry. A) S t r ucture of phosphoramidite B) F o ur monomers of nucleic acid phosphoramidite

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46 Figure 1 5. Automated oligonucleotide synthesis through phosphoramidite chemistry.

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47 Figure 1 6. 1D DNA self assembly. A) DNA double helix. B) Stick end cohesion. 5 Figure 1 7. 2D DNA nanostructures A) Sticky end assembly of branched DNA Holiday motif. 4 B) DX DNA tile and TX DNA tile 8 C) 4X4 tile r esulting in 2D square lattice. 13 D) DNA origami with 2D shapes. 14

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48 Figure 1 8. 3D DNA nanostructures. A) Cube and truncated octahedron. 25, 26 B) Octahedron. 27 C) Tetrahedron and bipyramid. 28, 29 D) Polyhedra. 30 E) Box with controlled lid. 31

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49 Figure 1 9. DNA nanomachines. A) B Z DNA tra nsistor. 32 B) DNA twe ezer. 33 C) DNA walker. 34

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50 Figure 1 10 Systematic Evolution of Ligands by EXponential e nrichment (SELEX)

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51 Figure 1 1 1 8 17 DNAzyme. A) Th e Secondary structure of 8 17. 52 B) The two step mechanism of substrate cleavage catalyzed by 8 17 DNAzyme and Pb 2+ 54

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52 Figure 1 12 Mechanism of action of photodynamic therapy (PDT).

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53 CHAPTER 2 APTAMER CROSS LINKED HYDROGEL AS A COL OR IMETRIC PLATFORM FOR VISUAL DETECTION Introduction Visual detection is an increasingly attractive method in many fields because both qualitative and semi quantitative assessment can be performed in real time without any advanced or complicated i nstr umentation. I t is especially, useful for rapid diagnostics in disaster situations, home healthcare settings and poorly equipped rural areas where low cost rapid ity and simplicity are essential. A variety of colorimetric reagents, such as visible dyes, 130, 131 polymers, 132, 133 enzymes, 134 136 and gold nanoparticles (AuNPs), 137, 138 can be used for visual detection of specific targets The c olor change s of these reagents is based on diverse yet selective molecular interactions Examples include stimuli induced release or absorbance of dye molecules, target binding in itiated color changing polym ers or molecular recognition trigger ed enzym atic reactions Meanwhile, as described in the previous chapter, hydrogels have attracted particular attention in the development of biosensor devices utilizing a broad spectrum of triggers, including changes in temperature, pH, ionic strength, electric field, or biological systems. However, most of these biosensing devices are transduced on the basis of mechanical work performed by gel swelling and shrinking or property changes o f free swelling gels, such as changes in optical transmission, 69 refractive index, 70 or resonance frequency, 71 most of which must rely on time consuming manipulation and sophisticated instruments. Herein, we propose a color i metric agent caging hydrogel as a novel visual detection platform that rel ie s on DNA base pair recognition and a ptamer target interactions for simple and rapid target detection by the eye

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54 Experimental Section Chemicals and Instrumentation All t he materials to synthes ize acrylic phosphoramidite were purchased from Aldrich Chemical, Inc. The materials for DNA synthesis, including CPG columns and reagents for DNA modification and coupling, were purchased from Glen Research Co. The DNA polymer conjugates were synthesized by a DNA synthesizer (Applied Biosystem s) with acrylic The reagents for hydrogel preparation were purchase d from Fisher Scientific Co. An ABI3400 DNA/RNA synthesizer (Applied Biosystems) was used fo r all the DNA relat ed synthesi s. Purifications were carried out on a ProStar HPLC system equipped with a gradient unit (Varian) and a C 18 column (Econosil, 5U, 250 4.6mm) (Alltech Associates). The concentrations of all DNAs were determined from the absorbance at 260nm usi ng a Cary Bio 300UV spectrometer (Varian). The gold nanoparticle absorbance at 520 nm was also determined by the same spectrometer. The gel pictures were taken by a Fuji F30 digital camera. Synthesis of Acrylic P hosphoramidite The acrylic phosphoramidite used in DNA sequences was synthesized in the lab by two steps (Figure 2 1) F irst, 6 amino 1 hexanol (9.32g, 0.08mol) and TEA (16.16g, 0.16m ol) in 100mL dichloromethane w ere cooled to 0 o C M ethacryloyl chloride (10g, 0.0957mol) was added slowly, and the reaction was stirred at 0 o C for 2 hour s after which 100mL of water was added to quench the reaction. The organic layer was washed with 5% HCl and dried. A fter evaporation of all solvent the crude 6 hydroxyhexyl methacrylamide was used for the next step w ithout further purification. To a solution containing 6 hydroxyhexyl methacrylamide ( 2 g, 10 .8 mmol) in a nhydrous

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55 CH 3 CN ( 4 0 mL) at 0 C, N,N' D iisopropylethylamine (DIPEA) ( 3 9 g, 3 0 .0 mmol) was added in 15 minutes. Then, 2 cyanoethyl diisopropyl chloropho sphoramidite (2 9 m l, 1 3 mmol) was added dropwise, and the reaction mixture was stirred at 0 C for 5 h. After removing the solvent, the residue was dissolved in ethyl acetate, and the organic phase was washed with NaHCO 3 solution and NaCl solution and dri ed over anhydrous magnesium sulfate. The solvent was evaporated, and the residue was purified by column chromatography (ethyl acetate/hexane/triethylamine 40:60: 3) and dried to afford the compound ( 3.33 g, 8.64 mmol, 80 %) as an colorless oil 1 H NMR (CDCl 3 ): 5.92 (br, 1H), 5.63 (m, 1H), 5.27 (m. 1H), 3.86 3.72 (m, 2H), 3.66 3.49 (m, 4H), 3.30 3.23 (m, 2H), 2.61 (t, 2H), 1.92 (m, 3H), 1.58 1.50 (m, 4H) 1.37 1.32 (m, 4H) 1.17 1.13 (m, 12H). 13C NMR (CDCl 3 ): 168.6, 140.4, 119.3, 118.0, 63.8, 63.6, 58.6, 58 .3, 43.2, 43.1, 39.8, 31.3, 29.7, 26.8, 25.8, 24.9, 24.8, 24.7, 19.0. 31P (CDC l 3 ): 14 8 Synthesis of DNA Sequences All the DNA sequences used in this project are listed in Table 2 1. The DNA product was synthesized on the ABI 3400 DNA synthesizer The DN A sequence was uploaded into a DNA synthesizer online. Except for normal DNA bases, 5' acrylic groups were also synthesized on the machine. The synthesis protocol was set up on synthesis, the DNA product was deprotected and cleaved from CPG by incubating with 2.5 mL am monium hydroxide for 17 hours in a 40 o C water bath. The cleaved DNA and 6. 25 mL ethanol, after which the sample was placed into a freezer at 20 o C for ethanol precipitation. Afterwards, the DNA prod uct was spun at 4000 rpm at 4 o C for 3 0

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56 minutes. The supernatant was removed, and the precipitated DNA product was dissolved in 500 L 0.1 M trithylamine acetate (TEAA, Glen Research Corp.) for HPLC purification. The HPLC purification was performed with a cleaned Alltech C 18 column on a Varian Prostar HPLC machine with an acetonitrile/ aqueous TEAA mobile phase The collected DNA produ ct was d ried and detritylated by dissolving and incubating in 200 L 80% acetic acid for 20 minutes. The detritylated ethanol and dried by a vacuum dryer. Acylite labeled DNA needed for the detritylation step and products could be used directly after HPLC. The purified probe was quantified by determining the UV absorption at 260 nm, after which the probe was dissolved in DNA grade water and stored in the freezer at 20 o C for future experiments. Hydrogel F ormation Stock sol ution s of acrylic modified sequences S A and S B were prepared separately with 4% acrylamide in 10 mM Tris HCl buffer (pH 8.0), 200mM NaCl and the solutions were kept in a desiccator connecting with vacuum pump for 5 min to remove the air. To this stock s olution was added 1.4% of freshly prepared initiator ( 0.5 mL H 2 O, 0.05 g ammonium persulfate ) and catalyst ( 0.5 mL H 2 O and 25 L TEMED ) The polymerization reaction also took place in the desiccator for another 20 min to form polymer strand A (PS A) and po lymer strand B (PS B). After that, PS A, PS B and L inker Apt were mixed in stoichiometric ratio and the hydrogel formed immediately S ynthesis and Modification of Colloidal Gold Nanoparticles Citrate stabilized AuNPs (13 1 nm) were prepared using publi shed procedures. 139 Mil lipore water (98 mL) was placed into the two neck flask and 2 mL of 50 mM HAuCl 4 solution were aded to give a final HAuCl 4 concentration of 1 mM. A fter equipping the flask with a condenser and a stopper, the flask was placed on a hot plate

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57 to reflux while st irring. When the solution began to reflux, the stopper was removed, 10 mL of 38.8 mM sodium citrate was quickly added, and the stopper was replaced The color changed from pale yellow to deep red in 1 min. T he system was allowed to reflux for another 20 min, and was then allowed to cool to room temperature with stirring. The diameter of the prepared nanoparticles was ~13 nm at a concentration of 13 nM To avid high salt concentration induced aggregation, the AuNPs were modified with bovine serum albumin (BSA) protein. 1 mL prepared AuNPs were added with 10 mg BSA and the mixture was gently shaken for 10 min. Then, AuNPs were centrifuged d own at 12,000 rpm for 5 min, washed twice with D. I water and resuspended in 100 L water with the concentration aroun d 100 nM Absorbance M easurement Then modified AuNPs were added to the sol system before adding sequence Linker Apt and mixed well with PS A and PS B. After introduction of Linker Apt, homogenous red hydrogel formed with AuNPs trapped inside and distribu ted well. A Cary Bio 300 UV spectrometer (Varian, Walnut Creek, CA) was used to monitor the AuNPs release process. In order to collect absorbance only from the released NPs, the light will be precluded from the gel itself. Th e absorbance was monitored i at 520 nm in the kinetics mode by taking the measurement s every 3 min. Results and Discussion Figure 2 2 i llustra tes the working principle of the visual detection method. Two pieces of DNA, strand A and strand B are grafted onto linear polyacrylamide polyme rs to form polymer strand s A and B (PS A and PS B), respectively. The sequence s of DNA strand s A and B are complementary to an adjacent area of a DNA aptamer sequence. When mixed in equ imolar amount s strand A and strand B grafted polymer are in

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58 transparen t liquid form. The addition of aptamer Linker Apt initiates hybridization of strand A and strand B with the aptamer sequence thus crosslinking the linear polyacrylamide polymers. As the hybridization proceeds, the crosslinking ratio of polyacrylamide incr ease s which results in a increase of viscosity of the polymer solution and eventual transformation into a gel 60, 63, 64 Upon the introduction of a target, the aptamer will bind with it, and the gel will be dissol ved as a result of reducing the crosslinking density by the competitive target aptamer binding. 65 If an enzyme is added prior to the gel formatiom it will be trap p ed inside the 3 D network of the hydrogel (represented as pink symbols in Figure 2 2 ) When target molecules are introduced to dissolve the gel, the e nzyme is released to t ake part in its catalytic role for signal amplification A cascade of events is thus set in motion whereby target binding triggers enzymatic reaction, which, in turn, causes color change for visual detection. Because the aptamer crosslinked hydrogel colori metric platform can be targeted to any ligand for which there is a corresponding aptamer, 35, 36 It will find many visual detection application s in a wide variety of fields There can be no argument that drug misuse i s a major challenge confronting public healt h and law enforcement. In this work, cocaine was used as the model target to test the new visual sensing method. A c ocaine aptamer has previously been obtained by through an in vitro selection process 1 40, 141 and has already been used for the design of several aptasensors. 142 146 The design of cocaine strands A, B and linker Apt have been adopted from the recent report of the Lu group using gold nanoparticles an d aptamer for colorimetric cocaine sensors. 143 The detailed sequences were listed in Table 2 1. The sequences with the same color are complementary.

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59 AuNP s for Principle Demonstration To systematically study the principles of the hydrogel platform and to optimize the s ystem AuNPs were trapped inside the hydrogel. AuNPs were adopted as indicating reagents or signal amplifying agent s based on their unique optical properties and chemical stability. AuNP s with diameter s of only a few nanomet ers can be easily obtained. Beca use this diameter range is similar to that of most enzymes (3 15nm) the behavior of hydrogel trapped enzymes can be extrapolated by studying that of gold nanoparticles. In addition and more importantly, the remarkably large extinction coefficient of AuNP s at the visible region ( around 520nm ) make s them a sensitive indicating reagent for visual detection. Thus, either the trapping or releasing of AuNPs by the aptamer crosslinked hydrogel through molecular recognition can be directly visualized by the chara cteristic red color. In this experiment, 13nm water soluble AuNPs were prepared based on an available protocol 139 and were modified with BSA to avoid aggregation caused by high salt concentration The modified AuNPs w ere added to the s ol system before adding Linker A pt and were mixed thoroughly with PS A and PS B. After introduction of Linker A pt, a homogeneous red colored hydrogel formed with evenly dispersed AuNPs trapped inside. After wash ing three times with buffer solution to remove surface bound AuNPs, t he gel was placed i n a buffer solution and was found to remain in gel form In buffer solution, the gel looked red while the upper buffer solution layer remained colorless. Upon the addition of the target, the gel dissolved and release d AuNPs to the upper layer buffer solut ion. As a result, the buffer solution turned from colorless to intense red, a change which can be easily seen by the naked eye.

