DNA-Conjugated Magnetic Nanoparticles for Bio-Analytical and Biomedical Applications

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DNA-Conjugated Magnetic Nanoparticles for Bio-Analytical and Biomedical Applications
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1 online resource (120 p.)
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english
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Bamrungsap,Suwussa
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University of Florida
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Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
Tan, Weihong
Committee Members:
Cao, Yun Wei
Fanucci, Gail E
Angerhofer, Alexander
Batich, Christopher D

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Chemistry -- Dissertations, Academic -- UF
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Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Magnetic nanoparticles (MNPs) can be synthesized to have various sizes, shapes, and compositions, providing distinctive magnetic, electronic and optical properties compared to their bulk counterparts. With the dimension similar to biomolecules, MNPs can be engineered to have specific properties and can be used to pursue an in-depth understanding of biochemical processes. Due to the strong and specific base-pairing of nucleic acids, as well as their ability to form self-assembled structural motifs, DNA conjugated MNPs can serve versatile functions including the investigation of biological activities and the construction of molecular machines. First, a magnetic nanosensor prepared by aptamer conjugated MNPs (ACMNPs), was developed based on magnetic relaxation switches (MRSw) mechanism. The detection can be achieved by switching between dispersed and aggregated of MNPs upon binding with target cells resulting in a change in proton spin-spin relaxation times (?T2). Specificity and sensitivity of nanosensors were capitalized by the ability of aptamers to specifically bind their targets and the large surface area of MNPs to accommodate multiple binding events. The detection as few as 10 target cells in buffer and 100 cells were obtained in biological samples. Furthermore, by using an array of ACMNPs, various cell types can be differentiated through recognition patterns, thus creating a cellular molecular profile and allowing clinicians to accurately identify cancer cells at the molecular and single cell level. Additionally, a nanosensor based on MRSw was also demonstrated for protein detection using Lysozyme (Lys) as a model protein. The nanosensor system consisted of MNPs aggregates prepared by the hybridization of MNPs conjugated with either aptamers or linker DNA that could hybridize to the extended part of Lys aptamers, leading to the cluster formation. In the presence of Lys, the aptamers bind with their targets, resulting in the disassembly of the clusters, and providing a change in T2 relaxation time. The high selectivity and sensitivity in nanomolar range of the detection was achieved in both buffer and human serum. The analysis of Lys in cancer cell lysates was also performed to validate the detection in real clinical samples. Another area of investigation was the development of a magnetically driven DNA nanomotor. In particular, DNA hairpins were conjugated to magnetic particles and the entire conjugation was immobilized to a solid support. The DNA hairpin can be interpreted as a nanomotor because it switched between ?closed? and ?open? states. The applied magnetic field triggered the change of molecular conformation or drove the movement of molecular probes between closed and open states, which can be observed by the change of fluorescence. By repetitive shrinking and extending movements, DNA hairpins were considered as a nanometer-scale motor. In summary, this research focuses mainly on the development of DNA-conjugated MNPs for the analysis of biological targets, and the construction of nanomachine. Successful outcomes form these investigations will lead to the improvement in bioanalytical science, biomedical applications, and bionanomaterial research.
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Suwussa Bamrungsap.
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 2012-08-31

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1 DNA CONJUGATED MAGNETIC NANOPARTICLES FOR BIO ANALYTICAL AND BIOMEDICAL APPLI C A TIONS By SUWUSSA BAMRUNGSAP A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIR EMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Suwussa Bamrungsap

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3 To m y f amily

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4 ACKNOWLEDGMENTS I am truly grateful to a long list of people. None of my success would have been possible without them. I wo uld like to express my gratitude to my research advisor, Dr. Weihong Tan, for providing continuous guidance, support, and opportunities for my research and life throughout my PhD studies. His valuable inspiration and advices encourage me to have scientific thought which will definitely have a great impact for my future career and life. I am pleased to thank my doctoral dissertation committee members, Dr. Alexander Angerhofer, Dr. Charles Cao, Dr. Gail Fanucci, and Dr. Christopher Batich for the helpful disc ussion and constructive comments. I would like to thank Dr. Benjamin Smith for all help and support during my five year at University of Florida. I also thank The Royal Thai government for the graduate stipend and tuition fees during my whole PhD studies. This dissertation is a result from successful collaboration with many scientists. I especially thank Dr. Yu Fen Huang, Dr. Joshua Smith, and Dr. Colin Medley for introducing me into the nanomaterial felids guiding, and training me in the early sta t es of r esearch. I also would like to express my appreciation to Dr. Kathryn Williams, Dr. Joseph Phillips, Dr. Ibrahim Shukoor, Dr. Kwame Sefah, Dr. Youngmi Kim, Dr. Arthur Hebard and Dr. Swadeshmukul Santra f or many helpful research discussions, experimental he lp, and all suggestions on different projects. I really appreciate Dr. Yan Chen, Dr. Zhi Zhu and Hui Wang as being supportive friends and colleagues, as well as their encouragement during my difficult times. It has been a great experience to spend several years in the Tan research group with both previous and current group members. I would like to thank Dr. Marie Carmen

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5 Estevez, Dr. Jilin Yan, Dr. Xiaoling Zhang, Dr. Liu Yang, Dr. Heipeng Liu, Dr. Jennifer Martin, Dr. Huaizhi Kang, Dalia Lopez Colon, Xiangl ing Xiong, Dimitri Van Simaeys, friendship. I also would like to express special thanks to my friends Jean Palmes, Preeyanan Sriwanayos, Aay Pawinee, Pattaraporn Vanachayangkul, Wit cha Imaram, and Werachart Ratanatharathorn for valuable friendship. I deeply appreciate them for being supportive during my difficult times, and sharing the good moments. Finally, without my family, especially my parents, Somkuan and Wasana none of this w ould have been possible. I sincerely thank for their continuous dedication, endless support, and constant encouragement. Their unconditional love and great attitude make me become who I am and keep me always moving on.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............. 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ .................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ..... 16 Impact of Nanoparticles for Bio analytical Applications ................................ ........... 16 Magnetic Iron Oxide Nanoparticle Fundamentals ................................ .................... 18 Synthesis of Iron Oxide Nanoparticles ................................ .............................. 19 Iron Oxide Nanoparticle Surface Modification and Bioconjugation .................... 21 Size, Shape, Magnetic properties and Surface Characterization ...................... 23 Magnetic Nanoparticles for Magnetic Resonance (MR) Based Assays ................... 24 Basic Concept of Magnetic Resonance Imaging (MRI) ................................ ..... 26 Magnetic nanoparticles (MNPs) and T 2 relaxation ................................ ...... 27 Magnetic Relaxation Switches (MRSw) ................................ ............................ 30 Mechanism of magnetic relaxation switches (MRSw) ................................ 31 Instrumentation for MRSw ................................ ................................ .......... 32 Nucleic Acid Probes ................................ ................................ ................................ 33 Selective Biorecognition Elements ................................ ................................ .... 34 DNA Molecular Machines ................................ ................................ .................. 37 DNA switches ................................ ................................ ............................. 37 DNA walkers ................................ ................................ ............................... 38 DNA motors ................................ ................................ ................................ 39 Fluorescence Techniques for Signal Transduction ................................ .................. 40 Jablonski Diagram ................................ ................................ ............................ 40 Fluorescence Quenching ................................ ................................ .................. 41 Fluorescence Resonance Energy Transfer (FRE T) ................................ .......... 43 2 M OLECULAR PROFILING OF CANCER CELLS USING APTAMER CONJUGATED MAGNETIC NANOPARTICLES ................................ ..................... 54 Introduction ................................ ................................ ................................ .............. 54 Experimental Section ................................ ................................ ............................... 56 Synthesis of DNA Aptamers ................................ ................................ .............. 56 Aptamer Nanoparticle Conjuga tion ................................ ................................ ... 57 Cells and Culture Conditions ................................ ................................ ............. 57 Determination of Conjugated Nanoparticle Cell Specific Targeting .................. 58 Sample Assays using Spin Spin Relaxation Time Measurement ...................... 59

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7 Results and Discussion ................................ ................................ .......................... 59 Magnetic Nanosensor Preparation ................................ ................................ .... 59 Specificity of ACMNPs ................................ ................................ ...................... 60 Cancer Cells Detection using Spin spin Relaxation Time Measureme nt .......... 60 Detection in Complex Biological Media ................................ ............................. 61 Detection in Mixture of Cells ................................ ................................ ............. 62 Cancer Cells Profiling ................................ ................................ ........................ 63 Conclusion ................................ ................................ ................................ ............... 64 3 DETECTION OF LYSOZYME USING MAGNETIC RELAXATION SWITCHES BASED ON APTAMER FUNCTIONALIZED SUPERPARAMAGN ETIC NANOPARTICLES ................................ ................................ ................................ .. 72 Introduction ................................ ................................ ................................ .............. 72 Experimental Section ................................ ................................ ............................... 74 Synthesis of DNA ................................ ................................ .............................. 74 Lys Nanosensor Preparation ................................ ................................ ............. 74 Magnetic Relaxation Measurement ................................ ................................ ... 75 Cell Samples Preparation ................................ ................................ ................. 75 Results and Discussion ................................ ................................ ........................... 76 Clusters Formation of Lys Nanosensor ................................ ............................. 77 Lys Induced Disassembly of Nanosensors ................................ ....................... 77 Selectivity and Specificity of Lys Nanosensors ................................ ................. 78 Quantitative Analysis of Lysozyme ................................ ................................ ... 79 Analysis of Lysozyme in Cell Lysates ................................ ............................... 80 Concl usion ................................ ................................ ................................ ............... 81 4 MAGNETICALLY DRIVEN SINGLE DNA NANOMOTOR ................................ ....... 87 Introduction ................................ ................................ ................................ .............. 87 Experimental Section ................................ ................................ ............................... 88 DNA Molecular Probes Synthesis ................................ ................................ ..... 88 Molecular Probes Assays ................................ ................................ .................. 89 Immobilization of DNA Molecular Probes on Glass Surface ............................. 90 Construction of DNA Nanomotor ................................ ................................ ....... 91 Results and Discussion ................................ ................................ ........................... 92 Signal Enhancement of the DNA Molecular Probes by Dnase I Cleavage ........ 93 DNA Nanomotor Driven by Magnetic Field ................................ ....................... 94 Conclusion ................................ ................................ ................................ ............... 98 5 SUMMARY AND FUTURE DIRECTIONS ................................ ............................. 105 The Developm ent of DNA Conjugated Magnetic Nanoparticles for Bio Analytical and Biomedical Applications ................................ ................................ .............. 105 Future Directions ................................ ................................ ................................ ... 107 LIST OF RE FERENCES ................................ ................................ .............................. 111

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8 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 120

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9 LIST OF TABLES Table page 2 1 Representative ce ll lines and binding affinities of their selected aptamers. ......... 71 3 1 List of DNA sequences. ................................ ................................ ....................... 86 3 2 The formation of magnetic clus ters upon hybridization between complementary strands ................................ ................................ ....................... 86 4 1 DNA hairpins and biotinylated li nker ................................ ................................ 104 4 2 The average signal en hancement of na nomotor after repeating several cycles. ................................ ................................ ................................ ............... 104