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60 The g reatest sensitivity of response in such a sensing scheme relies on optimizing the hydrogel pore size to maximize the diffus ion rate of target molecules into the gel for target recognition and rapid detection, while minimizing th e nonspecific leaking of cargo to avoid false positive results. The pore size of the gel is determined by the crosslinking ratio of DNA Accordingly f our hydrogel s with different DNA crosslinking densities (0.1 0.3, 0.5, 0.7 mM) were prepared and the gel stability as well as the kinetics of target triggered release of AuNPs from hydroge ls was investigated by both the naked eye and UV Vis spectrometry Leaking study The leaking reaction was studied by placing the gel in a buffer soluti on with gentle shaking and monitoring for 96 hours (Figure 2 3) With 0.1mM crosslinker ( too low ) the gel was not stable a nd 40% AuNPs were leaked out after the first tw o hours and kept leaking over time. For the other three types of gels with 0.3 mM, 0.5 mM and 0.7 mM crosslinker concentrations, the leaking was less than 5% in the first two hours, and was 23%, 15% and 10% respectively for 12hr. At the end of 4th day, t he leaking was 72%, 55% and 36% respectively. As expected, the stability of gel is highly dependent on the crosslinki ng density. During the duration time of the analysis, which normally completed within 2hr, the leaking from gel with more than 0.3 mM cros slinker concentration was considered acceptable. As a potential portable sensing device, the stability during the transportation and preservation i s also critical. So the gel stability without buffer solution on top was also examined Once made, sealed and kept at 4 o C, even a fter one week, the gel still had the same reactivity as the fresh made material indicating that without surrounding water the gel is quite stable and preserve s its function well.

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61 Target responsive kinetics study T he gel was prepared with AuNPs encapsulated and placed at the bottom of a quartz microcell with buffer solution on top. The AuNPs released to the buffer solution could be quantitatively monitored by the strong AuNPs absorption at 520 nm at each in terval. The absorption curves for the buffer solution s from the four types of hydrogels during the release of AuNPs are shown in Figure 2 4B The gels were monitored for 30 min before introducing 1mM target cocaine in order to check the encapsulating stability of the hydrogel The 0.1 mM hydrogel showed the most rapid response, but the least encapsulating stability. The 0.3mM and 0.5 mM hydrogels gave a similar response, with the 0.5mM hydrogel having lower background, as well as somewhat slower kinetics. As for the 0.7mM hydrogel, the response was much slower and the reaction did no t reach equilibrium during the monitoring period. The quantitative results indicate d 3.7 times signal to background for the 0.1mM hydrogel, 8.1 times for the 0.3mM hydrogel, 11 times for the 0.5mM hydrogel, and 7.7 times for the 0.7mM hydrogel. I f regarding 3 times higher than background is considered to be a readable signal, less than 10 min were required for each of the four types of gel to reach 3 times signal to background, which indicated the fast detect ion property. Figure 2 4A allows the photographs taken 30 min after introducing 1mM cocaine when the reactions were almost completed The tubes on the left are the control experiments under the same working condition s but without cocaine. Correlating wit h spectrometer data, leaking is a problem for the 0.1mM hydrogel, and the 0.7mM hydrogel has a slower response rate. In contrast the 0.3 mM and 0.5 mM hydrogels gave the best results. This difference among the four hydrogels clearly demonstrated

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62 the conce ntration dependent encapsulating and releasing capability upon target binding. That is, hydrogels with low concentration cross link er tend to d is solve much faster than hydrogels with high concentration cross link er but the formers have the stability probl em s On the other hand, hydrogels with high concentration cross link er may have slower kinetics for the gel sol transition thus prolong ing the detection time. As a consequence, the optimal condition was determined empirically to be the 0.5 mM DNA cross lin ker concentration, which was applied for all further experiment s The AuNP model also suggest ed that nano particle s or molecules with sizes of around 10 nm can be do ped inside the hy d rogel and subsequently release d Competition a ssay for AuNP hydrogel To d emonstrate the specificity of hydro gel response to cocaine, the competition assay was performed in a reverse manner As shown in Figure 2 5 four tubes were prepared. T ube 1 contained PS A, PS B and AuNPs without linker Apt, and n o gel formed. When buffer was introduced on top, AuNPs diffused evenly to generate a homogeneous red solution. In tube 2, PS A, PS B, AuNPs an d Linker Apt were mixed and a red gel formed. After adding buffer, two levels could be distinguished. In tube 3, before mixing with PS A/B, Linker Apt was first equilibrated with 1mM cocaine to form an Apt cocaine complex. Once formed, the complex was quite stable, and the aptamer could no longer crosslink PS A and PS B. As a result, tube 3 exhibited a result similar to that of tube 1 without Linker Apt, proving that the binding of cocaine with aptamer is the dominant reaction when competing w ith DNA hybridization. Tube 4 was a control experiment in which Linker Apt was first mixed with benzoylecgonine (BE), one of the cocaine analogs, and the n added to the PS A/B mixture. Gel still formed without the interference of BE, clearly demonstrating the selectivity of the aptamer.

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63 Enzymatic Reaction for Signal Amplification As a further step, we attempted to introduce enzyme into the gel system. A co mmon test for amylose is the dark blue color produced when amylose complexes with yellow iodine solution. On the other hand, amylase hydrolyzes amylose down into maltose which does not complex with iodine Even though these two phenomena are well known, n obody, to the best of our knowledge, has combined them into a colorimetric sensing platform. Therefore, the amylose I 2 amylase system was chosen because of the specificity of its color change, the fact that no toxic reagents are involved, and the simplicit y and cost effectiveness of its operation. More importantly, both amlyose and amylase are large polymers with high molecular weight. As a result, they can be separated physically by the hydrogel, with amylase trapped inside the gel and amylose outside the gel. Therefore, no amyl o se is digested by amylase unless the enzyme is released as a result of gel dissolution upon target recognition. However, once the target dissolves a certain area of hydrogel and releases enough amylase, even though the entire gel ma y not be completely dissolved, the color change would be sufficiently distinguishable to draw a clinically sound conclusion. Hence, the use of enzyme for signal amplification and colorimetric reaction deliver s a method for visual d etecti on with high sensit ivity. Because the complex formed between amylose and I 2 may affect the enzyme function, I 2 solution was introduced 10 minutes after adding cocaine as the final detection step. Target response in enzyme assay Similar to the trapping procedure for AuNPs th e amylase caged hydrogel was prepared by adding Linker A pt into a well mixed solution containing PS A PS B and amylase The l oading capacity of amylase was found to be as high as 2ug per 10 uL gel.

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64 After in troduction of linker Ap t, a colorless hydrogel fo rmed with evenly dispersed e nzyme trapped inside. N o change of catalytic activity of the enzyme was observed after trapping suggesting that the trapping process is very mild. The enzyme hydrogel response to cocaine was inves tigated in test tube s by the na ked eye, as shown in Figure 2 6 .T ubes 1 and 2, contained no amylase in the ge l Tube 1 contained gel on the bottle and amylose I 2 complex blue solution on top. No color change or gel dissolution was observed. In tube 2, 1 mM cocaine was introduced, and the gel was totally dissolved. Since no enzyme was trapped, only a homogeneous blue solution was observed In tube 3, gel was preloaded with amylase. However, without target, tube 3 behaved in a manner similar to that of tube 1 where amylase and amylose blue solutions were well separated by the gel. Then, different amounts of cocaine were introduced to the top solution of tubes 4 to 7. In tube 4 with cocaine, gel dissolved and solution was colorless. In tubes 5 and 6, a much smaller amount of cocaine was added which was not enough to completely dissolve the gel, and the solution was colorless after introducing I 2 This occurred because the gel partially dissolve d and release d sufficient enzyme to hydrolyze the amylose In this regard even 10 M cocaine (only 100 ng in these experimental condition s) could be detected directly by the n aked eye. T he cocaine concentration was lowered further to 2 M in tube 7. Although the blue did not fade completely, it could still be distinguishable from tube 3. C omparison of tube s 1 to 7 demonstrates how the introduction of enzyme reaction into this system amplifies the signal, enabling the detect ion of lower amount s of target directly by the naked eye, thus improving the overall sensitivity of this visual detection method.

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65 S pecificity test It has been reported that two cocaine metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME) have no affinity to cocaine aptamer 140, 141 and thus should not cause hydrogel dissolution Th erefore, these two metabolites were used as negative control s The results indicated that even challenged with 1mM, neither benzoylecgonine (BE) nor ecgonine methyl ester (EME) caused gel disso lution or color fading (Figure 2 7 ), which confirmed that the g el sol transition and enzymatic reaction w ere indeed triggered by cocaine aptamer recognition. It should be noted that this aptamer sequence has been found to bind with hydrophobic molecules such as steroids 147 and quinine. 148 To use the sensor developed for cocaine detection, one should consider the potential false positive signal caused by these interferences. A n aptamer w ith better selectivity is thus very desirab le. Conclusio n s In summary we have demonstrated the general design for a colorimetric visual detection platform based on an aptamer crosslinked hydrogel. Competitive binding of target to aptamer causes the reduction of crosslinking density thereby inducin g gel dissolution. Two sensing mechanisms have been proposed and examined Both of which employed the intrinsic advantage of hydrogel where large molecules can be trapped inside and separate d from the outside environment. Furthermore during the gel to sol transition upon target recognition the encapsulated molecules could be easily released and still function well. AuNPs are well known for their colorimetric detection property and were employed for system study because of their unique optical properties a nd chemical stability. Then, the simplest enzymatic reaction of amylase with amylose I 2 was employed to fur ther improve the sensitivity. It allows detection of l ess than 2 0 ng of

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66 cocaine by naked eye within 10 min The result is comparable to most sensitiv e aptamer methods 142 146, 149, 150 reported so far, but without any aid of sophisticated instrumentation. As no special features on the aptamers are required, this is a generic approach that can be applied with diff erent aptamer sequences for the detection of other molecules. Since the hydrogel is convenient fo r either micro or nano pattern ing this colorimetric visual detection platform can be further developed into lab on a chip devices for diversified application s, such as forensic analysis, medical diagnostics, and environmental monitoring.

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67 Figure 2 1 Synthesis of acrylic phosphoramidite. Table 2 1 Sequences of oligonucleotides for making hydrogel. Name Sequence Linke r Apt ACT CAT CTG TGA ATC TC G GGA GAC AAG GAT AAA TCC TTC AAT GAA GTG GGT CTC CC in Italic ) S A acrydite AAA A GT CTC CCG AGA T S B acrydite AAA A TC ACA GAT GAG T

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68 F igure 2 2 Working principle of DNA cross linked hydroge l for signal amplification and visual detection. Enzyme is trapped inside hydrogel cross linked by sandwiched hybridization of linker Apt sequence to DNA Strand A and Strand B grafted on linear polyacrylamide chains (PS A and PS B) Physical separation of substrate from enzyme by a 3 D network of hydrogel prevents enzymatic reaction from taking place. However, addition of a target to competitively bind Linker Apt dissolves the hydrogel, which releases the caged enzyme for enzymatic reaction, resulting in a color change.

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69 Figure 2 3. L eaking test of hydrogels with different crosslinking DNA concentrations over a 96 hour observation period The absorbance was monitored at 520 nm for AuNPs.

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70 Figure 2 4 Release of AuNPs from the hydrogel upon introduction of cocaine. A) Photograph of hydrogel before (left) and 30 min after (right) addition of 1 mM cocaine. Four hydrogels with different DNA cross linker concentration (0.1 mM to 0.7 mM) were prepared to study cargo release kinetics. AuNPs were trapped in DN A hydrogel with a cover layer of buffer B) Release kinetics of AuNPs from four types of hydrogels upon introduction of 1 mM cocaine at 30 min The absorbance was normalized by the maximum AuNP signal.

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71 Figure 2 5 Photograph of gels with entrapped Au NPs for competition assay to demonstrate that the aptamer target binding is the dominant reaction. Figure 2 6 Photograph of gel response to different concentration s of cocaine I 2 solution was always introduced 10 minutes after cocaine addition as t he last step to evaluate the results. Ez = amylase.

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72 Figure 2 7 Photograph of c ontrol test s for two cocaine analogs, benzoylecgonine (BE) and ecgonine methyl ester (EME) I 2 solution was always introduced 10 minutes after cocaine addition

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73 CHAPTER 3 DNAZYME CRO SS LINKED HYDROGEL FOR VISUAL DETECTION OF PB (II) Introduction Lead is a heavy metal which plays no known physiological role in human body It is severely toxic at high doses possibly due to its interference with different enzyme systems L e ad can bind to the thiol groups of enzyme s or displace other essential metal ions, thus affect ing a wide range of biological systems 151 Lead exposure can occur through a variety of sources, including air, bare soil home remedies, drinking water, toy jewelry, lead based paints and others. 151 153 Young children are particularly vulnerable to the detrimental effects of environmental pollutants especially lead. Ac cumulation of high level s of lead in children can cause irreversible brain damage, retard mental and physical development and lead to attention deficits and learning dis abilities While f or adults, high levels of lead can cause irritability, poor muscle coordination, k idney problems, and nerve damage to the sense organs 151 153 According to by the United States Environmental Protection Agency (EPA), the level of lead in the blood is considered toxic when it is higher than 0.1 mg/ L In drinking water system s the maximum allowable level of lead is 0.015 mg/L. Therefore as a matter of public health, the development of ultrasensitive assays f or the real time detection of lead is very important for water quality control clinical tox icology, and industrial monitoring Although current technologies such as atom ic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP MS) are ca pable of reach ing the required detection limit s, they often require expensive sop histicated instrumentation and complicate d sample preparation processes.