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10 LIST OF FIGURES Figure page 1 1 Nanoparticle size effects on magnetism. ................................ ............................. 45 1 2 Scheme representing the St ber process ................................ ........................... 45 1 3 Scheme representing a water in oil (W/O) microemulsion system. ..................... 46 1 4 Representative bioconjugation schemes for attaching biom olecules to NPs for bioanalysis ................................ ................................ ................................ ..... 46 1 5 Principal of magnetic resonance imaging (MRI) ................................ .................. 47 1 6 The role of magnetic nanoparticles as contrast agents ................................ ....... 47 1 7 Magnetic nanoparticle size and dopant effects on mass magnetization (Ms) and MRI contrast enhancement. ................................ ................................ ......... 48 1 8 Magnetic nanoparticle aggregation effects on MRI. ................................ ............ 49 1 9 Principle of magnetic relaxat ion switches (MRSw). ................................ ............. 49 1 10 DNA and RNA structures formed by phosphodiester linkages. ........................... 50 1 11 Structure of DNA double helix and base pairing. ................................ ................. 50 1 12 Schematic representation of DNA aptamer selectio n using the cell SELEX strategy ................................ ................................ ................................ ............... 51 1 13 DNA nanomachines ................................ ................................ ............................ 52 1 14 Jablonski diagram. ................................ ................ 53 1 15 Schematic representation of FRET donor and acc eptor spectra. ........................ 53 2 1 Schematic representation of magnetic nanosensor for cancer cell detection and profiling ................................ ................................ ................................ ......... 66 2 2 The specifi c recognition of ACMNPs to their target cells ................................ ..... 67 2 3 The optimization of magnetic nanosensor concentration in PBS. ....................... 67 2 4 sgc8c ACMNPs as a nanosensor for the detection of CEM cells in PBS ............ 68 2 5 The dynamic detection range of Ramos cells using magnetic nanosensor in PBS ................................ ................................ ................................ .................... 68

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11 2 6 sgc8c ACMNPs as a nanosensor for the detection of CEM cells in Fetal Bovine Serum (FBS) ................................ ................................ ........................... 69 2 7 The detection of target CEM cells in plasma and whole blood ............................ 69 2 8 The detection of target cells in complex biological media mimics detection in real clinical samples ................................ ................................ ............................ 70 2 9 The detection of mixed cell samples for th e development of rare tumor cells detection ................................ ................................ ................................ .............. 70 2 10 Profiling of cancer cells. ................................ ................................ ...................... 71 3 1 Schematic representation of the magnetic nanosensor for Lys detection based on MRSw. ................................ ................................ ................................ 82 3 2 Effect of incubating MNP Lys aptamer with MNP Linker at high concentration overnight ................................ ................................ ................................ ............. 82 3 3 A gradual change of 2 upon the addition of Lys ................................ .............. 83 3 4 Selectivity of the Lys nanosensor. ................................ ................................ ....... 83 3 5 Specificity of the Lys nanosensor. ................................ ................................ ....... 84 3 6 Changes in T2 relaxation time with increasing concentrations of Lys. ................ 84 3 7 The detection of Lys spiked human serum using relaxometry m easurements. ... 85 3 8 The detection of Lys spiked human serum using T 2 weighted MR images. ........ 85 3 9 Determination of Lys in cell lys ates. ................................ ................................ .... 86 4 1 Concept of magnetically driven DNA nanomotor ................................ ................. 99 4 2 Signal enhancement of DNA hairpins in solution after the additi on of DNase I. 100 4 3 Signal enhancement of DNA hairpins on the glass surface of micro channel after DNase I addition ................................ ................................ ....................... 101 4 4 C ycles of closed open state from DNA hairpins with 1.0 m magnetic beads. 101 4 5 Cycles of closed open state from DNA hairpins with 2.8 m magnetic beads. 102 4 6 Cycles of closed open state from DNA hairpins with 0.2 m magnetic beads. 102 4 7 Magnetic beads effect on the DNA nanomotor ................................ ................. 103

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12 5 1 Schematic representation of DNAzyme based magnetic nanosensor for Lys detection. ................................ ................................ ................................ ........... 110

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13 ABSTRACT OF DISSERTA TION PRESENTED TO TH E GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLME NT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DNA CONJUGATED MAGNETIC NANOPARTICLES FOR BIO ANAL Y TICAL AND BIOMEDICAL APPLICATIONS By Suwussa Bamrungsap August 2011 Chair: Weihong Tan Major: Chemistry Magneti c nanoparticles (MNPs) can be synthesized to have various sizes, shapes, and composition s providing distinctive magnetic, electronic and optical properties compared to their bulk counterparts. With the dimension similar to biomolecules, MNPs can be engine ered to have specific properties and can be used to pursue an in depth understanding of biochemical processes. Due to the strong and specific base pairing of nucleic acids, as well as their ability to form self assembled structural motifs, DNA conjugated M NPs can serve versatile functions including the investigation of biological activities and the construction of molecular machines. First, a magnetic nanosensor prepared by aptamer conjugated MNPs (ACMNPs), was developed based on magnetic relax ation switch es (MRSw) mechanism T he detection can be achieved by switching between dispersed and aggregated of MNPs upon binding with target cell s resulting in a change in proton spin spin relaxation times 2 ) Specificity and sensitivity of nanosensors were capitalized by the ability of aptamers to specifically bind their targets and the large surface area of MNPs to accommodate multiple binding events. The detection as few as 10 target c ells in buffer

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14 and 100 cells were obtained in biological samples Furthermore, by using an array of ACMNPs, various cell types can be differentiated through recognition patterns thus creating a cellular molecular profile and allow ing clinician s to accurat ely identify cancer cells at the molecular and single cell level. Additionally, a nanosensor based on MRSw was also demonstrate d for protein detection using Lysozyme (Lys) as a model protein. The nanosensor system consisted of MNPs aggregates prepared by the hybridiz ation of MNPs conjugated with either aptamers or linker DNA that could hybridize to the extended part of Lys aptamers le a d ing to the cluster formation. In the presence of Lys the aptamers bind with their targets, resulting in the disassembly of the clusters, and providing a change in T 2 relaxation time. The high selectivity and s ensitivity in nanomolar range of the detection was achieved in both buffer and human serum. The analysis of Lys in cancer cell lysates was also performed to validate t he detection in real clinical samples. Another area of investigation was the development of a magnetically driven DNA nanomotor. In particular, DNA hairpins were conjugated to magnetic particles and the entire conjugation was immobilized to a solid suppor t. The DNA hairpin can be The applied magnetic field triggered the change of molecular conformation or drove the movement of molecular probes between closed and open states, which can be observed by the change of fluorescence. By repetitive shrinking and extending movements, DNA hairpins were considered as a nanometer scale motor. In summary, this research focuses mainly on the development of DNA conjugated MNPs for the analy sis of biological targets, and the construction of nanomachine.

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15 Successful outcomes form these investigations will lead to the improvement in bioanalytical science, biomedical applications, and bionanomaterial research.

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16 CHAPTER 1 INTRODUCTION Impact of N anoparticles for Bio analytical Applications Over the past few decades, n anoparticles have received considerable attention in the translation of nanotechnology into advanced biomedical science and other biotechnological fields. Compared to their bulk count erparts, nanoparticles provide drastically different properties including (1) the small size in the sub 100 nanometer range resulting in high surface to volume ratios, (2) the size and shape dependent properties, such as the distinctive light scattering of gold nanoparticle and discrete luminescent behavior of quantum dots, and (3) the enhancement of physical and chemical properties of some materials in the nanoregime. 1 2 Additionally, with the similar dimension to biomolecules including oligonucleotides, peptides, proteins, and cells, nanoparticles can be engineered to construct versatile nano bio hybrid mate rials for the development of bioanalysis tools and the investigation of biological processes 3 4 The demand for ra pid and accurate disease screening has driven the development of novel bioassays having high sensitivity and selectivity with simple detection and sample preparation. The selectivity of the bioanalysis is achieved by molecular recognition properties such a s receptor ligand association, antibody antigen binding and oligonucleotide hybridization. In addition, the sensitivity is directly related to the transduction of the target probe into the reporting signal. Consequently, the design of hybrid materials tha t link molecular probes serving as recognition units and nanoparticles which can generate measurable signal s is very desirable Generally, the most common reporting signal s are based on optical techniques, including fluorescence, colorimetr y and Raman spe ctroscopy. 5 7 However, complex biological

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17 systems generate high background interference by scattering, absorption and autofluorescence, which lower the signal/ noise ratio and limit the sensitivity of detection. Therefore, sample purification and multiple preparation steps are needed re quiring complex instrumentation and time investment Alternative techniques which are light independent are of great interest to overcome these drawbacks. In recent years, considerable effort has been devoted to the design and construction of molecular machines. One of the most attractive materials chosen for construction of nanoscale machines is DNA due to the specific ba se pair formation, sequence programmability and feasible synthesis 8 9 DNA can be assembled to 2D or 3D structure and utilized as dynamic molecules that duplicate mechanical devices such as switches, walkers, and motors 10 12 In particular, DNA nanomotors can be achieved by confor mational change s in DNA induced by the energy input from sources such as DNA fuel strands and ATP molecules. 13 14 However, the addition of fuels results in the generati on of waste products, and the accumulation of waste strands after several cycles of operation results in decrease s motor efficiency. Therefore, alternative energy source such as electromagnetic field s are desired to eliminate the accumulation of waste products and to produce high efficiency nanomotors. The ultimate goal of this dissertation is the development of DNA conjugated magnetic nanoparticles for molecular detection and the construction of DNA n anomachines. The following section will discuss the synthesis, characterization and conjugation of magnetic nanoparticles. A brief discussion of nucleic acid probes as selective recognition elements including aptamer s and the systematic evolution of liga nds by exponential enrichment (SELEX) as well as DNA nanomachines will follow.

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18 Then, the principle of magnetic relaxation switches (MRSw), fluorescence spectroscopy, and fluorescence resonance energy transfer (FRET) will be reviewed. Finally, the overall focus of this dissertation will be outlined. M agnetic Iron Oxide Nanoparticle Fundamental s In bulk materials, magnetic properties can be determined by multiple parameters such as susceptibility, coercivity, composition, crystallographic structure and the presence of vacancies and defects. However, when the size of a magnetic material is reduced into the nanoregime, size is the dominant parameter because the properties of MNPs are strongly dependent on the ir dimension s. 15 16 Bulk magnetic materials posses multiple magnetic domains due to the different alignment of electron spin s generating varying magnetic moment s Wit h decreasing size of MNPs below their critical size they exist as a single domain with spin s align ed unidirectionally (Figure 1 1) However, when the size is further reduced, the thermal energy effect is significant in the nanoregime and it is sufficient to tilt the surface spin s of the MNPs S uch surface spin canting leads to a net magnetization of zero, and this behavior is called superparamagnetism. 17 18 Such particles can be magnetized in the presence of a magnetic field and then return to the original state in the absence of the external field. Superparamagnetic materials, especially iron oxide nanoparticl es (Fe 3 O 4 or Fe 2 O 3 ) are extremely attracti ve in biotechnological research, and t heir crucial characteristics avoid undesired particles aggregation. Thus, iron oxide particles have been used for numerous purposes, including biosensing bioseparation, magne tic r esonance imaging (MRI), drug delivery, hyperthermia therapy, and tissue engineering. 4 19 Typica lly, a siz e smaller than 100 nm and a narrow size distribution are

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19 required for biomedical applications. Therefore, the control of synthesis, surface modification, and biomolecular conjugation are extremely important. Synthesis of Iron Oxide Nanoparticles Iron oxide nanoparticles can be synthesized by various techniques: microemulsions, sol gel reactions, electrochemical methods, flow injection syntheses, and electrospray syntheses 18 20 However, the most common met hods of iron oxide nanoparticle synthesis are chemical coprecipitation and thermal decomposition reactions. The coprecipitation is the simplest way to prepare ir on oxide nanoparticles of magnetite Fe 3 O 4, or maghemite, Fe 2 O 3 Typ ically, they are prepared by aging stoichiometric mixture of ferrous and ferric salt s in aqueous medium according to the reaction Fe 2+ + 2Fe 3+ + 8OH Fe 3 O 4 + 4H 2 O (1 1) In general, complete precipitation of Fe 3 O 4 takes place at pH s between 8 and 14 with a stoichiometric ration of 2:1(Fe 3+ /Fe 2+ ) in a non oxidizing environment. 21 Fe 3 O 4 is not very stable and can be transformed to Fe 2 O 3 in the presence of oxygen as follows Fe 3 O 4 + 2H + Fe 2 O 3 + Fe 2+ + H 2 O (1 2) The advantages of the coprecipitation method are ease of synthesis and the capability for large scale production However, the drawback of this technique i s the non uniformity of the particles. During the process, two steps are involved: the nucleation occurs when the concentration of reactive components exceeds saturation and the subsequent slow growth of nuclei. In order to ac hieve monodisperse particles, nucleation should be avoided during the growth phase of nanocrystals. Generally, the size s and shape s of iron oxide nano particles can be tuned by adjusting the Fe 2+ /Fe 3+

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20 ratio, pH, ionic strength, tempe rature, and nature of the counterions The size of iron oxide particles increase s with the ratio of Fe 2+ / Fe 3+ 22 H igher pH s and ionic st rength s lead to the smaller size of particles because the chemical composition and electrostatic surface charge are determined by those parameters. It has also been found that the nucleation decreases when the temperature is increased 23 Iron oxide naoparticles prepared by the coprecipitation method can be dispersed in either aqueous media or a nonpolar solvent suitable for further surface modification. Superparamagnetic iron oxide nanoparticles can also be prep ared by thermal decomposition of iron organic precursors, such as iron pentaca r bonyl, Fe(CO) 5 iron acetylacetonate, Fe(acac) 3 or iron (III) chloride, FeCl 3 using organic solvents and surfactants. MNPs with a high level of size control and monodispersity can be obtained by the control of reaction conditions, including solvent, temperature, and reaction time. The nucleation steps may be faster than the growths steps at high temperature resulting in a decrease of particle size. However, a long reaction time facilitates the growth steps with other conditions held constant. For example, iron oleate can be formed by decomposition of Fe(CO) 5 in the present of oleic acid and octyl ether at 100C in an argon atmosphere. Then (CH 3 ) 3 NO is added and the solution is r efluxed at 300C for the oxidation st ep. These processes allow the production of highly crystalline and monodisperse ir on oxide nanoparticles with sizes from 4 to 16 nm. 24 High uniformity of iron oxide crystals with sizes ranging fr om 4 to 20 nm can also be achiev ed by a high temperature reaction of Fe(acac) 3 with 1,2 hexadecanediol in the presence of oleic acid and oleylamine. The hydrophobic particles are highly dispersed in organic solvents and can be further transformed into a hydrophilic phase by adding bipolar ligands.

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21 Iron Oxide Nanoparticle Surface Modification and Bioconjugation It is essential to modify iron oxide particle surface s in order to achieve stable magnet ic colloids that do not aggregate in magnetic field s and biological media. Stability of magnetic colloid results from the equilibrium between attractive and repulsive forces in suspension. In theory, interparticle interactions can be divided into four types: 1) van der Waals forces which are short range isotropic attractions, 2) electrostatic forces controlled by salt concentration, 3) dipolar forces generated between two particles, and 4) steric repulsion from coated particles 25 For iron oxide nanoparti cles, the surface contains iron atoms which act as Lewis acid s and can interact with molecules that donate a lone pair of electron. In aqueous solution, iron ions coordinate with water molecules which can be hydrolyzed resulting in hydroxyl groups on the p article surface. The hydroxyl groups allow iron oxide particles to be modified using several types of material s, including polymeric ligands, inorganic materials, and micellar coatings. Various polyme rs have been utilized to coat iron oxide nanoparticles either in situ or post synthesis coatings. In the first approach, nanoparticles are coated during the synthesis and the most common polymer used is dex tran which is a polysaccharide The possible mechanism of dextran adsorption on iron oxide might be the hydrogen bonding between hydroxyl groups on dextran and the particle surface. 26 The advantages of using dextran as a stabilizer are its non toxi city and biocompatibility. For the post synthesis coatings, ligands normally consist of two parts : the region that bind s to particle surface a nd the hydrophilic region that is expose d to aqueous media Polyethylene glycol (PEG) is commonly utilized for the post synthesis coating due to its hydrophilic and biocompatible properties. PEG can be modified with thiol, dopamine, phosphate, or siloxane groups in order to provide iron oxide particles with high stability

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22 and solubility as well as to block non specific ads orption of unwanted molecules on the particle surface. 27 Coating with silica is one of the most common strategies f o r modify ing the surface s of iron oxide nanoparticles. This coating not only provides high stability to particles but also facilitates further surface functionalization. S ilica coated M NPs can be prepared by two general routes: the St ber and reverse microemulsion methods. The St ber method is based on the formation of particles by hydrolysis of a silica precursor (e.g., tetraethylorthosilicate, TEOS) in ethanol media containing water a nd ammonia as the basic catalyst (Figure 1 2) MNPs have been incorporated in to the silica matrix and the coating process can be completed within a few hours. It was observed that lower concentration s of iron oxide nanoparticles induce larger colloids due to the presence of fewer number of particle seeds. 28 29 However, this route always yields large and non unif orm particles. The alt ernative method to prepare silica coated MNP is to use a micro emulsion or water in oil (W/O) microemulsion system, which has three main components: water, oil, and surfactant (Figure 1 3) 30 31 Water nanodroplets formed in the bulk o il phase act as a confined medium for the formation of discrete nanoparticle s. Iron oxide particles are en trapped in the water pool and t he size of particle s can be tuned by controlling the water to surfactant molar ratio (W o ). The microemulsion method takes 24 48 hours to complete and yields monodispersed and highly uniform particles. Another role of surface modification is to incorporate recognition elements, such as, antibodies, oligonucleotides, or peptides that specifically bind with target molecules for biological applications Typically, biomolecules can be functionalized on particle

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23 surface s either by p hysical adsorption or covalent attachment. Physical adsorption involves electrostatic, hydrophobic, hydrophilic, or van de Waals force s to associate the desire d molecules on to the particle surface. In general, covalent attachmen t of biorecognition elements is preferred, not only to avoid desorption from the surface, but also to control the number and orientation of the immobilized biorecognition probes 32 However, covalent attachment requires chemically reactive functional groups on the particle surface includi ng amine, carboxyl, or thiol. These groups can be attach ed to the surface either during or after post coating for particle stabilizati on. After naoparticle s are functionalized, they can act as scaffold s for biological moieties as demonstrated in the scheme (Figure 1 4) F or example, carboxyl modified particle s are suitable for covalent coupling of proteins and other amine containing bio molecules via cabodiimide chemistry Disulfide modified nanoparticle s can be immobili zed onto thiol functionalized particle s by disulfide coupling chemistry Amine modified nanoparticle s can be coupled with a wide variety of haptens and drugs via succinimi dyl esters and iso(thio)cyanates 33 After the bioconjugation step, the nanoparticles s can be separated from unbound biomolecules by centrifugat ion, dialysis, filtration or other laboratory techniques. Size, Shape, Magnetic properties and Surface Characterization Since many properties of nanoscale materials depend on their physical characteristic s it is necessary to define the size, shape, and po lydispersity of nanoparticl es. T ransmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) are the most common techniques that are utilize d for size determination. 34 The EM techniques can provide details about size, shape, and size distribution. However, sample preparation can induce aggregation and

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24 limited information is achieved for the dispersive nature of particles in solution. In addition, dynamic light scattering (DLS) is a common technique used to obtain information about both size s and dispersion of particles in suspension. The hydrodynamic radius corresponding to the sphere and the polydispersity of th e colloid is al so obtain ed. 35 X ray diffraction (XRD) can also be used to indicate crystalline structure s of nanoparticles. The proportion of iron oxide particles can be quantified by the peak intensities of the diffraction pattern compared to the reference peak intensities. Furthermore, the crystal size can also be calculated from the broadening of the XRD pattern. 36 37 Another important p arameter used for particle characterization is the surface charge or zeta potential ( ) which is affected by the environment including, pH and ionic strength. The magnitude of the zeta potential indicates the repulsive force between particles and can be used to predict the stability of a colloid Particles in suspension having high negative or positive zeta potential s tend to repel each other resulting in a low tendency to aggregat e Additionally, zeta potential measurement can be us ed to verify surface m odification or bioconjugation of the naoparticle surface. For example, during the synthesis and modification processes, nanoparticle surface charge will be changed according to the charge of the coordinated materials such as polymers DNA, or proteins. Magnetic Nanoparticle s for Magnetic Resonance (MR) Based Assays The ultimate goal of the development of biosensing platforms is to have far reaching implication in point of care clinical diagnostic s phamarceutical drug development, and proteome research. In order to achieve robust, versatile, and high throughput sensing platforms, the assay methodologies need to meet several

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25 requirements : 1) high sensitivity and specificity, 2) minimize d sample preparation, 3) capability to analyze diff erent types of target molecule s with the same format and instrument ; and 4) feasibility for both single tube and high throughput screening formats. 38 Magnetic nanoparticle based biosensors have gain ed considerable attention due to their unique advantages over other techniques. For example, magnetic nanopartic les can be easily and cheaply synthesi zed and modifi ed. They are physically and chemically stable, as well as biocompatible. Additionally, magnetic nanoparticles h ave strong magnetic properties which are not found in biological systems. With no magnetic b ackground in biological samples, magnetic nanoparticle based sensor s can perform highly sensitive measurements in turbid or obscured sample without any further processing. In contrast, optical techniques are always affected by autofluorescence, scattering, and absorption within samples. To date, a number of biosensors based on magnetic measurement have been developed. Several techniques including superconducting quantum interference device s (SQUID s ), 39 magnetoresistive sensor s 40 41 and Hall sensors 42 directly detect magnetic particles Another technique that has receives consi derable attention is nuclear magnetic resonance (NMR) spectroscopy which can be translated into an imaging technique called magnetic resonance imaging (MRI). Due to their noninvasive character, b oth conventional NMR and MRI have been widely used in medical applications for sample evaluation and characterization. In MRI magneti c nanoparticles are used as contrast agent s, which provide signal enhancement as a result of the interaction s with neighbori ng water protons. Recently, an other magnetic technique has

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26 been developed to produce a new approach for in vitro diagnostic s This new assay, term has led to a new platform for sensors with high sensitivity, selectivity, and feasibility in immuno and molecular diagnostics for point of care detection. Basic C oncept of Magnetic Resonance Imaging ( MR I) When the nuclei of protons are exposed to a strong magnetic field ( B 0 ) their spins align either parallel or antiparallel to the external magnetic field with a small excess al ligned parallel The aligned spin s precess with a net magnetic moment of M and a Lamor frequency of 0 = B 0 ( is a gyromagnetic ratio of proton = 2.67 x 10 8 rad s 1 T 1 ) as shown in Figure 1 5 A When a resonance radio frequency (RF) pulse is introduced p erpendicular to B 0 the protons absorb energy and are excited to the antiparallel state which has high energy. Net magnetization M as a vector has the components of M z and M xy (Figure 1 5 B ) By removal of the RF, the excited spins gradually relax to their i nitial or lower energy state. There are two different relaxation pathways: longitudinal or T 1 relaxation involves the recovery of magnetization ( M z ) to the initial state by transfering energy from the excited state to its surrounding (lattice) and transve rse or T 2 rela xation involves the disappearances of magnetization in the perpendicular plane ( M xy ) from the loss of phase coherence of the nuclei spins in the xy plane due to spin spin interaction s (Figure 1 5C and D ). The relaxation processes are expresse d as follows: M z = M (1 e t/T1 ) (longitudinal) (1 3) M xy = M 0 t + )e t/T2 (transverse) (1 4) where M is the net magnetization, 0 is the Lamor frequency of protons, and T 1 and T 2 are the longitudinal and transverse relaxation time s respectively.