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74 In the last decade, di fferent sensor concepts for Pb 2+ analysis have been reported, including 8 17 DNAzyme based sensor s As described in Chapter 1, 8 17 DNAzyme, has the RN A nuclease activit y to cleav e the ribonucle otide in the substrate and is highly specific for Pb 2+ as its cofactor. The 8 17 DNAzyme has been coupled with fluorescent signaling mechanisms to produce a Pb (II) specific biosensor with improved sensitivity and selectivit y. 48 However, future devices must link selectivity, speed, and simplicity with cost ef fectiveness. Visual detection may be an optimal choice to meet these requirements Since the DNAzyme is composed of two sequence s: the enzyme sequence with catalytic domain, and the substrate sequence with RNA cleavage site, we grafted the DNAzyme to the h ydrogel system described in C hapter 2 to design a D NA zyme cross linked h ydrogel system for lead ion visual detection Experiment al Section Chemicals and Instrumentation Acrylic phosphoramidite was synthesized by the same protocol described in Chapter 2 and all reagents used for its synthesis were purchased from Aldrich Chemical, Inc. The materials for DNA synthesis, including CPG c olumns, RNA phosphoramidite, and other reagents for DNA modification and coupling, were purchased from Glen Research Co. The DNA polymer conjugates were synthesized by a DNA synthesizer (App lied Biosystems) with acrylic en d. The reagents for hydrogel preparation were purchase d from Fisher Scientific An ABI3400 DNA/RNA synthesizer (Applied Biosystems) was used fo r all the DNA related synthesis The p urifications were carried out on a ProStar HPLC system equipped with a grad ient unit (Varian) and a C 18 column (Econosil, 5U, 250 4.6mm)

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75 (Alltech Associates) using acetonitrile/aqueous trimethylamine acetate as mobile phase The concentrations of all DNAs were determined from the absorbance at 260nm using a Cary Bio 300UV spec trometer (Varian). PAGE gel was performed using a Mini PROTEAN 3 Cell (Bio Rad, CA) with PowerPAC 300 as Gel electrophoresis power supply (Bio Rad, CA) The gel photographs were obtained by a Fuji F30 digital camera. Synthesis of DNAzyme All the DNA sequen ces used in this project are listed in Table 3 1. The DNA product was synthesized on the ABI 3400 DNA synthesizer Except for normal DNA bases, 5' acrylic groups were also synthesized on the instrument according to the specifications of acturers. Following on machine synthesis, the DNA pro duct was deprotected and purified using the method presented in Chapter 2. For the DNAzyme strand that containing a n O TriisopropylsilylOxyMethyl (TOM) group protected adeno sine phosphoramidite (A TOM phosphoramidite) was used to replace regular DNA phosphoramidite. A 15 min coupling time was used to improve the synthesis yield. After the synthesis, the DNA RNA hybrid sequence was cleaved from CPG by incubating with 1 .5 mL 30 % ammonium hydroxide / 40% methylamine (AMA) (1:1) solution for 10 min at 65 o C in a water bath. Starting with this step, all the pipets and tubes had to be sterile and handled carefully to avoid the RNA base digestion by the enzymatic contaminants The cle aved DNA product was transferred into a 15 mL centrifuge tube and mixed with 1 3.75 mL ethanol, after which the sample was placed into a freezer at 20 o C for ethanol pr ecipitation. Afterwards, the oligo prod uct was spun at 4000 rpm at 4 o C for 3 0 minutes. After the supernatant was r emove d, t he second deprotectio n procedure was performed to remove the TOM protecting group on the RNA base. T he precipitated

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76 oligo product was fully dissolved in 115 L DMSO and heated 65 o C for about 5 min to dissolve. 60 L triethylamine (TEA) was added to the DMSO/oligo solution and mix ed gently. Then 75 L triethylamine trihydrofluoride (TEA.3HF) was added and the mixture was heated at 65 o C for 2.5 hrs. Afterwards, the solution was passed through a desalting column with 0.2M tri e thylamine ace tate (TEAA) in RNase free water as eluen t for further HPLC purification The HPLC purification was performed with a cleaned Alltech C 18 column on a Varian Prostar HPLC The collected oligo product was freeze dried and the purified probe was dissolved in RNase free water and quantified by determ ining the UV absorption at 260 nm, after which the probe was split into several tubes to avoid contamination and stored in the freezer at 8 0 o C for future experiments. Hydrogel F ormation Stock solution s of acrylic modified substrate strand ( S DS ) and enzy me strand ( S E ) with certain concentration were prepared separately with 4% acrylamide in 10 mM Tris HCl buffer (pH 8.0), 200mM NaCl and the solutions were kept in a desiccator connected to a vacuum pump for 5 min to remove the air. To these stock soluti on s was added 1.4% of freshly prepared initiator consisting of 0.5 mL H 2 O, 0.05 g ammonium persulfate and catalyst consisting of 0.5 mL H 2 O and 25 L TEMED. The polymerization reaction also took place in the desiccator for another 20 min to form polymer st rand DS (PS DS ) and polymer str and 17E (PS E). After that, PS DS and PS E were mixed in a stoichiometric ratio and they immediately formed the hydrogel. PAGE for Probe Validation Polyacrylamide gel elect rophoresis was performed on a 1 2 % native gel in T B E buffer (89 m M Tris HCl, 89 m M boric acid 2 mM EDTA ) with 200 m M Na Cl and run at 4 o C for 60 min at 8 0 V Afterwards, the gel was stained with Stains All for 1hr in the dark

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77 and then illuminated with light to destain for 20 min. Then the images were obt ained under the visible light by a digital camera. Results and Discussion Figure 3 1 i llustra tes the working principle of the visual detection method. Two pieces of DNA, strand DS (S DS) containing the cleavage site and strand E (S E) containing the DNAzy me are grafted onto linear polyacrylamide polymers to form polymer strand DS and E (PS DS and PS E) respectively. The sequence s of DNA strand S DS and S E are complementary to each other with a catalytic loop left in S E When mixed in equal amount s the S DS and S E grafted polymer s i mmediately form a hydrogel Upon the introduction of the target Pb 2+ the already formed catalytic loop will bind with the Pb 2+ and the enzyme reaction will take place so that DNAzyme strand will cleave the rA nucleotide in the substrate strand. Due to the short sequence and low affinity after cleavage, the substrate and the DNAzyme will dissociate and t he gel will dissolve as a result o f the reduced crosslinking density Once the substrate and DNAzyme dissociate, the cataly tic loop can no longer hold Pb 2+ which can bind with another preformed catalytic loop and facilitate the cleavage of another substrate. If AuNPs are added prior to the addition of the aptamer, they will be trap p ed inside the 3 D network of the hydrogel. W hen target Pb 2+ is introduced to dissolve the gel, the AuNPs will be released and change the top solution from colorless to red Because our DNAzyme crosslinked hydrogel colorimetric platform can be applied to other DNAzymes we anticipate that it will fin d many visual detection application s for metal ions

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78 Sequence Design Strategy Although there have been many reports about 8 17 DNAzyme with optimized sequences, most of them were carried out in solution with low probe concentration. The situation in such h ydrogel system with relativel y high probe concentration may present a different situation. Thus, we first investigated sequence design to determine optimal pair of sequences. The design strategy is presented in Figure 3 2A. Before the introduction of targ ets, the hydrogel need s to be stable enough to hold the encapsulated AuNPs. However, in order to guarantee rapid and sensitive detection, the hydrogel has to respond to added target and dissolve quick ly. The former case requires that the S DS and S E have strong hybridization. For the latter case, to facilitate the gel dissolving, the cleavage product needs to have few er bases in order to leave the enzyme strand quickly. Substrate and DNAzyme bind through two portions of duplex, arm A and arm B shown in Fig ure 3 2B. After the cleavage, only a rm B determines the dissociation of S DS and S E thus the dissolving of hydrogel. The few er bases arm B has, the more rapid the dissociation will be. Without compromising the total binding stability of S DS and S E befo re cle avage, arm A should have extra base pairs compared to the original design. Based on this strategy, arm A was designed with 10 or 14 bp and ar m B with 5, 7, or 9 bp. Because i t is tedio us work to synthesize S DS with a RNA base in the middle the S D S was kept mostly unchanged, and S E was varied with different combination s of arm A length and arm B length. The sequences of S DS and S E are listed in Table 3 1. The sequences in orange and blue colors indicate the arm A and arm B respectively.

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79 The P erformance of DS 1 Related Probes To maximize probe performance, the design of the probes was optimized. The DS 1 with 10nt in arm A and 9nt in arm B was synthesized, as well as its corresponding enzyme strands E10 5, E10 7 and E10 9. The performance of th ese probes was examined to understand the system. PAGE for s ystem s tudy To validate that the synthesized probes have good response to Pb 2+ some tests were performed before making t hem into hydrogel Although fluorescence might be a more sensitive test, t here was no fluorescence labeling on these probes. Therefore, p olyacrylamide gel elect rophoresis (PAGE) is a simple and convenient option. The S DS and S E were mixed in stoichiometric ratio in buffer solution (10 mM Tris HCl, 200mM NaCl). Then different c oncentration s of Pb 2+ were added. The mixtures were incubated at room temperature for h alf an hour before running the PAGE gel. In Figure 3 3 three sets of probes were examined. Lane s 1 5 have DS 1/E10 5 Lane 1 is E10 5 alone as a control. In lane 2, DS 1/E10 5 formed a duplex an d showed a slower band than the one in lane 1. From lane 3 to lane 5, different concentrations of Pb 2+ were introduced. As seen in the figure, DS 1/E10 5 had almost completed cleavage with lowest Pb 2+ concentration (5 M) we test ed. The DS 1/E10 5 duplex band disappeared and two cleavage products can be seen: the duplex of half DS 1 with E10 5 showed a clear band and the cleaved strand with 1 4 nt had a s meared band on the bottom. Lane s 6 10 have DS 1/E10 7 with different concentr ations of Pb 2+ In this case, there was no significant cleavage with 5 M Pb 2+ Even with 50 M Pb 2+ the DS 1/E10 7 duplex band was still observable with around 70% reaction completed Lane s 11 15 have DS 1/E10 9. More than 50% of cleavage reaction was c ompleted with 5 M Pb 2+

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80 Lane 16 contains the control of DS 1 and lane 17 contains a DNA strand with 12 nt as ladder to indicate the position of the 14 nt cleavage product. From the gel results, with the same substrate DS 1, the reactivity is in the order : E10 5 E10 9 > E10 7. From the mechanism, two factors may i nfluence the reactivity. One is the stability of preformed catalytic domain for Pb 2+ to bind, and the other is the readiness of the cleaved product to release Pb 2+ for further catalytic function Althou gh the duplex formed between DS 1 and E10 5 is the least stable, the cleaved product has the fewest base pair s and can leave most rapidly so that Pb 2+ can be easily release d Strand E10 9 has the most stable duplex with DS 1 and the catalytic domain is wel l formed for the Pb 2+ to bind. However after the cleavage, the leaving strand does not release Pb 2+ as easily as that of E10 5 and enzyme activity is compromised. Both of E10 5 and E10 9 showed better performance than E10 7. Leakage test Stability is an i mportant criterion for a hydrogel detection system. The nonspecific leakage of encapsulated cargo would induce false positive results. Although E10 5 showed the best response toward Pb 2+ we still need ed to investigate whether the 5 nt in arm B bridged str ong ly enough to maintain the gel Three tubes were prepared: DS 1/E10 9, DS 1/10 7, and DS 1/10 5. AuNPs functionalized with BSA were introduced before gel formation and trapped inside the gel. After washing three times buffer was placed on top of the re d gel a nd the tubes were gently shaken for 30 minutes at ro om temperature. The results are shown in Figure 3 4A. After 30 minutes shaking, the gel made of DS 1/E10 9 was still stable enough to keep AuNPs inside the gel and the clear solution on top. The ge l mad e of DS 1/E10 7 had some leakage of AuNPs to the top solution. However the gel made of DS 1/E10 5

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81 showed significant leakage and was almost dissolved. These result s illustrate that although DS 1/E10 5 had best Pb 2+ response among the three sets of pr ob es, five base pairs are too few to hold the hydrogel. This result also agrees with the assumption that gel stability is directly related to the length of arm B. Hydrogel response to Pb 2+ Since DS 1/E10 5 had severe leaking problem s, only the gels made f rom DS 1/E10 9 and DS 1/E10 7 were tested with Pb 2+ From each sequence pair, two tubes were prepared 100uM Pb 2+ was introduced into one tube and the other was without Pb 2+ as negative control. All four tubes were gently shaken for half an hour before tak ing the picture. As presented in Figure 3 4B, with Pb 2+ the ge l made of DS 1/E10 9 was almost dissolved, and without Pb 2+ the gel was still stable enough to keep the AuNPs inside. For the gel made of DS 1/E10 7, with Pb 2+ the gel was completely dissolved to get a red homogeneous solution. However, without Pb 2+ some AuNPs leaked out. It appeared that DS 1/E10 9 wa s the best among this three pairs of probes. However, with 100 M Pb 2+ and 30 minutes rea ction time, the gel still did not completely dissolve At a lower Pb 2+ concentration, the response might be even worse and the requirement of rapid response will not be satisfied This is because the leaving strand of E10 9 has 9 nt, making it too stable to leave the enzyme strand quickly after cleavage. However as the experimental data demonstrated, simply shorten ing arm B of the enzyme strand leads to an un s table gel. In order to have rapid response without comprising the gel stability, another DNAzyme /substrate pair was designed with longer arm A and shorter arm B.