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27 The phase coherence in the xy plane disappears due to the di fference s of magnetic field s experienced by protons. The magnetic field difference s are generated by inhomogeneit ies in the applied field due to the system imperfection s, which can be reduce d by shimming coils, and the usage of spin echo sequence to revers e this effect. Furthermore, local magnetic field gradients can be induced by the differences in magnetic susceptibility between different tissues. Biological organs and tissues have a variety of aqueous environments in both density and homogeneity and the se generate contrast s and reveal anatomical information. MRI records these relaxation process es and reconstructs 3 D gray scale images. Areas with shorter T 1 relaxation are imaged as brighter contrast in T 1 weighted MRI, while areas with shorter T 2 relaxat ion are reported as darker contrast in T 2 weighted MRI. However, in some cases, the contrast between tissues is unable to provide precise imaging information due to the small differences with d to enhance local magnetic field gradients resulting in the high contrast MRI. Magnetic n anoparticles (MNPs) and T 2 r elaxation MNPs not only play a role as contrast agents to enhance contrast differences of biological targets from tissues but they also a ct as carrier s for ligands to provi de specific targeting. In an external field B 0 MNPs are magnetized with magnetic moment establishing local dipolar field s and perturbing their surrounding s Such induced field s generate local magnetic inhomogeneit ies and accelerate the dephasing rate of the Lamor precession of surrounding water molecules. Consequently, the T 2 relaxation time is reduced (Figure 1 6 ). According to the outer sphere theory, the relaxivity R 2 (1/T 2 ) of MNPs containing water solution is expr essed as follow: 43

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28 where M is the molarity of MNPs, r is the MNP radius, D is the diffusion coefficient of water, j n ( ) is the spectral density function, is transverse component of magnetic moment of the i is the gyromagnetic ratio of protons, N A and s and i a re the Lar mor angular precession freq uencies of the MNPs and the water proton magnetic moments, respectively. The spin spin or T 2 relaxation time is inversely proportional to the square of the magnetic moment () of M NPs as demonstrated in equation 1 5. As mentioned previously these values can be modulated by controlling characteristic s of the MNPs such as size, shape, composition, and crystallinity. Smaller particles with higher surface to volume ratio s possess weaker magnetic moment due to the significant canting effect on the surface. Su ch size effects have been demonstrated by Cheon et. al. for Fe 3 O 4 iron oxide nanoparticles. 16 It was reported that the mass magnetization value (M s ) increase from 68 to 191 emu/g(Fe) as th e particle size in creases from 6 to 12 nm (Figure 1 7 A ). Composition al effects also have been demonstrated by doping with different transition metal s i ncluding Mn Co, and Ni (Figure 1 7B ). For example, the replacement of octahedral site of Fe 2+ in 12 nm iron oxide particles with Mn 2+ Co 2+ or Ni 2+ results in a change of M s to 110, 99, to 85 emu/g, respectively. Higher magnetization of MNPs results in stronger contrast effects and larger R 2 rel axivity. As shown in Figure 1 7B doped iron oxide particles p osses a variety of R 2 ranging from 152 to 172, 218, and 358 mM 1 s 1 for Ni, Co, Fe, and Mn, respectively.

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29 When MNPs are in close proximity and agglomerate, their magnetic moments are coupled and generate stronger local magnetic field s (Figure 1 8 ). The ma gnetic coupling effect directly influences the MR contrast by accelerating the proton dephasing rate. The increase of R 2 relaxivity is expressed as: 43 where is magnetic moment of MNPs, N g is number of MNPs in agglomerates, L(x) is Langevin function, N A C A is the concentration of agglomerates, R A is radius of an agglomerate, and D is the water diffusion coefficient. According to equation 1 6 R 2 is proportional to and N g Consequently, the magnetic co upling effect significantly improves the sensitivity of magnetic resonance sensing where the MR signal contrast changes depending on the relative concentration of the assembled states of MNPs. Base d on this strategy, MNPs have been extensively used in can cer research. It is well known that nanoparticles can be accumulated spontaneously in tumor sites by the enhanced permeability and retention (EPR) effect, which enhances the accumulation of nanoparticles in tumor tissues that have abnormal blood vessels. 44 As a consequence, MNPs have been successfully used to image tumors without any targeting probes, called passive targeting 45 However, in order to improve imaging efficiency, surfaces of MNPs are conjugated to activ e targeting probes such as antibodies, proteins, and aptamers. For example, iron oxide nanoparticles are conjugated with specific antibodies to image tumors by the interactions with surface protein receptors that are overexpressed in gliomas 46 breast, 47 and colon cancers. Moreover, mul tifunctional nanoplatforms fabricated by co mbining various materials to perform different functions allow multimodal imaging, i ncluding simultaneous diagnosis

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30 and therapy. For example, Fe 3 O 4 Au dumbbell particles were applied to cancer targeted MR a n d reflection imaging. 48 Magnetic Relaxation Switches (MRSw) Recently, Weissleder and coworkers demonstrated MR based assays called Magnetic Relaxation Switches (MRSw) which allow detection by the change of T 2 ind uced by magnetic particles. The principle of MRSw a ssays is demonstrated in Figure 1 9. As illustrated, magnetic particles switch between the disp ersed and aggregated states when the target molecule bind w surfa ces. T he presence of multiple ligands on particle s induces multivalent affinity, resulting in multiple interactions between the conjugates and their corresponding target s. The change of spin spin or T 2 relaxation time is associated with the degre e of aggregation similar to convention al MRI. Magnetic particles used for MRSw assays are either MNPs or micrometer sized magnetic particles (MMPs). Since the dispersed and aggregated states of magnetic particles can be reversed by external stimuli such as pH, salt concentration, and concentration of competing analytes, the se assays are referred to as relaxation switches. The MRSw provides various unique properties (1) Because detection is not dependent on light (no fluorescence, absorbance, turbidity, o r chemiluminescence), analytes interfering with light do not affect the assay, and the experiment can be carried out in turbid and heterogeneous samples. (2) The washing step is not needed to remove unbound analytes, thereby minimizing the analysis time. ( 3) The assay does not require surface immobilization of biomolecules on a glass slide, thus minimizing the complexity of the preparation steps. (4) The assay is flexible and can detect various

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31 kinds of biomolecular interactions, including protein DNA inter actions, protein small molecule interactions, and protein protein interactions. Mechanism of m agnetic r elaxation s witches (MRSw) In MRSw assays, both MNPs and MMPs can be used to provide different T 2 relaxation time s for their respective dispersed and agg regated states. However, for Type I, MNP based system s T 2 decreases with the aggregation, while for Type II, MMP based system s T 2 increases with aggregation. 49 The basis of this can be described using the theory termed outer sphere relaxation theory. In this theory, two main parameters, D w and t D, are considered. D w is the difference in angular frequencies between the local field experienced by protons at the surfa c e of particles and in the bulk, and t D is the translation al diffusion time of water around the sphere of an aggregate. These parameters are given by: D w = m 0 M /3 (1 7) t D = R a 2 /D (1 8) where m 0 is the vacuum magnetic permeability, M is the particle magnetization, proton gyromagnetic ratio R a is the sphere radius and D is water diffusion coefficient. The outer sphere theory is applied when D w t D < 1, referred to as the motional average condition. 50 When MNPs aggregate (Type I), smaller numbers of larger magnetic field inhomogeneities are produced compare to the dispersed state. The larger homogeneities are more effective in the dephasing of water protons. In this condition, the relaxation rate R 2 (1/T 2 ) increases as th contrast, when MMPs aggregate (Type II), there are fewer clusters compared to the Type I case and spaces between them are large. Consequently, many water protons fail to diffuse in and out this inhomogeneit ies field during the measurement time. In this

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32 condition, the motional average is not fulfilled and t he relaxation rate decreases with the formation of aggregates. Here D w t D > 1 referred to diffusion limited case. MRSw based biosensors achieve the selectivity and specifi city for desired molecular targets by attachment of binding moieties such as antibodies or aptamers to magnetic particles. Therefore, the basis of the transition between dispersed and aggregated states is the affinity of ligands on the s for specific analytes. The density of ligands can be optimized to achieve fast reaction rates and high detection sensitivit ies 51 As in conventional MRI, o ther parameters such as particle types, sizes, and concentration a lso influence the change of T 2 Instrumentation for MRSw MRSw based sensors can benefit a variety of application s including home and clinical diagnostics, proteome research, bio warfare, and i ndustrial analysis. Such applications require simple, automated, robust, and high throughput instrumentation. A k ey component for facilitating successful applications is tailoring the processing and detection platforms. Currently, most MRSw assays in labor atory and industr ial scenarios depend on bench top relaxometers. High throughput MRSw assays also have been demonstrated using 384 well plates and an MR scanner for multiplex detections. 52 However, the bench top relaxometer and MR scanner are still impract ical for point of care detection due to their high cost and bulkiness. Recentl y, miniaturized MR relaxometer s have been fabricated by Lee and coworkers. 53 54 The system consists of a small palm sized permanent magnet and on board NMR electronics and planar microcoils with integ rated microfluidic channels. M ultiplexed detection of biomarkers was demonstrated for high thr oughput MRSw assays.

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33 MRSw based biosensors offer unique advantages over ot her techniques, such as a simple assay format, feasibility in biological detection without a separation or amplification step, high throughput capacity, and board applications to de tect and profile different types of targets. Taking these advantages, this technology has potential applications in biomedicines, cancer biology and proteomic research. Through the development of the chip based MR devices, clinic and other point of care di agnostics are possible. Nucleic Acid Probes Nucleic acids play important roles in transferring genetic information through generations and act as key e lements for proliferation. The main components of nucleic acid s consist of several nucleotides sharing t he same backbone structure but different bases (Figure 1 10 ). Various sequences generated from different assemblies of bases contribute to the specific interaction with complementary oligonucleotides forming a double helix structure. Furthermore, structura l folding of single stranded oligonucleotides provides molecular recognition abilities for a variety of biolo gical targets. Particularly, Watson Crick type hydrogen stacking bonds, and hydrophobic interactions enable the desi gn of molecular probes for signaling biomolecular interactions. Compared with traditional recognition, such as host guest chemistry, or protein interaction s, molecular recognition using nucleic acid s is flexible, and can b e e as ily modified. Consequently, v arious types of biosensors and medical diagnostic tools have been developed based upon the special recognition properties of nucleic acid s 55 58 Especially, when com bined with the extraordinary properties of nanoparticles, theses h ybrid materials demonstrate broa d application as powerful molecular recognition tools. 3 59 60

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34 In addition, nucleic acid s, especially DNA, are ideal building block s for the design and assembly of nanostructures due to the strong and highly specific base pairing of A T and G C interactions (Figure 1 11 ). Besides duplex formation, self assembled structural motifs of DNA are also available, such as G quadruplex es i m otif, or hairpin structure s 61 62 The diversity of structural patterns allo ws use of DN A as templates for the bottom up construction of 2D and 3D nanostructures. 63 64 Beyond the use of DNA for nano assembly, DNA can be utilized as dynamic molecules that duplicate mechanical devices e.g., switches, tweezer s, and motors. 65 68 Because these DNA nanomachines have the benefit s of precise controllability, biocompat ibility, and reproducibility, they can be utilized for the development of synthetic molecular machines. The following section s focus mainly on the use of nucleic acids as selective biorecogniti on elements. In particular, specific oligonucleotide s termed aptamer s and the selection process called Systematic Evolut ion of Ligands by Exponential enrichment (SELEX) will be discussed as well as DNA probes which can be designed, and engineered as molecular machines Selective B iorecognition E lements Several diseases, especially cancer, are associated with specific bioma rkers. The key to develop diagnostic and therapeutic tools is an understand ing of the molecular recognition of disease specific biomarkers. Since potential biomarkers encompass different types of molecules ranging from glycolipids to proteins, it is neces sary to identify molecular probes that are able to bind selectively with these molecules. However, long period s of time and considerable effort s are required for the identification and separation of biomarkers and their corresponding probes. The two most i mportant

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35 classes of biorecognition elements utilized as molecular probes are antibodies and aptamers. Antibodies are proteins produced by the immune system when triggered by foreign proteins or microorganisms defined as antigen s 69 Typically, antibodies consist of two basic structural units called the heavy and light chains. However, the antigen binding sites of each type of antibody are very unique and allow sel ective bind ing with a particular epitope on an antigen. Generally, antibodies can be generated by injecting antigen into animals and isolating the antibodies from their blood. After isolation and purification, antibodies are commonly used to identify, loca te, and separate both intracellular and extracellular proteins. They are also utilize d to differentiate cell types according to the expressed proteins. However, the application of antibodies as molecular recognition elements sometimes is limited due to the ir lack of reproducibility, sensitivity to environment al condition s difficulty in chemical modification, and s hort shelf life. Recently, a new type of selective recognition ligand s termed aptamer s ha s been introduced to identify wide variety of targets. A ptamers are single stranded oligonucleotides that can selectively recognize target cells, proteins, peptides small molecules, and ions. 70 71 The binding affinities of aptamers to thei r targets are comparable to those of antibodies and range from 10 12 M to 10 8 M. The high specificity of aptamers which can distinguish even homologous proteins, derives from their complex three dimensional structures. Aptamers have shown great promise in molecular recognition for diagnostic and therap eutic applications Moreover, aptamers

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36 have advantages over antibodies due to their reproducibility, ease of synthesis and modificatio n, low toxicity or immunogenicity, and long shelf life. A ptamers can be identified by an in vitro selection process called SELEX (Systematic Evolution of Ligands by Exponential enrichment ). 72 73 The potential sequences are selected from a pool of random sequences of synthetic DNA or RNA through many rounds of selective binding to the targets. Typically, aptamers have been selected using purified proteins or molecules as targets Recently, the Tan research group has developed a novel cell based aptamer process called cell SELEX. 58 74 Instead of using a single target molecule, whole cells are used as targets S elect ion of aptamers that recognize target cells but do not bind to control or non target cells provides several advantages. First, it is not necessary to know specific targets on the cell surface. As an alternative, different cell types are used in the selection process, so aptamers obtained from cell SELEX can be applied to differentiate different types of cells, especially cancer cells. Secondly, since the cell membrane surface is very complex and contains a large number of potential targets, aptamer s can be generated for different types of molecules, which may be express ed in different types and stages of disease s. T hus the select ed aptamers may be useful for the biomarker discovery or the study of disease development Furthermore, as live cells are used during the selection process, all target molecules are in their natural ly fold ed structure s Therefore, the ge nerated aptamers will recognize the actual conformation s of targets an important capability for in vivo applications The cell SELEX process starts with the selection using a large pool of library sequences ( 10 13 10 16 single stranded DNA oligomers) agai nst target or positive cells

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37 (Figure 1 12 ). Subsequently the negative or non target cells are used to exclude oligomers with non specific interaction s making the selected sequences specific to the target cells. The selected sequences are washed and purif ied in order to retain only the ones binding tightly to the target cells, and eliminate unbound or weakly bound sequences. Subsequently, the bound sequences are recollected and amplified by the polymerase chain reaction (PCR) for the next round of selectio n. After several rounds of repetitive selection, a group of aptamers will be obtain ed and further tasted for their affinity and specificity. DNA Molecular M achines Besides the fascinating properties in selective recognition of specific targets, DNA has al so gain ed attention in material science and nanotechnology. Based on the structural flexibility, and specific base pair formation, DNA is programmable and designable, and thus can be used as a structural building block Pioneered by Seeman et.al. in the 19 80s, 2D and 3D DNA nan ostructures have been assembled. 8 63 75 DNA ha s also been converted in to dynami c molecules that can perform nanoscale movements. Although, protein s are the naturally selected material s for motions in living organism, the simplicity of structure, d iversity of self assembly, and automated sy nthesis make DNA the most promising molecule that can duplicate machine functions. DNA based nanomachines can be delivered into several categories depending on their motions as switches, walkers, or motors. DNA s witches A m olecular switch or actuator is o ne of the simplest nanodevices which can switch between two conformations. One of the original DNA switches consists of double stranded DNA with the sequence (CG) n and can be flipped from the normal

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38 right handed helix (B DNA) to the left handed conformatio n (Z DNA ). This conformational change i s triggered by high salt concentration and low temperature (Figure 1 13 A). 76 77 The transition could be tracked by F rster Resonant Energy Transfer (FRET) since a reporter fluorophore was intercalated in each tile. Instead of environmental changes, the addition of DNA strands can also induce the switching of DNA nanostructures. A pair of DNA tweezers with two rigid do uble stranded arms was constructed (Figure 1 13 B). 10 The additional single stranded DNA can bind with the extended tails of both arms and transform the DNA tweezers from the the turning i t to the in itial open state DNA w alkers One of the most sophisticated tasks of DNA nanomachine design and construction is the contr ol of linear motion in a defined direction. To achieve this goal, a nother type of molecular device termed a DNA walker was created. Typically, a DNA walker is temporarily bound to a nucleic acid track. By sequential addition of DNA strands, dissociation and re association of the walker to the next single stranded a nchor occurred (Figure 1 13 C). 12 By appropriate design of the hybridizing nucleic acid allows stepwise translocation in unidirectional movement. Labeling of the footholds with a fluorophore and quencher facilitates the real time visualization of the and the movement can also be further confirmed using gel electrophore sis.

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39 DNA m otors Biologica l motors that use free energy fro m hydrolysis of adenosine tri phosphate (ATP) to drive the movement can move with very fast speeds ( up to 60 ms 1 ) and long travelling distances ( up to 1m ) 78 Inspired by biological motors such as myosin, kinesin, and dynein that move along cytoskeleton networks, DNA nanomotors have been constructed. Previously, three different energy sources for synthetic DNA nanomotors including hydrolysis of ATP and the DNA backbone as well as DNA hybridization, have been explored. Yin and co workers demonstrated a na nomotor that passes its cargo autonomously from one anchor to the next by enzymatic li gation and hydrolysis (Figure1 13 D). 13 The designed sequenc es were recognized by restriction enzyme s to provide uni directional movement and prevent dissociation of the cargo from the track. In the mean time, DNA nanomotors driven by the catalysis of DNA hybridization as an energy s ource were developed (Figure 1 13 E). 79 Generally, thei r operating cycles involve a conformational change triggered by the addition of fuel DNA which is later removed by hybridization with the complementary anti fuel strands. Th e duplex between fuel and anti fuel strands are waste products generated in ever y cycle of operations. The entire processes are driven by the decrease in free energy on forming additional base pairs much like ATP hydrolysis. 80 Furthermore, various DNA nanomotors have been developed using alternative energy sources such as ions, protons, light, and small molecules. 81 84 In addition to the construct ion of stable, powerful, and simple DNA nanodevices, the most important requirement is to achieve machi nes that can do practical work. C urrent ly DNA has been proven to be a po tential material for the realization of synthetic molecular machines. DNA devices ha ve been de sign ed to perform functions such as

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40 sensing molecules, directing chemical reactions, driving objects, and controlli ng released molecules. A future accomplishment cou ld be the combination of a DNA computer, DNA nano mechanics, and DNA nano electronics, etc. The further development of this field may have great impact in nanobiotechnology and biomedical applications. Fluorescence Techniques for Signal Transduction Du ring the few past decades, fluorescence has played significant roles in biological sciences. Fluorescence spectroscopy is considered to be a primary research tool in biotechnology, biomedical diagnostic s and nanotechnology. Because of its high sensitivity ease of sample handling, and multiplexing capability, fluorescence is widely used for signal transduction as well as cellular and molecular imaging. Fluorescence results from a multi stage process involving fluorescent substances called fluorophores. J ablonski Diagram The processes involved in fluorescence are illustrated by the Jablonski diagram 85 (Figure 1 14 ) which is named after Professor Alexander Jablonski. In particular, the singlet ground, first, and second electronic states are depicted by S0, S1, S2, respectively, while T1 stands for the triplet state. Each of t hese electronic energy levels consists of discrete vibrational energy level s with vibrational quantum numbers 0, 1, 2, etc. A fluorophore is typically excited by photons to a higher vibrational level of either S1 or S2 depending on the magnitude of the ab sorbed energy. The excitation process happens in a ver y short time (ca 10 15 s ). After light ab sorption, some molecules in excited state, S1 for example, rapidly relax to the lowest vibrational level of S1. This process is called internal conversion which g enerally occurs within 10 12 s. In this process, energy is transferred as heat by collisions with surrounding molecules. The

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41 excited molecules can t h en relax from the lowest energy vibrational state of S1 to ground state S0 with emission of photons referred to f luorescence. The average time for a molecule to stay in an excited state is referred to as the fluorescence lifetime, t ypically about 10 8 s. Due to the closely spaced vibrational energy level s of the ground state coupled with thermal motion, a wide ra nge of photon energies is produced during emission. Consequently, fluorescence is normally observed as emission over a band of wavelengths instead of a sharp line. Most fluorophores can repeat excitation and emission up to hundreds or thousands times befor e the excited state molecules are destroyed. Molecules in the S1 state can also undergo a spin conversion to the first triplet state T1 by a process called intersystem crossing. The relaxation from T1 to S0 with emission of photons is referred to as phos phorescence. Generally, the transition from the triplet excited state to the singlet ground state is forbidden. Therefore, rate constants for phosphorescene are several orders of magnitude smaller than those of fluorescence. Additionally, a small fraction of energy is always lost during vibration al relaxation resulting in an energy difference between the absorbed and emitted energy. Thus, the emission spectrum of a fluorophore generally appears at longer waveleng th or lower energy than absorption. This phe nomenon called the allows the spectral separation of the excitation and emission of photons for sensitive studies. Fluorescence Quenching Fluorescence quenching refers to any process that decrease s the fluorescence intensity of a sample an d can be caused by a variety of molecular interaction s Basically, there are two main types of quenching: collision or dynamic quenching and static quenching. 85 Both dynamic and static quenching requires molecular contact between fluorophore and quencher.

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42 In the case of dynamic quenching, the quencher diffuses to the fluoroph ore while it is in the excited state. By the contact, the fluorophore relaxes to the ground state without emission of a photon. In general, dynamic quenching occurs without a chemical reaction. The collisional quenching of fluorescence can be described by the Stern Volmer equation: F 0 /F = 1+ k q 0 [Q] = 1+ K[Q] (1 9 ) where F 0 and F are the fluorescence intensities in the absence and presence of quencher, respectively; k q is the bimolecular quenching constant; 0 is the lifetime of the fluorophore in the absence of quencher, and Q is the concentration of quencher. The Stern Volmer quenching constant is given by K= k q 0 One of the best known dynamic quencher s is molecular oxygen which can quench most of fluorophores. Other types of collisional quenchers include hydrogen peroxide, nitric oxide, nitroxide. 86 87 For static quenching, the mechanism relates to th e formation of a nonfluorescent complex between the fluorophore and the quencher. When the complex absorbs light, it returns to the ground state immediately without photon emission. The effect of static quenching is related to the observed fluorescence int ensities by : F 0 /F = 1+ K[Q] = 1+[FQ]/[F][Q] (1 10 ) where K is the complex formation constant; [FQ], [F], and [Q] are the concentrations of the complex, fluorophore, and quencher, respectively. The most effective method to distinguish static and dynam ic quenching is the measurement of fluorescence lifetime. In static quenching, the complex fluorophores are nonfluorescent, and the only observed fluorescence comes from the uncomplexed

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43 fluorophores, which are unperturbed. Therefore, the lifetime remains t he same as before quenching. In contrast, dynamic quenching involves depopulation of excited state resulting in the equivalent ratio between lifetime and fluorescence intensity (F 0 /F = 0 / ). Another additional method to distinguish static and dynamic que nching is measurement of the absorption spectra of the fluorophores. For collisional quenching, only the excited states of fluorophores are affected, hence there is no change in adsorption spectra. While the complex formation of static quenching causes gro und state perturbations, resulting in changes in absorption bahavior Quenching plays a significant role in sensors for molecular biology. A wide variety of analytes including oxygen, ions, and heavy metals have been sensed using the fluo rescence quenchi ng mechanism. 88 89 Fluorescence quenching can also be utilized to determine the fraction and conformatio nal change of proteins. 90 91 Moreover, target DNA has been analyzed through fluorescence quenching using a variety of oligonucleotide probes such as molecular beacon s (MB), ribozyme s and DNAzyme s Many fluorophore/quencher pairs are intercalated in th e se probes to track the conformational changes due t o the binding of analytes. E xamples of well known fluoro phore/quencher pairs include tetramethylrhodamine(TMR)/DABCYL, fluorescein/DABCYL, EDANS/DABCYL, fluorescein/TMR 92 Fluorescence Resonance Energy Transfer (FRET) Fluorecence resonance energy transfer (FRET) is an energy transfer process between two fluorescent molecules. Th is phenomenon occurs between a donor (D) molecule in the excited state and an acceptor (A) molecule in the ground state. 85 Generally, the donor molecules emit at shorter wavelength overlap ing with the absorption spectrum of the acceptor. The energy transfer occurs through a long range