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82 The Performance of DS 2/E14 7 Probes In order to have rapid gel dissolution in the presence of Pb 2+ we still believe 7 nt in leaving strand should have better response than that of 9 nt. However, to keep the hydrogel stable with shorter arm B, we elongated the arm A from 10 bp to 14 bp to offset the loss in stability from arm B As a result, the new set of probes DS 2/E14 7 (Table 3 1) was synthesized and tested. PAGE for validation After the synthesis and purification, the PAGE experiment was per formed to validate the probe response to Pb 2+ and the results are shown in Figure 3 5. Lane 1 and lane 8 showed the strand E14 7 and DS 2, respectively, as control s. Lane 2 contained DS 2/E14 7 without Pb 2+ an d only one duplex band was observed In lane s 3 7, different concentration s of Pb 2+ were introduced, from 50 nM to 200 M. The cleavage band appeared from 5 M Pb 2+ which achieved similar response as the first sets of probes in solution. This experiment demonstrated that the new probe DS 2/E14 7 also has good response to Pb 2+ and can be used for further hydrogel application s Hydrogel response to Pb 2+ Using this new set of probes, the hydrogel response to Pb 2+ was investigated by visually observing the reaction in a series of tubes (Figure 3 6). In tu be 1, there was o nly the DS 2/E14 7 with AuNPs trapped inside. Without Pb 2+ during the 30 minute reaction time there was no leakage observed. The red gel was sitting on the bottom with clear solution on top. From tube s 2 to 5, different concentration s of Pb 2+ were introduced into the upper solution. In tube 2, only 2 M Pb 2+ was added, which was not enough to completely dissolve the gel, and the solution was slight red but still distinguishable from tube 1. With increasing concentration s of Pb 2+ in tube 3 to 5, more

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83 gel dissolved and the red color of the top solution became more intense. In tube 4 with 200 M Pb 2+ the gel was almost completely dissolved. In this regard, even 2 M Pb 2+ could be detected directly with the naked eye, without any signal ampli fication Conclusion s In conclusion, we have extended our hydrogel visual detection work and demonstrated another general design for metal ion detection based on DNAzyme crosslinked hydrogel s The target specific enzymatic cleavage shortens the hybridizati on length and causes the reduction of crosslinking density thereby ind ucing gel dissolution. AuNPs which are well known for their unique optical properties and chemical stability, were employed to trap inside hydrogel We were able to use this simple sys tem to detect 2 M Pb 2+ by n aked eye w ithout the aid of sophisticated instrumentation. If we further apply the enzyme amplifica tion used in Chapter 2, we may be able to lower the detection limit down to the nM range with the naked eye, which is our future goal As no special feature s of DNAzymes are required, this may be a generic approach that can be applied with different DNAzyme sequences for the de tection of metal ions.

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84 Table 3 1 Sequences of DNAzymes and substrates Name Sequence DS 1 acr ydite AAAA ACT CAC TAT rA GGA AGA GAT G E 10 5 acrydite AAAA C ATC TCT TCT CCG AGC CGG TCG AA A TA G T E 10 7 acrydite AAAA C ATC TCT TCT CCG AGC CGG TCG AA ATA GTG A E 10 9 acrydite AAAA C ATC TCT TCT CCG AGC CGG TCG AA ATA GTG AGT DS 2 acrydite AAAA ACT CAC TAT rA GGA AGA GAT GTC AC E 14 7 acrydite AAAA GT GAC ATC TCT TCT CCG AGC CGG TCG AA ATA GTG A Figure 3 1. Working Princip le of DNAzyme cross linked hydrogel for Pb 2+ visual detection. AuNPs are trapped inside hydr ogel cross linked by hydridization of substrate and enzyme strand s grafted on to linear polyacrylamide chains (PS DS and PS E). Addition of target Pb 2+ catalyzes the cleavage reaction and dissolves the hydrogel, which releases the caged AuNPs for visual det ection.

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85 F igure 3 2. S eq uence design strategy. A) S chema tic illustration of the reaction. B) The actual sequences of S DS and S E

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86 Figure 3 3. Polyacrylamide g el electrophoresis (PAGE) results of DS 1 related probes when treated with diffe rent concentration s of Pb 2+

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87 Figure 3 4. Photograph of DS 1 h ydrogel with entrapped AuNPs. A) The AuNP leaking study of hydrogels made of DS 1 with E10 9, E10 7 and E10 5. B) The hydrogels with and without the addition of 100 M Pb 2+ for 30 minutes

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88 Figure 3 5. Polyacrylamide g el electrophoresis (PAGE) results of DS 2/E14 7 treated with different concentrations of Pb 2+ Figure 3 6. Photograph of DS 2/E14 7 gel response to different concentrations of Pb 2+

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89 CHAPTER 4 REGULATION OF SINGLE T OXYGEN GENERATION US ING SINGLE WALLED CARBON NANOTUBES Introduction Singlet oxygen ( 1 O 2 ) is one of the most important cytotoxic agents generated during photodynamic therapy (PDT), which is gaining wide acceptance as an alternative noninvasive treatment of cancers 125, 126 As described in Chapter 1, PDT involves a two step process whereby a nontoxic photosensitizer is delivered to an organism and then activated by an appropriate harmless light source. The photosensit izer, generally a chemical, transfers the light energy to tissue oxygen to generate highly reactive 1 O 2 an aggressive chemical species which can react rapidly with cellular molecules and mediate cellular toxicity to cause cell damage, ultimately leading to cell death. 127, 154 Because the lifetime and diffusion distance of 1 O 2 is very limited, a controllable singlet oxygen generation (SOG) with high selectivity and localization capability would lead to more efficie nt and reliable PDT as well as fewer side effect s This is where careful molecular engineering can play a major role in designing PDT Several research groups have now taken this approach to develop selective PDT agent s that can be triggered by protease d igestion, 154, 155 pH change 156 or DNA hybridization. 157 For instance, Zhang and coworkers 154 have reported a photodynamic molecular beacon in which a photosensitizer and a 1 O 2 quencher were ke pt in close proximity by a disease specific peptide sequence. Upon enzyme cleavage of the peptide the photosensitizer was freed from the quencher leading to an increase in the amount of SOG. Cl et al. 157 linked a photosensitizer on 15 mer DNA sequence (P DNA) and a quencher on its complementary strand (Q DNA) to quench the SOG. Upon the introduction of third

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90 strand that can displace and release P DN A from the P Q pair, the SOG was restored to 85% of original efficiency Comparing with protease digestion and DNA hyb ridization strategies w e believe that ap ta mer s can be more effectively used to control 1 O 2 generation upon target binding. The targets o f aptamers range from small molecules to proteins, and even to disease cells 158 Meanwhile aptamers intrinsically are DNA that ha ve a large variety of adaptability for molecular engineering, making the design of c ontrollable PDT feasible. In this work, we propose a new molecular design for regulating SOG by single wall ed carbon nanotube s (SWNT s ) SWNTs have already proven to be efficient quenchers in fluorescence probe design 94 99 Based on these findings and the fact that both the fluorescence process and SOG share a similar photophysical mechanism, we anticipated that SWNT s could quench SOG, thus replacing organic molecular quencher s Meanwhile, the interaction s of SWNT s wi th biomolecules, such as proteins and DNA 89, 100, 102, 112, 159 161 have been intensively studied and applied to biosensing 100, 112, 159 and as intracellular transport ers 160, 161 Especially, ssDNA can interact noncovalently with SWNT s such that ssDNA will wrap onto the SWNT surface by stacking interaction s to protect DNA probes from digestion by nuclease As a result, ssDNA aptamer labeled with photosensitizer has been adopted to react with SWNTs for PDT regulation as well as probe protection and potential delivery. Experimental Section Chemicals and Instrumentation The materials for DNA synthesis, including CPG columns and reagents for DNA modification and coupling, were purchased from Glen Research Co. Chlorine e6 (Ce6) was purchased from Frontier Scientific, Inc. Dicyclohexylcarbodiimide (DCC ) and

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91 N Hydroxysuccinimide (NHS ) used for Ce6 coupling were purchased from Si gma Aldrich Inc. The singlet oxygen sensor green (S OSG ) used for detecting 1 O 2 was purchased from Invitrogen Inc. An ABI3400 DNA/RNA synthesizer (Applied Biosystems) was used fo r all the DNA related synthesi s. Purifications were carried out on a ProStar H PLC system equipped with a gradient unit (Varian) and a C 18 column (Econosil, 5U, 250 4.6mm) (Alltech Associates) with acetonitrile/aqueous triethylamine acetate as the mobile phase The concentrations of all DNAs were determined from the absorbance at 260nm using a Cary Bio 300UV spectrometer (Varian). All fluorescence measurements were performed using a Fluorolog (Jobin Yvon Horiba) or microp late reader (TECAN). A 100 cro cuvette was used for the experiments on Fluorolog, and the sampling volume w 96 well plate (Nunc) contained the samples in the microplate reader. The microplate reader was also utilize d for measuring the absorbance in the MTS assay. PAGE gel was usin g a Mini PROTEAN 3 Cell (Bio Rad ) with PowerPAC 300 as the electroph oresis power supply (Bio Rad ) The image was recorded by a Typhoon 9410 variable mode imager (GE Healthcare) Synthesis of Aptamer Photosensitizer (AP) Probe synthesis involved two major st eps: the on machine DNA synthesis and the off machine coupling of Ce6. The DNA product was synthesized on the ABI 3400 DNA synthesizer using the procedure s described in Chapter 2

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92 After synthesis, purification and detritylation, t h e DNA product was then ready for off machine coupling of Ce6. As shown in Figure 4 1, e ach Ce6 molecule has three carboxyl groups, which m ay DNA product. To improve the coupling efficiency and reduce the multiple coupling products, the Ce6 was added in 10 fold excess in the coupling reaction. The Ce6 ( 10 ) was mixed with an equimolar amount of DCC and NHS N,N Dimethylformamide (DMF) for the activation reaction with 1 hour stirring. The purified DNA product (1 ) 3 solution and m ixed with the activated Ce6 for coupling. The coupling reaction was performed with strong stirring for at least 8 hours before ethanol precipitation. To remove the uncoupled reagents, the product was precipitated three times with the addition of 100 M TEAA buffer for HPLC purification. To eliminate the residual free unconjugated chemicals, the reaction product was HPLC purified twice The purified probe was quantified by d etermining the UV absorption at 260 nm, after which the probe was dissolved in DNA grade water and stored in the freezer at 20 o C for future experiments. Preparation of Single Walled Carbon Nanotube Single walled carbon nanotubes (SWNT s ) made through the Hipco process were treated by sonication and acid reflux as described previously to afford short, (~2 00 nm) water soluble nanotubes with acidic surface groups. 101 Fluorescence E xperiment The fluorescence emission of aptamer photosensitizer (AP) was scanned from 600 nm to 800 nm with the excitation at 404 nm, which is th e maximum absorption of

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93 Ce6. To evaluate the SOG of probe samples, the singlet oxygen sensor green (S OSG ), which is high ly selective for 1 O 2 was introduced at the concentration of 2 .0 1 O 2 was generated by irradiation at 404 nm, the maximum absorpt ion of Ce6, for 10.5 minutes. The SOSG fluorescence was read out with the excitation at 494 nm with SOG was evaluated by the SOSG fluorescence enhancement compared with the b ackground and control sample s AP SWNT Response t owards T hrombin Probes were prepared as a 200 nM solution in 10mM Tris HCl buffer containing 5 mM MgCl 2 and then mixed with the oxidized nanotubes for 1 2 h. To extend the lifetime of singlet oxygen and in crease the sensitivity of SOG assay, all buffers and samples were prepared using deuterium oxide. The thrombin concentration ranged in samples temperature, the mixture was centrifuged for 5 min at 14 000 rpm to remove most of the SWNT s which might ha ve some effect on SOG detection by SOSG. Gel E lectrophoresis for Demonstration Polyacrylamide gel elect rophoresis was performed on a 10 % native gel in TB E buffer (89 m M Tri s HCl, 89 m M boric acid 2 mM EDTA ) with 10 m M K Cl and run for 50 min at 100 V The fluorescence images of gels were re corded by Ce6 fluorescence with the excitation at 457 nm and the emission at 670 nm on a Typhoon 9410 variable mode imager. Throm bin and control protein IgG are not fluorescent themselves. The only fluorescence signal comes from Ce6 on the 3 end of DNA. After that, g els were stained using the protein stain Coomassie Blue (Bio R a d) to image the position of proteins by

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94 im merging the gels in t o Coomassie Blue solution for 1 hr and washing with D.I. water for 0.5 hr. Then the images were obtained under the visible light with a digital camera. The P hototoxicity of AP SWNT toward L iving C ells The Ramos cells (ATCC) were cultured in the RPMI 1640 cell culture media (ATCC) containing 10% fetal bovine serum (FBS) (heat inactivated, GIBCO) and 100 IU/mL penicillin streptomycin (Cellgro). The cells were harvested and spun at 950 rpm for 3 minutes to remove the supernatant before washing with PBS wash b uffer PBS containing 4.5 g/L glucose and 5 mM MgCl 2 ). Then, the cells were resuspended in RPMI 1640 supplemented with 100 IU/mL penicillin streptomycin and 2.5 mM MgCl 2 and counted using a hemac ytometer (Hausser Scientific) with an Olympus IX70 microscop e. The cells were diluted to 1. 5 million/mL and split into a 96 well cell culture cluster at 20,000/well, 100 The irradiation experiment was performed at room temperature during 3 hours of irradiation under a white fluorescent lamp. After irr adiation, a ll cell samples were incubated in darkness for 40 hours before the cell viability test. A sample of CellTiter 96 AQ ueous One Solution Reagent [ The Cell Proliferation Assay (Promega) ] wer e added to each well. T he plate s were incubated for 2 hours at 37 o C in a humidified, 5% CO 2 atmosphere. T he absorbance at 490 nm was recorded using a 96 well plate reader. DP SWNT Response towards cDNA The DNA photosensitizer (DP) p robes were prepared as a 200 nM solution in 10mM Tris HCl buffer containing 5 mM MgCl 2 and then mixed with the oxidized nanotubes for 1 2 h. To extend the lifetime of singlet oxygen and increase the sensitivity of the SOG assay, all buffers and samples were prepared using deuterium

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95 oxide. After allowing this complex to hybridize a nd bind with cDNA or rDNA for ~3 h at room temperature, the mixture was centrifuged for 5 min at 14 000 rpm to remove most of the SWNT which may have some effect s on SOG detection by SOSG. Results and Discussion Figure 4 2 shows how the significant feature s of SWNT, aptamer, and a photosensitizer were combined to form a simple, but efficient and elegant PDT agent named AP SWNT. T he aptamer was developed previously by an in vitro process known as S ELEX A photosensitizer is covalently attached to one end of the DNA aptamer which wraps onto the SWNT surface. In the absence of a target, the close proximity of the photosensitizer to the SWNT surface causes efficient quenching of SOG. Importantly, the conformation of the probe can be altered upon target binding Thus, in the presence of its target, the binding between the aptamer and target molecule will disturb the DNA interaction with SWNTs and cause the DNA to fall off the SWNT surface, resulting in a restoration of SOG for PDT applications SOG can thus be r egulated by target binding. T hrombin aptamer s have been widely investigated for the past decade. Alpha thrombin is a trypsin like serine protease that has many effects in the coagulation cascade and relates to a multitude of diseases. For these reasons, hu man thrombin (Tmb) aptamer was chosen as the m odel probe to investigate the photodynamic process that takes place under the conditions suggested above. Chlorin e6 (Ce6), a second generation and easily modifiable photosensitizer was selected because of its h igh photosensitizing efficacy and low dark toxicity. Since it is highly selective for 1 O 2 singlet oxygen sensor green (SOSG) was employed to quantify SOG by fluorescence enhancement.

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96 Fluorescence and SOG Q uenching by SWNTs The Ce6 was covalently linked to DNA aptamer ; therefore, the stronger binding affinity of DNA with SWNTs should enable high quenching efficiency. As shown in Figure 4 3 A, more than 98% quenching of Ce6 fluorescence was observed for AP. Further, we tested the quenching efficiency for SOG by mixing all the samples with 2.0 minutes. The SOG was evaluated in terms of signal to background ratio (S/B) by comparing the SOSG fluorescence enhancement after subtract ing the buffer background. Compared with buffer solution, the SOG was significantly quenched by SWNTs (Figure 4 3 B), clearly demonstrating the extremely strong quenching of SWNTs to fluorescence and SOG. Fluorescence E nhancement and SOG upon Target Binding The basis for target recognition by aptamer s is the tertiary structure of single stranded oligonucleotides, which is presumed to undergo a conformation al alteration of t he probe upon target binding. This may disturb the DNA interaction with SWNTs and cau se the DNA detach from the SWNT surface, resulting in the restoration of fluorescence and SOG. The results in Figure 4 4A confirmed our assumption that the target binding indeed can cause the conformation al change and induce fluorescence restoration. The C e6 fluorescence increased up to 20 Thrombin As shown in Figure 4 4B the SOSG fluorescence of AP SWNT did not change much compared to buffer solution. However, the SOSG fluorescence of AP SWNT exhibited a 13 fold enhancem demonstrates that SWNTs can efficiently turn off SO G and, more importantly, that SOG

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97 c an be reversibly mediated by the target binding event i.e. the binding between the aptam er and thrombin Quantitative Resp onse to Target The SOG of AP SWNTs with a series of thrombin concentration s was also investigated. As shown in Figure 4 5 an almost linear increase of SOSG fluorescence intensity was observed over the thrombin concentration range of 0.1 1.6 th e fluorescence intensity reached a plateau, even when the concentration of thrombin SWNTs, no more fluorescence increase was observed. During the effective concentration range, the SOG of AP SWNTs can be quantitatively mediated by thrombin concentration, indicating the potential of this probe as a quantitative SOG controlling PDT agent. Gel Electrophoresis for Proving the Different States of AP SWNT To investigate the process further we performed p ol yacrylamide gel electrophoresis to observe the different States of the AP SWNT complex. Ce6 Fluorescence was used to indicate the DNA bands and Coomassie Blue was u se d for protein band identification. Six samples were prepared as follows: lane 1, AP only; lane 2, AP with thrombin protein; lane 3, AP with IgG protein; lanes 4 6 supernate of samples after centrifugation to remove SWNTs. Lane 4 was from AP SWNT; lane 5 was from AP SWNT incubated with thrombin protein; lane 6 was from AP SWNT incubated with I gG protein. The amount of DNA was around 20 pmol, and t he amount of proteins was ten times higher As shown in Figure 4 6 A, AP (lane 1) binding to the thrombin resulted in a strong band shift (lane 2), while there was no binding to Ig G (lane 3). When incub ated with SWNTs, AP attached to the SWNT surface by

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98 remove the SWNTs, AP stayed with SWNT precipitate, and there was no obvi ous band in lane 4. When thrombin was added to the AP SWNT complex, thrombin bound with AP and release d it from SWNT surface. After centrifugation, we observed a similar result in lane 5 to that of lane 2, proving that thrombin indeed bind s with AP to produce the dissociation product as AP Tmb. When thrombin was replaced with control protein IgG no band appeared in lane 6, which implied that most AP was kept on the SWNT surface in the precipitate, because without specific binding IgG cannot release AP from SWNTs. Figure 4 6 B is the photo taken af ter the gel was stained by Coom a ssie blue f or 1 hr. The blue band indicate s the position of proteins. IgG stay ed close to the wells during the electrophoresis, while some thrombin moved out of the wells because the binding with DNA g ave them some negative charges. The position s of thrombin in lane s 2 an d 5 were overlapped very well with DNA in Figure 3 6 A, confirm ing the formation of the AP Tmb complex. S pecificity of AP SWNTs again Proteins As noted above, aptamers have high binding affinity and specificity, and our results showed that AP SWNT maintain ed this advantage and presented excellent specific response towards thrombin. As shown in Figure 4 7 when tested with bovine serum albumin (BSA), protein A, protein L, NeutrAvidin and IgG at the concentration of SWNT gave only a small SOSG fluorescence response to these proteins compared with that of AP SWNT only. However, significant SOSG demonstrated that the production of 1 O 2 by our AP SWNT could only be triggere d by the specific targe t protein, without the interference of other proteins

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99 P hototoxicity of AP SWNT toward Living Cells We use d Ramos cells (CRL 1596, B lymphocyte, human to test the phototoxicity of our AP SWNT. The cells were dilut ed to 1.5 million/ml and split into a 96 well cell culture cluster at 20,000/well, 100 Six groups of cell samples were set up as follows: group 1, cells only; group 2, cells plus 3 Tmb; group 3, cells plus 3 Tmb and 2 ; group 4, cells incubated with 0.3 ; group 5, cells incubated with 0.3 AP SWNT; and group 6, cells p uls both Tmb and AP SWNT. Each group had triple parallel samples. The irradiation experiment was performed at room temperature during 3 hours of irradiation under a white fluorescent lamp. After irradiation, all cell samples were incubated in darkness for 40 hours before the cell viability test. The MTS assay was then performed as a cell viability test I n Figure 4 8 the cell viability decreased to 30% when cells were incubated w ith AP SWNT and Tmb (group 6). This percentage is comparable to that of cells incubated with AP only (group 4) Meanwhile, about 70% cell viability was observed in group 5 containing only AP SWNT. Compared with the cells alone cells with Tmb, or cells with both Tmb and SWNT, w hich resulted in about 100%, 95 % and 97% cell viability in sample 1, 2 and 3 respectively, the AP SWNT presented limited phototoxicity in group 5 but considerably enhanced toxicity with the addition of Tmb in group 6 These results clearly demonstrated that the phototoxicity of AP SWNT toward living cells can be mediated by T hrombin protein F l uorescence R esponse and SOG of DP SWNT towards cDNA Based on the Watson Crick base pairing principle, if the aptamers are changed to some specific DNA or RNA sequences, the selective PDT can also be realized in another w ay. S ome preliminary experiments were performed to demonstrate this

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100 possibility Following the same procedure, we synthesized another DNA sequence with 35 bases and labeled with Ce6, named as DP SWNT. T he response of DP SWNT towards its complementary DNA ( cDNA) was investigated. As shown in Figure 3 9, t he Ce6 fluorescence increased greatly cDNA ; however, there was little response to 2.0 random DNA ( rDNA ) The case was the same for SOG regulation. This demonstrates that a ssD NA to dsDNA conformation change can differentiate the inte raction with SWNTs, thus mediating the SOG The cDNA concentration was varied has also been examined (Figure 3 10), and a similar concentration dependent phenomena was observed. Conclusion s In summary we have succe ssfully constructed a novel PDT based on SWNTs that can b e selectively triggered by target proteins To the best of our knowledge, this is the first study to report on the quenching of SOG by SWNT s and the restoration by specific target proteins. The AP SWNT design is based on the attachment of a DNA aptamer and photosensitizer on SWNTs with the aptamer interact ing noncovalently with SWNTs stacking between nucleotide bases and SWNT sidewalls. This AP SWNT design has several significant advantages. First, it can be a general approach for SOG regulation by a variety of targets. T here is no strict requirement for the design : no need for ha irpin structure 5 or peptide self folding 3 D ifferent aptamer sequences can be used for a variety of targets For example, we have also design ed other ssDNA with the same scheme and all of them performed for the regulation of SOG by their corresponding tar gets such as proteins and cDNA T hese features establish the universality and simplicity of AP SWNT as a PDT agent. Second the SOG of AP SWNT can be specifically triggered by a target of interest By simply changing the sequences of the

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101 ssDNA to a specif ic aptamer, a selective PDT agent can be created to trigger SOG with that specific target. This is important because m any biomolecules, such as thrombin in our model play significant roles in life processes and various diseases. It should be noted that, i n the absence of a target, even with light irradiation, only minimal 1 O 2 would be generated. This can reduce side effects Third SWNTs em ployed as cargo carriers for probe delivery can protect recognition ligands, such as ssDNA aptamers, from enzymatic di gestion or degradation in the biological environment. SWNTs can also be used for multiple ssDNA probes for high capacity in the delivery of PDT agents or in multivalent binding and delivery. Overall, this molecular engineering approach could have the poten tial to generate a wide range of PDT agent s for selective and controllable treatments

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102 Table 4 1 Sequences of oligonucleotides synthesized. Name Sequence Aptamer Photosensitizer(AP) GGT TGG TGT GGT TGG Ce6 DNA Photosensitizer (DP) TCT CTC AGT CCG TGG TAG GGC AGG TTG GGG TGA CT Ce6 Complementary DNA (cDNA) AGT CAC CCC AAC CTG CCC TAC CAC GGA CTG AGA GA Random DNA (rDNA) 5' AGA GAA CCT GGG GGA GTA TTG CGG AGG AAG GT 3' Figure 4 1. Synthesis of Chlorin e6 conjugated DNA.

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103 Figure 4 2. Schematic of aptamer photosensitizer SWNT complex and the regulation of SOG upon target binding: (I) AP and SWNTs were mixed together to form AP SWNT complex. The ssDNA aptamer is wrapped on the surface of SWNTs, which brings the photosensi tizer close to the SWNT to quench SOG. (II) Target binding with am aptamer can disturb the interaction between AP and SWNTs, resulting in the restoration of SOG. Figure 4 3. F luorescen ce and SOG quenching by SWNTs. A) The Ce6 fluorescence spect ra of buffer, AP and AP SWNT B) The SOSG signal readout after 10.5 minutes of irradiation with light at 404 nm.

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104 Figure 4 4. F luorescence a nd SOG regulation by thrombin. A) The Ce6 fluorescence spectra of the buffer, AP SWNT, and AP SWNT + T mb. B) The SOSG signal readout after 10.5 minutes of irradiation with excitation at 404 nm. Figure 4 5. Ce6 and SOSG response to thrombin concentration. A) With increasing of thrombin concentration, the Ce6 fluoresc ence gradually increased. B) The SOSG signal plotted as the function of thrombin concentration. The purple

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105 Figure 4 6. Polyacrylamide g el electrophoresis results. A) F luorescence image of the PAG E gel recorded by Ce6 fluoresc ence to show the DNA position. B) The photo of the PAGE gel after staining by Coomassie blue to indicate the position of proteins as blue band s Figure 4 7. SOG specificity. SOG selectivity of AP SWNT towards different proteins: thrombin, bovine serum albumin (BSA), protein A (PA), protein L (PL), NeutrAvidin (NA) and IgG. The SOSG fluorescence signals were normalized to that of AP SWNT.

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106 Figure 4 8 The cell viability a ffected by the phototoxicity of AP SWNT, determined by CellTiter 96 AQueous One Solution Reagent and plate reader. Figure 4 9. F luorescence a nd SOG regulation by cDNA. A) Ce6 f luorescence spectra o f the buffer, DP SWNT, DP SWNT + cDNA and DP SWNT + rDNA. The DP Ce6 fluores cence increased significantly. B) The SOSG signals were normalized to that of DP SWNT. The same trend as Ce6 fluorescence was observed for SOG.