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44 dipole dipole mechanism between the dono rs and acceptors without photon transfer. The rate of energy transfer depends upon the extent of spectral overlap of the donor emission with the absorption spectrum of the acceptor, the distance between the donor and acceptor, the relative orientation of t he donor and acceptor transition dipoles, and the quantum yield of the donor. Typically, the energy transfer occurs within a distance of 100. The FRET efficiency is described as followed: E = R 0 6 /( R 0 6 + r 6 ) (1 11 ) where the F rster radius R 0 is the distance at which energy transfer is 50% efficient, and r is the distance between donor and acceptor. As shown in equation 1 11 FRET is a distance dependent process. Generally, F rster distance s rang e from 20 6 0 which is compara ble to the size of biological macromolecules. Consequently, FRET has been widely used as a spectroscopic ruler 93 to measure the distance between two sites on a macromolecule, especially a protein. FRET has been used to measure conformational change of proteins, distance between a site on a protein and a membrane surface, association between protein subunits, and association of membrane bound proteins. 94 96 Furthermore, FRET is extensively used in oligonucleotide analysis. Due to the ability to form three dimensional structure s of DNA and RNA, FRET is used to track the conformational change s Based on this strategy, a number of molecular probes molecular beacon for example, has been developed using a DNA labeled with a donor/acceptor pair. 97 99 DNA hybri dization and many bioaffinity reactions are observed using FRET measurement s

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45 Figure 1 1 Nanoparticle size effects on magnetism. (A) C anted spins appear on the surface surrounding core magnetic atoms. (B) Relationship between surface to volume rati o and size, canted surface spins, net magnetic moment, and T 2 contrast effect. Figure 1 2 S cheme representing the St ber process

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46 Figure 1 3 S cheme representing a water in oil (W/O) microemulsion system Figure 1 4 Representative bioconjugati on schemes for attaching biomolecules to NPs for bioanalysis. 33

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47 Figure 1 5. Principal of magnetic resonance imaging (MRI). 100 A) Spins align parallel or antiparallel to the magnetic field ; small excel parallel to B 0 produces net magnetization in the Z direction. B) After the RF pulse magnetization of spins changes. C) T 1 relaxation and D) T 2 relaxation Figure 1 6. The role of magnetic nanoparticles as contrast agent s 4 A) T 2 relaxation mode without MNPs. B) T 2 relaxation in the presence of MNPs

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48 Figure 1 7. Magnetic nanoparticle size and dopant effects on mass magnetization (Ms) and MRI contrast enhancement 4

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49 Figure 1 8. Magnetic nanoparticle aggregation effects on MRI 4 Figure 1 9. Principle of magnetic relaxation switches (MRSw ).

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50 Figure1 10 DNA and RNA structure s formed by phosphodiester linkage s Figure 1 11 Structure of DNA double helix and base pairing.

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51 Figure 1 12 Schematic representation of DNA aptamer selection using the cell SELEX strategy. 74

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52 Figure 1 13 DNA nanomachines (A) DNA Switch 77 (B) DNA Tweezers. 10 (C ) DNA Walker. 12 (D E) DNA Motors. 13 79

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53 Figure 1 14 Jablonski diagram. Figure 1 1 5 Sche matic representation of FRET donor and acceptor spectra.

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54 CHAPTER 2 MOLECULAR PROFILING OF CANCER CELLS USIN G APTAMER CONJUGATED MAGNETIC NANOPARTICL ES Introduction Because each type of cancer cell has specific intracellular or extracellular biomarkers wh ich distinguish it from non cancerous cells, a detection method must be able to recognize the given biomarker and bind to it with high sensitivity and specificity. Recently, a new class of ligands, known as aptamers, has been isolated and identified for su ch specific tumor cell recognition. Aptamers are single stranded polynucleotides, which recognize specific molecular targets with high affinity and selectivity. 101 They are obtained through an in vitro selection process, systematic evolution of ligands by exponential enrichment (SELEX), against a variety of targets, including ions, proteins, and cells. 102 103 Typically, antibodies are used to detect protein targets, but aptamers have several demonstrated advantages over antibodies, such as ease of manipulation, reproducible synthesis, good stability and non toxicity. Moreover, these properties make aptamers excellent candidates for biochemical sensors, signal transduction, and targeted therapeutic applications. 56 104 105 To date, several types of nanoparticles have been developed for point of care applications using opt ical signals. However, many of these nanoscale materials, such as quantum dots and dye doped or gold nanoparticles, are subject to significant background interference by scattering, absorption or autofluorescence within samples, which limits high sensitivi ty detection. In contrast, the signals from magnetic nanoparticles ( MNPs ) are not present in biological samples, resulting in the absence of background noise and, hence, high detection sensitivity. Specifically, the conjugation between aptamers and MNPs, or aptamer conjugated magnetic nanoparticles

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55 (ACMNPs), constitute s a novel kind of magnetic nanosensor, combining the specific binding ability of aptamers and their easy bioconjugation to solid surfaces, and the large surface areas of MNPs for multivalent interactions. The detection mechanism of ACMNPs in solution is based on the change of spin spin relaxation time, or 2 of protons in water The assembly of ACMNPs upon specific binding with their target cells leads to cluster formation thereby inducing coupling of magnetic spin moments generating strong local magnetic fields ( Figure 2 1A ). 38 49 106 At the same time, s uch local magnetic fields generate inhomogene i ties that accel erate the spin dephasing of surrounding water protons, which results in a decrease in the proton spin spin relaxation time (T 2 ). Moreover, MNPs are known to enhance the magnetic resonance (MR) signal of protons from surrounding water molecules. 107 109 Under these circumstances, aggregation can be detected by a change in proton 2 ), corresponding to the binding event between ligand conjugated MNPs and target molecules. This phenomenon has led to the development of magnetic relaxation switches (MRSw) for the detection of molecular targets, such as DNA, RNA, proteins, bacteria, viruses, small molecules, and enzymatic activity. 110 114 Based on previous studies, some cancer biomarkers are not restricted to a single cell type; rather, they are expressed in different cell types or at different developmental stat es of cancer cells. 115 Thus, multiple cell types at different physiological stages of cancer may show binding towards the same ligand, but with different affinities, depending on the level of biomarker expression. A methodology able to analyze various cancer cells, both qualitatively and quantitatively, can lead to the development of a cancer cell profile and thus greater understanding of cancer pathogenesis and the

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56 potential efficacy of new therapeutic modalities. By using an array of ACMNPs, various cell types can be differentiated throug h pattern recognition (Figure 2 1B ). A distinct pattern of responses generated from a set of ACMNPs would provide a cellular profile allowing clinicians to accurately classify and identify cancer cells at the molecular level. In the following discussion, we will demonstrate the specificity of ACMNPs to their targ et, explain and demonstrate the viability of MRSw, particularly in complex media, and show that ACMNPs arrays can recognize and differentiate various cell types and, by so doing, can create a distinct pattern recognition profile for various cancer cells. E xperimental Section Synthesis of DNA A ptamers The aptamers with strong affinities toward their intact tumor cells were selected by cell SELEX and were chosen as demonstrated in Table 2 1. All aptamers were synthesized using standard phosphoramidite chemist ry with an ABI3400 DNA/RNA synthesizer (Applied Biosystems, CA). Biotin core pore glass (CPG) from Glen Research was used for the synthesis. After the synthesis, the aptamers were deprotected in concentrated AMA (1:1 mixture of ammonium hydroxide and aqueo us methylamine) solution at 65C for 30 min prior to further purification with reversed phase high pressure liquid chromatography (RP HPLC). RP HPLC was performed on a ProStar HPLC Station (Varian, CA) equipped with a fluorescent and a photodiode array det ector using a C 18 column (Econosil, C18, 5 M, 250 x 4.6 mm) from Al l tech (Deerfield, IL). The eluent was 100mM triethylamine acetic acid buffer (TEAA, pH 7.5) and acetonitrile (0 30min, 10 100%). The collected DNA products were dried and detritylated wit h acetic acid. The detritylated aptamers were precipitated with ethanol and dried with a vacuum drier. The purified aptamers were then dissolved in DNA grade

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57 water and quantified by determining the UV absorption at 260 nm using a UV Vis spectrometer (Cary Bio 300, Varian, CA). Aptamer Nanoparticle C onjugation In order to prepare aptamer conjugated magnetic nanoparticles (ACMNPs), 30 nm streptavidin coated iron oxide nanoparticles (Ocean Nanotech) were dispersed at 0.1 mg/mL in 100mM phosphate buffered salin e (PBS), pH 7.4. An excess amount of biotin labeled aptamer was then added to the streptavidin coated MNPs solution. The mixture was vortexed at room temperature for 1 h followed by three washings with PBS buffer using centrifugation at 14000 rpm to remove any aptamers that did not conjugate to the MNPs. Zeta potential measurements were performed using Brookhaven ZetaPlus at ACMNPs were dispersed in PBS and stored at 4C at a concentration of 0.1 mg/mL. Cells and C ulture C onditions The cell lines listed in Table 2 1 were obtained from the American Type Culture Collection (ATCC). CEM, Ramos, and DLD1 cells were cultured in RPMI 1640 medium (ATCC). K562 cells were maintained i n culture with IMDM (ATCC). HCT116 cells were (ATCC). All media for cancer cells were supplemented with 10% heat inactivated FBS and 100U/mL penicillin streptomycin. HBE135 E 6E7, Normal Bronchial lung cell line, was maintained in Keratin Serum Free Medium supplemented with 5 ng/mL human recombinant Epidermal Growth Factor (EGF), 0.05mg/ml bovine pituitary extract (Invitrogen), 0.005 mg/ml insulin and 500ng/mL hydrocortisone. A ll cultured cells were grown in a humidified incubator at 37C under a 5% CO 2 atmosphere. In order to obtain single cell suspensions for the binding studies of adherent cells, cells were cultured

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58 overnight in low density and treated with non enzymatic cell dissociation solution (MP Biomedicals) for 5min. Cells were aspirated several times, and the single cells were pelleted and washed twice before use in the binding assays. Cell suspensions were centrifuged at 1000 rpm for 5 min, and the pellet was resuspen ded in 2mL of washing buffer. Ten microliter aliquots of the cell suspension were mixed with 10 L trypan blue solution. Cell quantification was performed using a hemacytometer (Hausser Scientific) and a microscope (Olympus). After determining the cell con centration, serial dilution of cells was prepared in PBS, FBS, plasma, or whole blood and used immediately after preparation. Determination of Conjugated Nanoparticle C ell S pecific T argeting To demonstrate specific targeting, CEM cells with their correspon ding aptamer, fluorescien (FAM) labeled sgc8c, were used, and FAM labeled TDO5 was selected as a negative control. The sgc8c ACMNPs were incubated with approximately one million CEM cells with the final concentration of 30g Fe/mL at 4C for 20 min in PBS. Similarly, TDO5 ACMNPs were also incubated with CEM cells as a negative control. After incubation, the cells were washed twice to remove unbound ACMNPs and resuspended in PBS. The binding of aptamer conjugated nanoparticles with target cells was investiga ted using a laser scanning confocal microscope setup consisting of an Olympus IX 81 inverted microscope with an Olympus Fluoview 500 confocal scanning system and a HeNe laser with a photomultiplier tube (PMT) for the detection. The cellular images were tak en with a 20x objective. The ACMNPs were excited at 488 nm ex for FAM), and the emission was detected with a 505 525 nm band pass filter.

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59 Sample A ssays using Spin Spin Relaxation Time M easurement To determine the specificity and sensitivity of the detect ion, 50L aliquots of CEM cell suspensions with different numbers of cells (1 to 106 cells) were incubated with 200 L of sgc8c ACMNPs solution in PBS ([Fe] = 10g/mL) at 4C for 40 min at the final volume of 250 L. Similarly, as a negative control, TDO5 ACMNPs were also incubated with the cells. The spin spin relaxation times (T 2 ) were measured at 1.5 T by an mq60 NMR analyzer (Minispec, Bruker, Germany), operating at 37C without a washing step. In order to mimic real clinical samples, which normally con tain thousands of different species, similar experiments were also performed in FBS, plasma and whole blood from Innovative Research. To generate the profiling of cancer cells, all cell types listed in Table 1 were dispersed in PBS such that each sample wo uld contain only one cell type and approximately 1000 cells. Each type of ACMNPs was incubated with each cell sample individually using the same conditions mentioned above, followed by the spin spin relaxation time measurement. Results and Discussion Magne tic Nanosensor Preparation The magnetic nanosensor was prepared by conjugating streptavidin coated iron oxide nanoparticles with biotin labeled aptamers. The streptavidin coated MNPs have an average hydrodynamic diameter of about 30 nm and a zeta potential 32.4 3.7 mV. The conjugation of aptamers to the MNPs results in an increase of negative 41.8 2.6 mV for sgc8c ACMNPs ) arising from the large negative charge of the DNA aptamers The la rge surface area of MNPs allows the attachment of multiple aptamers, which result in

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60 ACMNPs were stable and well dispersed without precipitation after storage at 4C for several months. S pecificity of ACMNPs Although ACMNPs have been previously used for cancer cell separation 116 117 it is necessary to confirm that the aptamers remain viable in terms of their ability to specifically recognize their targets after conjugation. Therefore, CEM cells and their corresponding aptamer, FAM labeled sgc8c ACMNPs, were selected for the demonstration and FAM labeled TDO5 ACMNPs were used as a negative control. Since our aptamers were labeled with fluorescent molecules, a fluorescence confocal microscope was used to validate the target specificity of the aptamer conjugate. The binding between the sgc8 c ACMNPs and CEM cells was demonstrated by bright fluorescence, while the control, TDO5 ACMNPs, showed only minimal fluorescence signal ( Figure 2 2A and Figure 2 2B ). To further validate the specificity, the binding of TDO5 ACMNPs to their corresponding ta rget, Ramos cells, was also investigated. There was also a significant difference in the amount of fluorescence signal seen between the images of the target and control ( Figure 2 2C and Figure 2 2D ). This observation demonstrated that the ACMNPs maintain t he same biological recognition to their targets as the free aptamers. Cancer Cells Detection using Spin spin Relaxation Time Measurement After using the fluorescence technique to demonstrate the specificity of ACMNPs to their targets, the use of MRSw to de tect cancer cells was investigated. The first assay was perfo rmed to detect CEM cells in PBS The results showed that 10 g Fe/mL was the optimal concentration for the detection of target cells, since lower concentrations generated significant errors in mea surement, while higher concentrations limited the

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61 detection threshold (Figure 2 3) When sgc8c ACMNPs were mixed with CEM cells, a decrease of T 2 was observed as the number of target cells increased. To verify whether the change of T 2 resulted from specifi c aptamer mediated interaction and not nonspecific aggregation of MNPs, TDO5 ACMNPs were also incubated with CEM cells as a control, followed by the relaxation time measurements. To determine the binding, 2 was defined as follows: 2 = T 2 sample T 2 nonspiked (2 1) where T 2 sample is the average T 2 relaxation time of ACMNPs in the presence of target cells and T 2 nonspiked is the average T 2 relaxation time of ACMNPs in the absence of target cells. Figu re 2 4 shows a wide dynamic range of detection and excellent 2 using sgc8c ACMNPs, whereas 2 of the control had no significant change. I n addition, fewer than 10 target cells in 250 L of PBS could be detected without any amplification method. The detection of Ramos cells was also demonstrated using their corresponding aptamer, TDO5 ACMNPs. The incubation of Ramos cells with TDO5 ACMNPs led to proportional 2 with increasing number of ce lls, while the mixture of Ramos cells and sgc8c 2 changes ( Figure 2 5) These results agreed with our fluorescence assays, as described above, and confirmed the specific recognition of ACMNPs, making this a viable and practical technique for the sensitive detection of cancer cells. Detection in Complex Biological Media To further assess the potential of this technique, detection in FBS, plasma, and whole blood samples was also performed. These assays were meant to mimic real clinical samples, which normally contain thousands of different species. The detection

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62 and quantification of CEM cells in FBS was demons trated by incubating CEM spiked FBS with sgc8c 2 was also proportional to the number of target cells, while the control showed only negligible changes ( Figure 2 6 ). It is important to note that this nanosensor can detect as few as 1 0 cells in 250 L of serum, which is much lower than the detection limits of conventional fluorescence or colorimetric based methods. 118 119 Although the detection of a few target cells has been demonstrated by the chip based Diagnostic Magnetic Resonance ( DMR ) system 53 our nanosensor requires no microfabrication, and the washing step is eliminated. Similarly, CEM spiked plasma or whole blood was incubated with sgc8c ACMNPs, followed by T 2 measurement. The results revealed that the detection and quantification of target cell s can also be achieved in both plasma and whole blood ( Figure 2 7 ). Nonspecific interactions in complex media containing thousands of proteins caused unwanted T 2 sample and T 2 nonspiked The low T 2 nonspiked value generated a high er background, resulting 2 for detection in complex media compared to detection in buffer with the same concentration of target cells. Nonetheless, we were able to detect as few as 100 target cells in all biological complex media ( Figure 2 8 ). Detection in Mixture of Cells T he detection of mixtures of targets and non targets (CEM and Ramos cells, respectively) with different ratios was also demonstrated One hundred CEM cells were mixed with non target Ramos cells at different ratios: 1:1, 1:2, 1:5, 1:10, 1:50, and 1:100, respectively. s gc8c ACMNPs were used to detect the target cells, and random sequenced DNA conjugated with MNPs was a lso used as a negative control. The results showed t hat the target CEM cells can be detected in mixtures of CEM and

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63 2 similar to those observed in the presence of target CEM cells only ( Figure 2 9 ) For a large number of non target cells, which may hinder the binding between the ACMNPs an 2 was observed. However, the detection in mixtures in which the ratio between target and non target cells was as small as 1:100 was achieved This result shows promise for detection in complex biological matri c es. S uccessful detection of target cells i n FBS, plasma, whole blood and mixtures of cells indicates that this method can be used for cellular detection in real clinical applications. Cancer Cells Profiling With the successful detection of target cancer cells with high specificity and sensitivity, the use of ACMNPs to monitor the interactions between different ACMNPs and multiple cell lines was investigated. The use of aptamers as specific recognition elements provides both qualitative and quantitative informat ion about cell surface receptors. For example, s gc8 c aptamer was used to recognize PTK7 receptors on leukemia cancer cells with precise quantitation. 120 121 However, by using an array of ACMNPs combined with the use of the MRSw technique, as described above, recognition patterns were generated resulting in the differentiation of various cell types and, in turn, a cancer cell profile that could be utilized to identify and classify cancer cells more precisely than might otherwise be achieved by a single specific probe. The target cells were chosen to represent a variety of cancer cell types: a normal lung cell line and six types of representative cancer cells, as listed in Table 1. One thousand cells of each cell line were spiked in PBS, incubated with each ACMNP individually, followed by a T 2 relaxation time measurement similar to that of the previous assays. The s ix cancer cell lines showed a large variation in T 2 reductions upon mixing

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64 with different ACMNPs (Figure 2 10 2 upon the incubation of multiple cell types with different ACMNPs can be explained by the different affinities of the aptame rs to their target and non target cells. The aptamers, which have high affinity 2 while the non target cells showed no significant difference in T 2 Based on the MR re sponse, sgc8c ACMNPs not only showed strong binding with their target, CEM, but also with the DLD1 and HCT116 cell lines. It was previously described that the target of sgc8c, PTK7, is also expressed in colorectal cancers. 115 As expected, the binding of sgc8c to both colorectal cancer cell lines was observed. Similarly, KCHA10 aptamer was found to inte ract with most colorectal cell lines, 58 resulting in the recognition of KCHA10 ACMNPs to both HCT116 and DLD1. The other aptamers, KDED2 3, TD05, T2 KK1B10, and TSL11a, which have recognition to only single cell types, demonstrated strong specificity to their targets. Significantly, none of ACMNPs had any interaction with the normal cell line, indicating that targets of these aptamers are related only to cancer. This result also confirmed that the binding based on the interaction of aptamer cell surface receptors involved and excluded nonspecific interactions. With the low background observed, the detection and profiling can be potentially used for analysis of cli nical specimens, which normally contain both diseased and normal cells. Compared to o ther methods, for example, flow cytometry or cell morphology analysis, MRSw also offers the advantages of simplicity, minimal detection time, and low detection limits. Con clusion In summary, we have developed a rapid and sensitive nanosensor for the detection of cancer cells as well as a method of profiling cancer cells based on MRSw.

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65 The ACMNPs were found to maintain their biological recognition and provide a multivalent effect, resulting in strong interaction with their target cells. Significantly, high sensitivity and specificity could be achieved by this nanosensor for the sample assays in complex biological systems including serum, plasma, and whole blood. An array of ACMNPs was utilized qualitatively and quantitatively to generate a profile for multiple types of cancer cells. The nanosensor allowed not only the identification of cancer cells but also the differentiation between cancerous and normal cells. Combined wit h the ease of operating magnetic relaxation instrumentation, the ACMNPs based nanosensor could become a useful tool for reliable and sensitive detection as well as cancer screening for clinical use. Furthermore, this demonstration provides strong evidence that the ACMNPs nanosensor has the potential to help identify the protein receptors on the surfaces of various cancer cell types for more informed cancer studies and biomarker discovery.

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66 Figure 2 1. Schematic representation of magnetic nanosen sor for cancer cell detection and profiling. (A ) The magnetic nanoparticles were conjugated with DNA aptamers having specific binding to their target cells. Addition of target cells results in an aggregation of magnetic nanoparticles, decreasing the T 2 rel axation time of adjacent water protons. (B ) Distinct recognition pattern generated from different affinities of aptamers to multiple cell types. An array of ACMNPs can be used to differentiate various cell types providing a molecular profile for qualitativ e and quantitative identification of cancer cells.

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67 Figure 2 2 The specific recognition of ACMNPs to their target cells. Confocal images (left: fluorescence image, right: transmission image) of CEM cells labeled by ( A ) FAM labeled TDO5 ACMNPs and ( B ) F AM labeled sgc8c ACMNPs; Ramo s cells labeled by (C ) FAM labeled sgc8c ACMNPs and (D ) FAM labeled TDO5 ACMNPs. The binding between ACMNPs and their target cells was demonstrated by the bright fluorescence, while the control showed minimal fluorescence signa l. Figure 2 3 The optimization of magnetic nanosensor concentration in PBS. The optimal concentration of ACMNPs for the detection of target cells was determined by using ~100 CEM cells as a target. The result showed that 10 g Fe/mL is the optimal conce ntration. The higher concentrations of ACMNPs limited the detection threshold, while the lower concentrations generated significant error for the measurement.

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68 Figure 2 4 sgc8c ACMNPs as a nanosensor for the detection of CEM cells in PBS The detection w as demonstrated after 40 min incubation at 4C TDO5 ACMNPs were used as a negative control. The result showed a wide dynamic range, with a limit of detection as low as 10 cells in 250 L of sample volume Figure 2 5. The dynamic detection range of Ramos cells using magnetic nanosensor in PBS The detection of Ramos cells was demonstrated by using TDO5 ACMNPs after 40 min incubation at 4C. Sgc8c ACMNPs were used as a negative control. The result showed a wide dynamic range, with a limit of detection as l ow as 10 cells in 250 L of sample volume, similar to the detection of CEM cells.

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69 Figure 2 6 sgc8c ACMNPs as a nanosensor for the detection of CEM cells in Fetal Bovine Serum (FBS) The detection was demonstrated after 40 min incubation at 4C in 250 L of sample volume TDO5 ACMNPs were used as a negative control. Figure 2 7. The detection of target CEM cells in Human plasma (A ) and whole blood ( B ). The results demonstrated that as few as 100 cells can be detected in CEM spiked plasma or whole blo od with low background.

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70 Figure 2 8. T he detection of target cells in complex biological media mimics detection in real clinical samples. All the measurements in complex media were p erformed using ~100 cells in a 250 L sample volume. Figure 2 9. Th e detection of mixed cell samples for the develop ment of rare tumor cells detection. sgc8c ACMNPs were incubated in a mixture of cells including CEM, target cells, Ramos, and non target cells, at different ratios.