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107 Figure 4 10. Ce6 and SOSG response to cDNA concentration. A) The Ce6 fluorescence spectra of buffer and DP SWNT with dif ferent concentrations of cDNA. B) The SOSG signal plotted as the function of cDNA concentration. The

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108 CHAPTER 5 SSDNA /GRAPHENE OXIDE FOR MOLECULAR MEDIATION OF MRNA IMAGING AND POTENTIAL PHOTODYNAMIC THERAPY Introduction Over the past decade, increasing evidence has revealed that nucleic acid molecu les are responsible for a wide range of cellular functions, such as regulation/silencing of gene expression, structural support for molecular machines, and precise control of cell behaviors. The ability to detect gene expression, especially in real time an d with a degree of sensitivity suitable to monitor minor changes at the single cell level, will have considerable value in basic biomedical research, drug discovery and medical diagnostics. DNA hybridization and biomolecular interaction studies are major t ools for the diagnosis of genetic di seases, since the hybridization of a nucleic acid strand to its complementary target is one of the most specific known molecular recognition events. Progress in genetic research has also revealed that many diseases are c haracterized by abnormal gene expression, which can be used to better understand disease on a molecular basis. Thus, using antisense oligonucleotide (AS ON) and short interfering RNA (siRNA) molecules to disrupt these aberrantly expressed genes at the leve l of mRNA transcription has evolved rapidly over the past few years. These nucleic acid molecules could also be regarded as molecular biomarkers for the disease diagnosis and therapy. Photodynamic therapy (PDT) is a minimally invasive cancer treatment rely ing on in situ generation of highly reactive singlet oxygen ( 1 O 2 ) by activation of a photosensitizer with appropriate light irradiation. 1 O 2 is a highly oxidative reagent that can react with the cellular components such as lipids, amino acids and nucleic a cids, inducing cell damage, ultimately leading to cell death. Because of its noninvasive nature, the site

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109 selectivity, cost effectiveness, the minimal recovery time, and its compatibility with other cancer treatments, PDT has gained worldwide attention wit h increasing applications in clinical use. However, it is important to note that the toxic reagent photosensitizer can also serv e as a fluorescence imaging agent that fluoresce in the visible region upon excitation with the appropriate wavelength. Although the quantum yield is lower than traditional fluorescent dyes, photosensitizer can realize both fluorescent imaging and photodynamic therapy a t the same time. Meanwhile, rapid development in nanoscience and nanotechnology has results in the success synthe sis and characterization of various inorganic nanomaterials, as well as their wide applications in bioanalytical and biomedical areas Especially, graphene, a single atom thick, two dimensional carbon nanostructure, has attracted tremendous attention since its first isolation in 2004. 114 Because of its remarkable electronic, mechanical and thermal properties, graphene has been widely applied in nanoelectronic devices, transparent conductors, and nanocomposites. Recently, its biological app lications became highly attractive and have been explored f or biosensing and biomedicine. 119, 123 Similar to its sister nanomaterial carbon nanotube, graphene oxide (GO) was also reported as a superquencher for vari ous fluorophores with the long range nanoscale energy transfer property. It can strongly bind single stranded oligonucleotides from nuclease cleavage. 124 Moreover, according to the first in vivo behavior study done by K. Yang et al recently, 162 GO has highly efficient tumor passi ve targeting property in mice, which is unique to carbon nanotube. The GO also showed relatively low retention in reticuloendothelial system and no obvious side effects.

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110 I nspired by the cha rming pro perties of GO we design ed a phtosensitzer labeled ssDNA/ GO nanocomp lex for intracellular mRNA image and activatable photodynamic therapy. GO can not only act as a superquencher for photosensitizer quenching, but also help the ssDNA intracellular delivery and resist to nuclease degradation as well as nonspecific protein binding. Experimental Section Chemicals and Instrumentation The materials for DNA synthesis, including CPG columns and reagents for DNA modification and coupling, were purchased from Glen Research Co. Chlorine e6 (Ce6) was purchased from Frontier Scientific, Inc. Dicyclohexylcarbodiimide (DCC ) and N Hydroxysuccinimide (NHS ) used for Ce6 coupling were purchased from Sigma Aldrich Inc. The Ce6 labeled ssDNA was synthesized as described in Chapter 4. The singlet oxygen sensor green (S OSG ) used fo r detecting 1 O 2 was purchased from Invitrogen Inc. An ABI3400 DNA/RNA synthesizer (Applied Biosystems) was used fo r all the DNA related synthesi s. Purifications were carried out on a ProStar HPLC system equipped with a gradient unit (Varian) and a C 18 col umn (Econosil, 5U, 250 4.6mm) (Alltech Associates) with acetonitrile/aqueous triethylamine acetate as the mobile phase The concentrations of all DNAs were determined from the absorbance at 260nm using a Cary Bio 300UV spectrometer (Varian). All fluoresc ence me asurements were performed on a Fluorolog Tau 3 spectrofluorometer (Jobin Yvon, Edison, NJ), using a quartz fluorescence cell with an optical path length of 1.0 cm. PAGE gel was using a Mini PROTEAN 3 Cell (Bio Rad ) with PowerPAC 300 as the electroph oresis power

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11 1 supply (Bio Rad ) Cell images were conducted with a confocal microscope setup consisting of an Olympus IX 81 inverted microscope with an Olympus FluoView 500 confocal scanning system. Preparation of Graphene Oxide Graphene oxide (GO) was synth esized from natural graphite po wder by modified Hummers method. 163, 164 Prior to the experiments, the GO powder was dissolved in Milli Q water and then sonicated for 5 h to give a homogeneous brown solution The TEM image was taken to show the morphology of the soluble GO (Figure 5 1). Fluorescence E xperiment The fluorescence emission of m Ce6 was scanned from 600 nm to 800 nm with the excitation at 404 nm, which is the maximum absorption of Ce6. To evaluate the SOG of probe samples, the singlet oxygen sensor green (S OSG ), which is high selective for 1 O 2 was introduced at the concentration of 2 .0 by irradiation at 404 nm, the maximum absorption of Ce6, for 10.5 minutes. The SOSG fluorescence was read out with the excitation at 494 nm with maximum at 534 nm after G was evaluated by the SOSG fluorescence enhancement compared with the background or control sample. Gel E lectrophoresis for Demonstration Polyacrylamide gel elect rophoresis was performed on a 1 2 % native gel in TB E buffer (89 m M Tris HCl, 89 m M boric aci d 2 mM EDTA ) with 5 m M MgCl 2 and run at 4 o C for 60 min at 80 V Afterwards, the gel was stained with Stains All for 1hr in dark and then shining with light to destain for 20 min. Then the images were obtained under the visible light with a digital camera

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112 Results and Discussion Here, we take c raf 1 mRNA as a model, which is a well known antisense therapeutic target due to its role in the activation of MAP kinase pathways and malignant transformation. 165 As shown in Figure 5 2 we simply conjugated a photosensitizer Chlorin e6 (Ce6) t o one end of a short single stranded antisense oligonucleotide, named as m Ce6. When incubated with graphene oxide (GO), m Ce6 bases and GO, which brought the Ce6 close to the surface of GO and got completely quenched. Upon the target introduction and duplex formation, the interaction of m Ce6 with GO would be disturbed and fall off the GO surface, resulting in Ce6 fluorescence restoration and singlet oxygen generation (SOG ). Here, GO can serve as a transporter and protector for delivering m Ce6 into targeted tumor cells, as well as a superquenche r to both fluorescence and SOG. Quenching E fficiency of GO The quenching ability of GO was estimated by measuring the fluorescence intensity of 200nM m Ce6 solution with the additional of GO. T he buffer condition, especially the cations, was found to affect the quenching efficiency in a large degree. As shown in Figure 5 3 A, by fixing the probe and GO concentration buffers containin g different salt types and concentrations were tested It turned out that 5 mM MgCl 2 in 10 mM Tris HCl buffer gave the best quenching efficiency, which was applied for further experiments. Afterwards, different concentrations of GO, ranging from 5 to 35 ug /mL, was treated with the probes to get an optimal GO concentration. The kinetics data showed that the quenching process is pretty fast, within seconds. In order to get stable data, every spectrum was obtained two minutes after addition of GO. I n Figure 5 3 B and

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113 C, with the increase of GO concentration the Ce6 fluorescence intensity dramatically decreased. Here, 30ug/mL was chosen as optimized GO concentration because fluorescence intensity was only 0.5% of the original one. Restoration of Fluorescence an d SOG by cDNA The propensities of the binding interactions between GO and ssDNA/dsDNA are signifi cantly different. The ssDNA can tightly attach onto the GO surface. When binds to its target complementary DNA (cDNA) it undergoes a ce rtain conformation al ch ange to dsDNA which weakens its interaction with the GO or even results in dissociation from GO inducing the restoration of fluorescence and SOG. The results in Figure 5 4 A confirmed our assumption that the target cDNA binding indeed can cause the confor mation change and induce fluorescence restoration. The Ce 6 fluorescence increased up to around one hundred fold after the addition of 400 nM cDNA As shown in Figure 5 4 B, the SOSG fluorescence of m Ce6/GO did not change much compared to buffer solution. H owev er, the SOSG fluorescence of m Ce6/GO exhibited over one hunderd fold enhancement upon introduction of 400 nM cDNA. This demonstrates that GO s can efficiently turn off SOG and, more importantly, that it can be reversibly mediated by its target binding event i.e. the hydridization between ssDNA and its cDNA Quantitative Response to cDNA The fluorescence of m Ce6/GO with a series of cDNA concentration s has also been investigated. As shown in Figure 5 5 A, the Ce6 fluorescence gradually enhanced with the increasing of cDNA concentration. I n Figure 5 5 B the SOSG fluorescence intensity showed similar behavior. Especially, an almost linear increase was observed during the cDNA concentration range of 5 100 nM (inner graph of Figure 5 5 B) During the certain concentration range, the SOG of m Ce6/GO can be quantitati vely mediated

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114 by cDNA concentration, which suggested the potential of this probe as a quantitative SOG controlling PDT agent. Detection Selectivity of m Ce6/GO The hairpin structure is known to ha ve better sel ectivity to its target sequence than that of ssDNA. To examine whether it is the similar case on GO surface, we synthesized a hairpin DNA labeled on one end with Ce6, named hp Ce6. In this study, we compared the single base mis match (1bm) sele ctivity of GO quenched ssDNA and hpDNA. We treated both m Ce6/GO and hp Ce6/GO with a series of cDNA concentration s, 1bm cDNA, and random DNA ( rDNA ) from 5 nM to 1 M. The results in Figure 5 6 revealed that on the GO surface, the ssDNA and hpDNA have ver y similar selectivity to its single base mismatch target. On the GO surface, the conformational restriction for selectivity is mainly due to GO itself, and the difference s between ssDNA and hpDNA are minimal. It demonstrated that our m Ce6/GO complex not o nly had the structural simplicity but also kept the good selectivity. Gel Electrophoresis for System Demonstration To demonstrate that the reaction happened as we hypothesized that ssDNA aborbs onto GO and cDNA forms duplex to compete it away from GO, we performed p olyacrylamide gel electrophoresis to prove the different States of m Ce6/GO complex. For 12% native PAGE gel, the m Ce6/GO cannot run into the gel because of the large size of GO. If duplex forms and falls off the GO surface, the duplex band co uld be observed in gel. Five samples were prepared as follows: lane 1, m Ce6 only; lane 2, m Ce6/cDNA in duplex form ; lane 3, m Ce6/GO ; l ane 4, m Ce6/GO treated with cDNA; lane 5, m Ce6/GO treated with rDNA.

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115 As shown in Figure 5 7 lane 1 and lane 2 indica ted the ssDNA and dsDNA position as control. W hen incubating with GO m Ce6 attached to the GO surface by and the complex is too big to go inside the gel. There is no obvious band in lane 3. When adding cDNA to the m Ce6/GO complex, cDNA can hybridize with m Ce6 and release it from GO surface. W e o bserved the duplex band in lane 4 as that of l ane 2, which proved that cDNA did bind with m Ce6 and make it dissociate from GO When treating the complex with rDNA no duplex band showed up in lane 5 and only the ssDNA band of rDNA which implied m Ce6 was kept on GO surface and without specific bind ing rDNA cannot release m Ce6 from GO Confocal Image for I nternalization D emonstration We further investigated the ability of m Ce6/GO to enter cells and detect mRNA targets. The MDA MB 231 cells, a human breast epithelial adenocarcinoma cell line, whic h over express c raf 1 mRNA, were used as a model. As a control, another ssDNA probe with Ce6 labeling containing a noncomplementary sequences was used, named as r Ce6. We hypothesized that the m Ce6/GO complex can translocate across the cell membrane s and deliver m Ce6 into cells. Upon encountering with target mRNA, m Ce6 will hybridize with the target mRNA and be release d from GO surface, leading to a fluorescence restoration which could be detected by confocal microscopy. As shown in Figure 5 8 A and B, the cells treated with m Ce6/GO complex for 3hr at 37 o C, had a high fluorescence signal inside the cells. For comparison, the control probe r Ce6/GO complex was tested under the same conditions, and there was a significantly lower fluorescence signal (Fig ure 5 8 C and D). We also tested the free m Ce6 treated cells without GO under the same conditions. There was no detectable signal by confocal microscopy. The self delivery of ssDNA into the cells normally needs

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116 a long incubation time. These results prove t hat GO can efficiently deliver DNA probe into cells to detect the target mRNA and the m Ce6/GO complex could be a good fluorescence imaging agent for mRNA. Conclusion s In summary, we have demonstrated that GO s like carbon nanotubes, can be used as a novel PDT drug and intracellular imaging agent The m Ce6/GO design is based on the attachment of a ssDNA and photosensitizer on GO, with the ssDNA interacting noncovalently with GOs by stacking. Later on, when the ssDNA probe meet s with its complementary DNA or mRNA targets, the conformational change from ssDNA to dsDNA disturbs the interaction between probes and GOs, inducing the probe detachment from GOs as well as fluorescence and S OG recovery. The quenching efficiency of GOs was demonstrated to be over 99% and the fluorescence recovery was more than 100 fold. Because of the self internalization capability of GOs the preliminary cell imaging experiment has been performed and indicat ed m Ce6/GO as a good intracellular mRNA detection and imaging agent. The in vitro and in vivo toxicity demonstration is our future goal to prove its potential as a PDT drug.

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117 Table 5 1 Sequences of oligonucleotides synthesized. Name Sequence m Ce6 Ce6 TCC CGC CTG TGA CAT GCA TT r Ce6 Ce6 NNN NNN NNN NNN NNN NNN NN cDNA 5' AAT GCA TGT CAC AGG CGG GA 3' 1bm cDNA 5' AAT GCA TGT AAC AGG CGG GA 3' rDNA AAT CAA CTG GGA GAA TGT AAC TG Figure 5 1. TEM image of soluble GO.

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118 Figure 5 2. Schematic representation of photosensitizer ssDNA /GO complex and the regulation of SOG upon target binding. The photosensitizer ssDNA attaches onto GO to get photosensitizer quenched. Upon target mRNA binding, the duplex leaves away from GO and restores the SOG. Figure 5 3. Optimizati on of GO quenching conditions. A) Investigation of GO quenching i n different buffer conditions. B) With the increasing of GO concentration, the fluorescence intensity of mj Ce6 gradually decreased. C) The fluorescence intensity of m Ce6 versus the concentration of GO.

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119 Figure 5 4. Fluorescence and SOG regulation by target cDNA. A) The Ce6 fluorescence spectra of the buffer GO, m Ce6/GO, and m Ce6/GO + cDNA. B) The SOSG signal readout after 10.5 minute s of irradiation with excitation at 404 nm. Figure 5 5. Ce6 fluorescence and SOSG response to cDNA concentration. A) With the increase of cDNA concentration, the Ce6 flu orescence gradually increased. B) The SOSG signal plotted as the function of cD NA concentration.

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120 Figure 5 6. Polyacrylamide g el electrophoresis results Gel was stained with stains all to show DNA pos i tion.

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121 Figure 5 7. Intracelluar imaging of ssDNA/GO complex. Confocal fluorescence microcopy of MDA MB 231 cells treated with m Ce6/GO (A and B) and control r Ce6/GO (C and D). Ce6 fluorescence field (left) and bright filed Ce6 fluorescence overlay (right) are shown.

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122 CHAPTER 6 DNA SCAFFO LD AS LOGIC SWITCHBO AR D FOR SMART DETECTION Intr o duction Logic gates are devices that per form logic operations on one or more inputs and produce a single output They are the fundamental components of the digital circuits, which process binary data encoded, in electrical signals. Logic gates can be classified as single input and multiple input logic gates. The symbol and the truth table of the several basic logic gates are listed in Figure 6 1. For example, the NOT is a single input logic gate that always opposes the input whatever the input is. The AND gate converts two inputs into signal outp ut, in which the output is 1 only if its two inputs are both 1. Combinational logic circuits can be assembled by connecting the input and output terminals of these basic logic gates. The modern computers have hundreds of millions of such logic gates connec ted into very complex circuits. By analogy, in recent decades, considerable efforts have been focused on using logic gates for information processing in chemical molecular systems such as in physiology, medicine and biotechnology as sensors and diagnostic tools As a result, logic gates have been constructed from numerous molecular settings, including small molecules, 166 supramolecular complexes, 167 nucleic acids, 168 enzymes, 169 peptides, 170 and oth ers. Among versatile materials, DNA, because of its sequence specific recognition property, provides intriguing possibilities for the construction of logic gates at molecular and multi level networks. For example, Stefanovic and coworkers did extensively s tudy on ribozyme based logic gates. 171 Winfree and coworkers constructed molecular log ic gates with logic cascading, restoration and modularity. 168 Willner and coworkers built a DNA computing circuits using a library of DNAzyme subunits. 169 The specific DNA

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123 recognition can not only produce the addressable hybridization, but also can disrupt DNA interactions through the process of strand displacement (Figure 6 2) If the coming strand AB is totally complementary to target strand (Figure 6 2A), AB can bind to single of and proceeds to invade and displace the shorter length hybridized sequence A via a three way branch migration. However, if the coming strand is which is shorter than AB two cases might happen in Figure 6 2B. After the strand invasion, if A still has enough base pairing with it can still keep hybridizing with and form th e three strand structure. Otherwise, the interaction between A and is unstable, strand A will dissociate from resulting in a duplex and ssDNA. Various DNA based molecular machines have been made based on this concept, such as DNA tweezers 33 and DNA walkers. 34 This concept is also well suited for use in the DNA logic gates design. 172 While these strategies are elegant, there is still room for improvement. Here, we propose the construction of logic switchboard using combinatorial self assembly of DNA nan otiles into m icrometer sized 2D arrays The nanotiles we used are adopted from the work of Yan and coworkers. 13 This nanotile, named 4X4 tile, contains four four arm DNA branched junctions pointing in four directions. It has a square aspect ratio and readily self assembles into 2D lattice forms. It can periodi cally carry various molecules, such as proteins, gold nanoparticles, and nucleic acid probes. 13, 17, 23 M oreover, with the fluorescent dye labeling, the nanoarrays can be visualized under fluorescence microscopy, as a feasible and convenient signal readout. 23 As a result, we took the advantage of this nanotile as DNA scaffold to build our logic switchboard on it to achieve basic and multi level logic operations for s mart DNA detecti on

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124 Experimental Section Chemicals and Instrumentation The longest DNA (100mer) and the DNA oligos w ith labeling were synthesized in our lab and followed the method mentioned in previous chapter. The rest of the plain DNA oligos were purchased from Integra ted DNA technologies ( www.idtdna.com ). The 50bp mini DNA ladder was purchased from Fisher Scientific Co. An ABI3400 DNA/RNA synthesizer (Applied Biosystems) was used fo r DNA related synthesi s. Purifications were carried out on a ProStar HPLC system equipped with a gradient unit (Varian) and a C 18 column (Econosil, 5U, 250 4.6mm) (Alltech Associates) with acetonitrile/aqueous triethylamine acetate as the mobile phase The concentrations of all DNAs were determined from the absorbance at 260nm using a Cary Bio 300UV spectrometer (Varian). All fluorescence me asurements were performed on a Fluorolog Tau 3 spectrofluorometer (Jobin Yvon, Edison, NJ), using a quartz fluorescence cell with an optical path length of 1.0 cm. PA GE gel was running on Mini PROTEAN 3 Cell (Bio Rad ) with PowerPAC 300 as Gel electroph oresis power supply (Bio Rad ) Self assembly of Nan otiles To assemble the nanotiles, the strands invol ved in each tile (see Figure 6 3 ) were mixed separately in differen t tubes in equal molar ratio (all 2 M) in 1XTAE Mg buffer (40 mM Tris acetic acid buffer, pH 8.0, Mg(Ac) 2 12.5mM), then the mixtures were heated to 95 o C and cooled down slowly (over 24 hours) to room temperature. Then the nanotile was kept at 4 o C for futu re use.

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125 Gel E lectrophoresis for Structure Validation To validate the nanotile structure, p olyacrylamide gel elect rophoresis was performed on a 6% native gel in TA E Mg buffer and run at 4 o C for 2.5hr at 80 V Afterw ards, the gel was stained with s tai ns all for 1hr in dark and then shining with lamp to destain for 20 min. The image w as taken under the visible light with a digital camera. Fluorescence E xperiment The dabcyl labeled a nd fluorescein labeled reporter strands were mixed with 1:1 ratio in TAE M g buffer. The time based fluorescence measurement with the addition of nanotiles and inputs was recorded with excitation at 488nm and emission at 516nm Results and Discussion The overall programme was illustrated in figure 6 4 Two types of nanotiles are constructed based on 4X4 tile the sensor tiles and report er tiles. Once mixed, these two types of tiles can associate with each other in an alternative arrangement to form 2D arrays. The reporter tile has organic dyes on it for encoding. The sensor tile h as attached recogniti on elements, such as cDNA or aptamer. On ce the target binds, the sensor tile releases a single stranded DNA, which can i nteract with the nearby reporter tile by a strand displacement reaction to release a dye labeled short sequence, th us causing a color change of the entire array. By carefully designing t he communication between sensor tile and reporter tile, the logic gate functions such as AND and OR can be achieved with readout by confocal fluorescence microscope. Furthe rmore, by enc oding the reporter tiles with different dyes, multiple color readouts and multi level networks can be constructed. To validate the system and perform sequence optimization, at first step, we only constructed the sensing part into nanotile For the reporte r, fluorophore and quencher

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126 labeled ssDNA were directly used, instead of putting them into nanotile. Fluorescence microscopy was applied as detection method to optimize the system. Two basic logic gates, OR and AND have been constructed and demonstrated. OR Gate Construction and Validation In OR gate, according to the truth table, the output is 1 if one or both the inputs to the gate are 1. If neither input is 1, the output is 0. Based on this working principle, o ur OR gat e design was shown in Figure 6 5 On the sensor tile, two sensing elements were constructed on it: OR 1 hybridized with OR AX, and OR 2 hybridized with OR AY. Input 1 can hybridize with OR 1 and release the OR AX by strand displacement. The released OR AX can interact with reporter and und ergo another strand displacement reaction to separate the fluorophore labeled strand (OR F) from quencher labeled strand (OR Q) thus inducing the fluorescent signal. It is the same case for input 2 that reacts with OR 2. The addition of either input 1 or input 2 displaces either the OR AX or OR AY output strands, respectively. Both of the outputs can react with reporter bind with OR Q and release OR F to general fluorescent signal. The sequence design for the nano tile was dr a w n in Figure 6 3 A. The strand s were mixed in equal molar ratio and annealed to form nanotile. And the sequences for sensor and reporter part has been optimized and listed in Table 6 1. PAGE for structure validation The replacement of original strands to OR 1 and OR 2 might have some strand invasion effects to the whole structure. As a result, w e constructed three different nanotiles, the nanotile s only containing OR 1 or Or 2 and the nanotile containing both OR 1 and OR 2 in one single tile. To validation the formation of nanotile s w e performed gel el ectrophoresis. 6% native PAGE gel was prepared in order to have large pore size

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127 that the nanotiles can go through. The experiment was carried out at 4 o C and low voltage 80V to avoid structural decomposition due to elevated temperature dur ing the process. The results were shown in Figure 6 6. Lane 1 is 100mer ssDNA as a control Lane L on the right is the 50bp mini DNA ladder to indicate the nanostructure size. Lane 2 is nanotile with only OR 1; lane 3 is nanotile with only OR 2; and lane 4 is nanotile with both OR 1 and OR 2. In all these three lanes, a clear band with molecular weight around 200 bp could be observed, which is coherent with the calculated molecular weight and demonstrate the success of nanotile construction. OR operation p roved by fluorescent measurement The reporter was generated by mixing strand OR F and strand OR Q with 1:1 ra tio. Afterwards, the annealed OR nano tile was introduced and mixture was regarded as OR gate solution. No fluores c ence change was observed, indicat ing no cross reacti on between reporters and sensor tiles. The logic operation was initiated by adding input strands to the OR gate solution and time based fluorescence measurement was applied to record the results. As shown in Figure 6 7A to C, with the a ddition of input 1, input 2 or input 1&2, the fluorescent signals increased immediately and reached the maximum for about half an hour. The results were summarized as column graph in Figure 6 7E. With either or both inputs as 1, the readout of output is 1. When both inputs are present, the effective outputs to react with reporter are doubled. However, equimolar quantities of the gate solutions are used, so the maximum output of each gate is limited to a single equivalent even in the presence of excess input s. The signal for input 1&2 is only slightly higher

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128 than that of any single input. Overall, the fluorescent analysis of the resulting output revealed proper implementation of the OR gate behavior ( Figure 6 7D ) AND Gate Construction and Validation In the A ND logic system, the output is 1 only when both of the input values are 1. The designing of the AND gate was illustrated in Figure 6 8. The input 1 hybr idizes with the AND 1 on the sensor tile and releases the AND AX strand, which can hybridize with o f the quencher strand (AND Q). However, the fluorophore strand (AND F) can still hybridize with the AND Q through the portion and keep at the stage of three strand structure, at which the fluorophore is still in close proximity of quencher and gets que nched. In the same case, when only input 2 are introduced, it releases the AND BY st rand from AND 2, which can hybridize with of AND Q and leave the part for AND F to hybridize. As a result of three strand structure, the florescence signal is stil l low However, when both input 1 and input 2 coexist they react with sensor tile and release AND AX and AND BY, respectively. Then, AND AX and AND BY cooperatively interact with reporter and separate the AND F from the AND Q, generating strong fluorescen ce signal Thus, the AND gate has been generated. The sequence design for the nanotile was drawn in Figure 6 3B. The strands were mixed in equal molar ratio and annealed to form the nanotile. And the sequences for sensor and reporter part has been optimize d and listed in Table 6 2. AND operation proved by fluorescent measurement The reporter was prepared by mixing AND Q and AND F in 1:1 ratio. Then, the annealed AND nanotile was introduced and the mixture was regarded as AND gate solution. Firstly, fluore scence results confirmed that on interaction between reporters and AND sensor tiles. The logic operation was initiated by adding input strands to the

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129 AND gate solution and time based fluorescence measurement was applied to record the results. In Figure 6 9A, with the addition of input 1, the fluorescen c e signal slightly increase and kept constant after few minutes. Then the input 2 was introduced, and the fluorescence signal dramatically increased. By reversing the additional order in Figure 6 9B, the inpu addition of input 1 generated fluorescent enhancement The results were summarized as column graph in Figure 6 9D Adding either input 1 or input 2 did not result in an increase of fluoresc ence intensity. Only when both inputs coexist, they operated effectively to cause the separation of quencher and fluorophore, inducing fluorescence signal. Overall, the fluorescent analysis of the resulting output revealed proper implementation of AND gate behavior (Figure 6 9C ). Conclusion s In summary, we have successfully constructed the DNA nanotile and demonstrated its application in logic operation. By smartly designing the DNA sequences to control the communication s between sensor tile and reporter bas ed on strand displacement principle, we realized the implementation of OR and AND logic gates. Upon the response to the inputs, the logic gates can release ssDNA outputs that react with reporters and generate certain fluorescence signals. This is the first step on the way to our ultimate goal: using DNA scaffold as logic switchboard. In the next step, we will design the reporter onto the report nanotile and assemble the sensor tile and reporter tile together into DNA 2D nanoarray. Then, fluorescence micros cope will be applied as signal readout. These gates will also be wired together to form multi level circuits, since the output of one gate can serve as the

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130 input to another. On the other side, the inputs of logic gates should not be limited only with oligo nucleiotides. By introducing aptamer strands into the sensor tile s, we will be able to extend the scope to other biomolecules. Example for a potential application, it can be used as a smart sensing and diagnostic system to interpret complicated and abnorma l re sults from a medical examination

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131 Figure 6 1. The symbol s and truth table s of basic logic gates.

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132 Figure 6 2. Sequence dir ected DNA strand displacement. A) Full length complement strand AB leads to the i rreversible displacement of strand A via a three way brand migration mechanism. B) For shorter length complement strand two situations might happen. (1) Strand A keeps hybridize with strand and form three strand structure. (2) Strand A releases from the because of the unstable duplex formation.

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133 Figure 6 3. Strand structure and DN A sequences used in nanotiles. A) OR gate nanotile. B) AND gate nanotile.

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134 Figure 6 4. The design strategy of DNA scaffold based logic switchboard.

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135 Figure 6 5. The principle of OR gate based on DNA sensor nanotile s and duplex reporter s

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136 Tab le 6 1. Sequences of the sensor and reporter part for OR gate. Name Sequences OR 1 CAG GCT ACG GCA CGT AGA GCA TCA CC ATG ATC CTG CTCAG TT AACG CCT ACGATGG ACACGCCGACC OR AX GG CTGAG CAG GAT CAT OR 2 GAGC GCAACC TGCCTGGCAAG ACTC AA CTCAG CCG TCT TCC A GAC AAG AGT GCA GGG OR AY GGA AGA CGG CTGAG CA OR Q ATG ATC CTG CTCAG CCG T (Q) CTTCC OR F F ACG GCT GAG CAG G Input 1 CAG GAT CAT GGT GAT GCT CTA CGT GCC GTA GCC TG Input 2 CCC TGC ACT CTT GTC TGG AAG ACG G Figure 6 6. The PAGE gel experiment results for nanotile structure validation.

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137 Figure 6 7. Fluorescence measurement resu lts of OR nano tile logic gate. A C) T he time based measurement with input 1, input 2 or input 1&2. D) Symbol and truth table of OR logic gate. E) Fluorescence intensities are summarized as a truth table.

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138 Figure 6 8. The principle of AND gate based on DNA sensor nanotile s and duplex reporter s

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139 Tab le 6 2. Sequences of the sensor and reporter part for AND gate. Name Sequences AND 1 CAG GCT ACG GCA CGT AGA GCA TCA CC ATG ATC CTG CTC AG TT AACG CCTACGATGG ACACGCCGACC AND AX GAGGG CTG AG CAG GAT CAT 3 AND 2 CCG TCT TCC AGA CAA G AGT GCA GGG TAT CC AA CAGAGG ACTACTCATCCGT 3 AND BY GTTC GGA TA CCC TGC ACT AND Q ATG ATC CTG CTC AG CTT CG Q AGT GCA GGG TAT CC GAACC AND F GGTTCGGATA CC F CGAAGCTGAG CA Input 1 CAG GAT CAT GG T GAT GCT CTA CGT GCC GTA GCC TG Input 2 CCC TGC ACT CTT GTC TGG AAG ACG G Figure 6 9. Fluorescence measurement resul ts of AND nanotile logic gate. A) and B) Time based measurement with addition of input 1 & 2 and input 2&1. C ) Symbol and t ruth table of OR logic gate. D ) Fluorescence intensities are summarized as a truth table.

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140 CHAPTER 7 SUMMARY AND FUTURE D IRECTIONS Target Responsive DNA Switches for Analytical and Biomedical Applications DNA is the molecule that nature uses as genetic material with its high fidelity base pair recognition as the chemical foundation. Nevertheless, biology is no longer the only branch of science where DNA is finding a significant role. Many different types of DNA probes and architectures have been propose d and developed as biosensors and biomaterials. As an ideal building block, DNA is renowned for its predictability and specificity of Watson Crick base pairing that enables the recognition and highly selective binding of complementary DNA strands. Meanwhil e, upon certain molecular engineering process, some sequence specific DNA, well known as aptamer and DNAzyme can also effectively recognize other molecules such as ions and proteins. Combining with other significant features such as simplicity of synthesi s, diversity of modification, and compatibility with many detection techniques, DNA, provides a new opportunity and design medium for molecular engineering, which will fundamentally change the field related to molecular probes. The overall direction of th is doctoral research has been dedicated to combine the selectivity of DNA with other functional materials for the development s and application s of novel probes, named DNA switches, for target responsive reactions in different systems. T hree key projects ha ve been presented: 1) the design and construction of functional DNA cross linked hydrogel for visual detection ; 2) the establishment of a safer and more efficient photodynamic therapy based on DNA and carbon nanomaterials ; 3) the molecule engineering of DN A nanostructure as logic switchboard for smart diagnosis

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141 Functional DNA Cross linked Hydrogel for Visual D etection Visual detection is an increasingly attractive method, especially for rapid diagnostics in disaster situation, home healthcare settings, and in poorly equipped rural areas, where low cost, rapidity, and simplicity are essential. W e grafted the DNA recognition into hydrogel and built such a colorimetirc detection platform. The hydrogel was made of polyacrylamide with DNA strands covalently atta ched, and the polymers were cross linked into gel by DNA hybridization. AuNPs or enzyme s were trapped inside the 3D network of the hydrogel. In Chapter 2, aptamer target recognition was applied for molecule sensing wi th cocaine and its aptamer as a model. Upon introduction of a target, the cross linker aptamer inside the gel bound with it, and the gel dissolved as a result of reducing the crosslinking density by competitive target aptamer binding. The released AuNPs or enzyme s took part in their role for si gnal amplification. In Chapter 3, we further extended the system with DNAzyme for Pb 2+ detection. The hydrogel was formed through the cross linking by hybridization of substrate and enzyme strands grafted onto linear polyacrylamide chains. The addition of target Pb 2+ bound with enzyme strand and catalyzed the cleavage reaction of substrate and dissolved the gel. The trapped AuNPs were released to give the visual detection readout. Due to abundance of aptamer and DNAzyme candidates, this hydrogel platform is a generic approach for visual detection of various molecules with high sensitivity and selectivity. Regulation of Singlet Oxygen Generation Using Carbon Nanomaterials Photodynamic therapy (PDT) is gaining wide acceptance as an alternative noninvasive tre atment of cancers. However, it often suffers from non specific treatment that causes low efficiency to injured areas and high side effects to healthy organs. To overcome these drawbacks, two contro llable PDT design were performed. In Chapter 3,

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142 the PDT des ign was based on covalently linking a photosensitizer with a n aptamer then wrapping onto the surface of SWNTs SWNTs were demonstrated to be great quenchers to singlet oxygen generation (SOG). The target binding events disturb ed the DNA interaction with th e SWNTs and cause d the aptamer to fall off the SWNT surface, resulting in the restoration of SOG. Moreover, derived from same family, graphene oxide (GO) has been reported to have similar properties as SWNT s with enhanced tumor passive targeting and less t oxicity. In Chapter 4, we designed another PDT by ssDNA /GO for mRNA imaging and therapy. Similar effect has been achieved with the mRNA triggered SOG These studies validated t he potential of our design as novel PDT agent s with the regulation by target mol ecules, enhanced specificity, and efficac y of therapeutic function, directing the development of PDT to be safer and more selective. DNA Scaffold as a Logic Switchboard for S mart D etection Logic diagnosis becomes increasingly valuable with the development of modern medical technology, since more complicated and abnormal results from a medical examination need to be interpreted. In Chapter 5, we gave the first attempt of using highly developed DNA nanotechnology to construct DNA nanoscaffold as a logic switc hboard for smart detection. The sensing elements were built on a 4X4 DNA nanotile. By smartly designing the DNA sequences to control the communication between sensor tile and reporter based on strand displace ment principle, implementation of OR and AND log ic gates have been successfully realized These results demonstrated the feasibility and capability of DNA nanotiles as scaffold to construct logic operation on it and further development of multi level circuits is promising.

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143 In summary, this research ha s mainly focused on the molecular engineering of DNA along or with other functional materials for more sensitive and selective detection and therapy A successful outcome from these studies will lead to advanced nucleic acid based probes for biological sci ences, biomedical research, and therapeutic and clinical applications Future Directions Hydrogel Lab on chip Device on Paper for Visual Detection The future direction of hydrogel based visual detection will be the transformation of the test tube based as say to a lab on chip device. In order to fulfill the requirement for remote regions, such as those found in less industrialized countries, in emergency situations or in home health care settings, the analysis must be affordable, sensitive, specific, user f riendly, equipment free, rapid and robust. Since the first proposal of using paper to build lab on chip device by Whitesides and coworkers, 135 paper has aroused great attention in this field. Paper has several advantages as an ideal material for constructing such devices: 1) paper is thin and lightweight; 2) paper is compatible with fluidic samples; 3) modification is easy; 4 ) paper is usually white and a good medium for colorimetric tests; 5) paper is disposable. Currently, we are developing a simple paper based hydrogel visual detection platform as shown in Figure 7 1. The gel and sensing principle is the same as the one dev eloped in Chapter 2 and 3. The gel is spotted on certain area of the strip paper. By heating the paper, the gel can be dissolved and absorbed into the paper, where it eventually stiffens. The gel here can work as a filter to block large sensing molecules f rom moving along the strip. Without targets, the sensing molecules can only go along the strip with water till the gel area and stop there. With targets, the gel can be dissolved

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144 and the sensing molecules will move up, showing a dif ferent color pattern to that without target The successful development of this cost efficient and user friendly device has the potential to bring the attempt in lab to our daily use. DNA Scaffold as a Logic Switchboard for S mart D etection So far, we demonstrated the basic conce pt of using DNA nanotile for building logic gates, which is the starting point of this project. In the next step, we will assemble the sensor tile and reporter tile together into DNA 2D nanoarray. Multicolor nanotiles will also be introduced. Then, fluores cence microscope will be use d as signal readout. These gates will be further wired together to form multi level circuits, since the output of one gate can serve as the input to another. On the other side, the inputs of logic gates should not be limited onl y with oligonucleiotides. By introducing aptamer strands into the sensor tile s, we will be able to extend the scope to other biomolecules. To build up a complete set of logic gates and multi level network for smart detection and diagnostics is our ultimate goal.

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145 Figure 7 1. Illustration of paper based lab on chip device with different color patterns for buffer and target solution. A) Constru ction of hydrogel paper strip. B) Dipping the strip in buffer solution, the sensing molecules will move up u ntil gel region. C) Dipping the strip in target solution, target dissolves the gel, and sensing molecules can move up cross the gel region.

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146 LIST OF REFERENCE S 1. Watson, J. D.; Crick, F. H. C., Molecular Structure of Nucleic Acids a Structure for Deoxyribose Nucleic Acid. Nature 1953, 171 (4356), 737 738. 2. Chargaff, E.; Zamenhof, S.; Green, C., Composition of Human Desoxypentose Nucleic Acid. Nature 1950, 165 (4202), 756 757. 3. Tanaka, T.; Letsinger, R. L., Syringe Method for Step wise Chemical Synthesis of Oligonucleotides. Nucleic Acids Research 1982, 10 (10), 3249 3260. 4. Seeman, N. C., Nucleic Acid Junctions and Lattices. Journal of Theoretical Biology 1982, 99 (2), 237 247. 5. Seeman, N. C., DNA in a material world. Nature 200 3, 421 (6921), 427 431. 6. Lin, C. X.; Liu, Y.; Rinker, S.; Yan, H., DNA tile based self assembly: Building complex nanoarchitectures. Chemphyschem 2006, 7 (8), 1641 1647. 7. Yang, D. Y.; Campolongo, M. J.; Tran, T. N. N.; Ruiz, R. C. H.; Kahn, J. S.; Luo, D., Novel DNA materials and their applications. Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology 2010, 2 (6), 648 669. 8. Fu, T. J.; Seeman, N. C., DNA Double Crossover Molecules. Biochemistry 1993, 32 (13), 3211 3220. 9. LaBean, T. H.; Yan, H.; Kopatsch, J.; Liu, F. R.; Winfree, E.; Reif, J. H.; Seeman, N. C., Construction, analysis, ligation, and self assembly of DNA triple crossover complexes. Journal of the American Chemical Society 2000, 122 (9), 1848 1860. 10. Liu, Y.; Lin, C. X.; L i, H. Y.; Yan, H., Protein nanoarrays Aptamer directed self assembly of protein arrays on a DNA nanostructure. Angewandte Chemie International Edition 2005, 44 (28), 4333 4338. 11. Winfree, E.; Liu, F. R.; Wenzler, L. A.; Seeman, N. C., Design and self a ssembly of two dimensional DNA crystals. Nature 1998, 394 (6693), 539 544. 12. Liu, D.; Park, S. H.; Reif, J. H.; LaBean, T. H., DNA nanotubes self assembled from triple crossover tiles as templates for conductive nanowires. Proceedings of the National Aca demy of Sciences of the United States of America 2004, 101 (3), 717 722. 13. Yan, H.; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H., DNA templated self assembly of protein arrays and highly conductive nanowires. Science 2003, 301 (5641), 1882 18 84. 14. Rothemund, P. W. K., Folding DNA to create nanoscale shapes and patterns. Nature 2006, 440 (7082), 297 302.

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160 BIOGRAPHICAL SKETCH Zhi Zhu was born in Shaowu, Fu jian Province, China, in 1984. She attended the Shaowu No. 1 Middle School for her secondary education. She obtained the 9 th ranking in Fujian out of nearly 180,000 students in the National University Entrance Examination and attended Peking University to study c hemistry As an undergraduate student, Zhi to carry out scientific research as a chemist. With great interest in chemistry aroused by undergraduate study and research, d irectly after obtaining her Bachelor of Science degree, Zhi came to the United States in the fall of 2006 to pursue her Ph.D. deg ree in chemistry under the s upervision of Dr. Weihong Tan at the University of Florida. Zhi Zhu received her Doctor of Philosophy degree in analytical c hemistry from the University of Florida in May of 2011.