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71 Figure 2 10. Profiling of cancer cel ls. The changes of T 2 relaxation times obtained by incubating different ACMNPs with multiple cancer cell types or control normal cells. The measurement was performed on ~1000 cells in a 250L sample volume. Table 2 1. Representative cell lines and binding affinities of their selected aptamers. Cell lines Type of cell Aptamer Kd(nM) CEM Leukemia sgc8c 58 ,a 0.8 Ramos Leukemia TDO5 122 75.0 K562 Leukemia T2 KK1B10 123 ,b 30.0 DLD1 Colon KDED2a 3 124 29.2 HCT116 Colon KCHA10 124 21.3 LH8 6 Liver TLS11a 125 ,c 7.0 HBE135 E6E7 d Lung N/A N/A a sgc8c is a truncated DNA of sgc8. b T2 KK1B10 is a truncated DNA of KK1B10. c TLS11a was origina lly develope d for liver cancer. d No rmal cell line.

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72 CHAPTER 3 DETECTION OF LYSOZYM E USING MAGNETIC RELAXATION SWITCHES B ASED ON APTAMER FUNCTIONALIZ ED SUPERPARAMAGNETIC NANOPARTICLES Introduction Over the past few decades, nanoparticles (NPs) have received consider able attention in advanced biomedical science. The unique characteristics of NPs, such as large surface to volume ratio and size dependent optical and magnetic properties, hold promise for the development of highly sensitive and selective diagnostic tools for clinical use. Specific ligands can be conjugated to a variety of nanoparticles to provide specificity and multivalent affinity. The interaction of NP ligand conjugates with their target molecules can be transduced into a reporting signal which can be d etected by fluorescence, colorimetric, and Raman spectroscopy. 33 126 127 However, many of these NPs, such as quantum dots (QDs) and dye doped or gold NPs, cause high background interference in biological media by scattering, absorption or autofluorescence, thus limiting highly sensitive detection. It has been observed that magnetic NPs (MNPs) have the ability to enhance the magnetic resonance (MR) signal of protons from surrounding water molecules. 107 108 111 Aggregation of MNPs induces the coupling of magnetic spin moments and generates strong local magnetic fields. Such local magnetic field inhomogeneities accelerate the dephasing of adjacent water protons, resulting in a decrease of transverse or spin spin relaxation times (T 2 ). Thus, both aggregation and dissociation of MNP clusters can be detected by a 2 ) by using NMR, magnetic resonance imaging (MRI), or relaxometry. This phenomenon has led to the development of magnetic relaxation switches (MRSw), in which the self assembly of MNPs, or disassembly of pre existing m agnetic clusters, corresponds to the presence or

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73 absence of specific targets, respectively. Such reversible systems can be designed to detect a variety of targets, such as DNA, RNA, bacteria, viruses, and small molecules, with the MNPs going either from th e dispersed to aggregated state or vice versa. 111 114 Additionally, these magnetic properties are not exhibited in biological samples in the absence of MNPs, resul ting in a reduction of background noise, thereby increasing detection sensitivity. Protein dysfunction in the context of cell regulation and signal transduction is always associated with the development of diseases, especially cancer. 128 130 A dvances in protein detection and quantification are essential for pharmaceutical and biomedical research. Recently, aptamers, which comprise a new class of ligands, have been isolated and identified to recognize a variety of chemical and biological molecules with high affinity and selectivity. 101 Aptamers are obtained through an in vitro selection process, which is known as systematic evolution of ligands by exponential enrichment (SELEX), against a variety of targets, such as ions, proteins, and cells. 102 103 Aptamers have several advantages over antibod ies, such as ease of manipulation, reproducible synthesis, good stability against biodegradation, and non toxicity. In the follow ing discussion, we will demonstrate an aptamer based sensor for protein detection based on MRSw, using lysozyme (Lys) and anti Lys aptamers as models. Lys is a highly isoelectric point enzyme (pI~11) that contains 129 amino acids. Generally, a low concentr ation of Lys is distributed in body tissues and secretions. However, it was reported that elevated levels of Lys in serum, urine, and cells are related to many diseases, such as leukemia 131 renal diseases 132 and meningitis. 133 Therefore, the detection and quantification of Lys is very important. In order to

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74 demonstrate the feasibility of our magnetic nanosensors for real clinical analysis, the detection of Lys in serum and cell lysates was also investigated. Experimental Section S ynthesis of DNA All DNA samples as demonstrated in Table 3 1 were synthesized using standard phosphoramidite chemistry with an ABI3400 DNA/RNA synthesizer (Applied Biosystems, CA). Biotin core pore glass (CPG) f rom Glen Research was used for the synthesis. After the synthesis, the aptamers were deprotected in concentrated AMA (1:1 mixture of ammonium hydroxide and aqueous methylamine) solution at 65C for 30 min, prior to further purification with reversed phase high pressure liquid chromatography (RP HPLC) using a ProStar HPLC Station (Varian, CA) equipped with a fluorescent and a photodiode array detector and a C 18 reversed phase column (Econosil, C18, 5 M, 250 x 4.6 mm) from Alltech (Deerfield, IL). The eluen t was 100mM triethylamine acetic acid buffer (TEAA, pH 7.5) and acetonitrile (0 30min, 10 100%). The collected DNA products were dried and detritylated with acetic acid. The detritylated aptamers were precipitated with ethanol and dried using a vacuum drie r. The purified aptamers were then dissolved with DNA grade water and quantified by determining the UV absorption at 260 nm using a UV Vis spectrometer (Cary Bio 300, Varian). Lys Nanosensor Preparation In order to prepare Lys nanosensors, 30 nm streptavid in coated iron oxide nanoparticles (Ocean Nanotech) were dispersed at 0.1 mg/mL in 100 mM phosphate buffered saline (PBS), pH 7.4. An excess amount of biotin labeled Lys aptamer or linker DNA was then added to separate samples of the streptavidin coated MN Ps. The mixtures were vortexed at room temperature for 1 h followed by washing 3x with PBS

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75 buffer using centrifugation at 14000 rpm to remove any DNA that did not conjugate to the MNPs. The conjugates were dispersed in PBS and stored at 4C at a concentrat ion of 0.1 mg/mL. Equimolar amounts of MNP Lys aptamer and MNP Linker were mixed together and dispersed in PBS buffer at a final concentration of 12 g Fe/mL, leading to cluster formation within 20 min. The spin spin relaxation times (T 2 ) were measured at 1.5 T by mq60 NMR analyzer (Minispec, Bruker, Germany), operating at 37C to confirm the aggregation of nanosensors. Magnetic Relaxation Measurement The Lys nanosensor was prepared as mentioned in the previous section. A stock solution of protein (0.1mM) w as prepared in deionized water and diluted in PBS as necessary. Fifty L aliquots of Lys with different concentrations were incubated with 200 L nanosensor mixture in PBS at room temperature at a final volume of 250 L and [Fe] = 12 g/mL. The spin spin r elaxation times (T 2 ) were measured after 40 min of incubation without a washing step. The samples with human serum (Innovative Research) were prepared by adding protein and nanosensors to 100% human serum using the same concentration of nanosensors. T 2 wei ghted MR images were obtained by using a 11 T NMR instrument with a spin echo pulse sequence, variable echo time (TE) of 50 100 ms and repetition time (TR) of 3000 ms. Cell Samples Preparation All cancer cell lines were obtained from the American Type Cult ure Collection (ATCC). CEM and Ramos cells were cultured in RPMI 1640 medium (ATCC). K562 cells were maintained in culture with IMDM (ATCC). All media for cancer cells were supplemented with 10% heat inactivated FBS and 100U/mL penicillin streptomycin. All cultured cells were grown in a humidified incubator at 37C under a 5% CO 2

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76 atmosphere. The normal white blood cells (WBC) were separated from whole blood samples (Innovative Research) and transferred into 15 mL tubes. To isolate the WBC, blood was centrif uged at 2000 rpm for 10 min at room temperature. This procedure separated the blood specimen into three layers: an upper plasma layer, a lower red blood cell (RBC) layer, and a thin interface buffy coat containing the WBC. With a transfer pipette, the plas ma was first removed, and then the buffy coat was carefully aspirated into a separated tube. Cell suspensions were centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in 2mL of washing buffer. After washing 2x, ten microliter aliquots of the cell suspension were mixed with 10 L trypan blue solution. Cell quantification was performed using a hemocytometer (Hausser Scientific) and a microscope (Olympus). Ten million cells of each cell type were resuspended in 250 L of 1% ice cold CHAPS ( 3 [(3 Cholamidopropyl) dimethylammonio] 1 propanesulfonate) lysis buffer. The lysates were incubated for 30 min on ice and centrifuged 20 min at 12000 rpm at 4 C. The lysates were transferred to clean centrifuge tubes for Lys assay or frozen at 80 C. Fifty L aliquots, which contained lysate from 110 6 cells, were incubated with nanosensor solution in PBS using the same conditions mentioned above, followed by the spin spin relaxation time measurement. Results and Discussion The MRSw mechanism for the Lys senso r is based on analyte induced disassembly of MNPs, an event which results in an increase in T 2 as explained above. As shown in Figure 3 1, iron oxide nanoparticles are conjugated with either Lys aptamer or linker DNA. In the absence of Lys, the linker can hybridize with part of the aptamer (7 bases of the aptamer plus a 5 base extension) to form clusters (short T 2 ). However, in the presence of Lys, the aptamer undergoes a structural change in order to bind with

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77 the target, resulting in base pair disruption The five remaining base pairs between MNP Lys aptamer and MNP Linker are not strong enough to hold the cluster together at room temperature, leading to the disassembly of the clusters (longer T 2 ). Therefore, the Lys induced disassembly of the clusters ca n be monitored by this increase in T 2 Clusters Formation of Lys N anosensor S treptavidin coated iron oxide nanoparticles were conjugated with either biotin labeled aptamers or biotin labeled linker. To prepare Lys nanosensor, equimolar of MNP Lys aptamer w as incubated with MNP Linker ( [Fe] = 12 g/mL) followed by T 2 measurement. Within 5 minutes, the nanosensor showed a decrease in T 2 indicating the formation of clusters upon the hybridization between the complementary strands ( Table 3 2 ). In contrast, the T 2 of either the individual MNP Lys aptamer or MNP Linker showed no significant change. The T 2 of the nanosensor remained constant after 10 min of incubation, indicating that the formation of nanosensors could be completed in a short time. The aggregation was confirmed again by mixing a high concentration of MNP Lys aptamer and MNP Linker at [Fe] = 100 g/mL and incubating overnight at 4C. Precipitation of the nanosensor was observed as a consequence of the formation of large clusters ( Figure 3 2 ). Howeve r, the same effect was not observed for individual MNP Lys or MNP Linker. Lys Induced Disassembly of N anosensors To avoid precipitation t he Lys assay was performed with the nanosensor at a low concentration ([Fe] = 12 g/mL). Two hundred fifty nM of Lys w as added into the prepared nanosensor, followed by T 2 measurements at 5 minute intervals after adding the target. For each time interval, the change in T 2 2 ) was calculated by the following equation:

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78 2 = T 2 sample T 2 blank (3 1) where T 2 sample is the average T 2 relaxation time of three replicates of the nanosensor after protein addition and T 2 blank is the average T 2 relaxation time of three repl icates of the nanosensor without target protein 2 was observed from 5 to 20 min after Lys addition ( Figure 3 3 ), due to disassembly of the magnetic clusters after aptamer binding to the Lys. The signal reached a maximum within 20 m in, demonstrating the rapid detection of Lys by the nanosensor. Selectivity and Specificity of Lys Nanosensors To assess the selectivity of detection, the change in T 2 was measured within 40 min after adding 50 nM of Lys or 50 nM of some possible interferi ng proteins, such as insulin, avidin, trypsin, thrombin, BSA, and streptavidin. As shown in Figure 3 4 a significant increase in T 2 upon adding Lys was observed, while the other proteins showed no significant change in T 2 values. The result showed high ly sozyme selectivity against other proteins, which may have otherwise have interfered with detection in biological samples. To confirm that the detection resulted from specific binding between the target protein Lys and nanosensors rather than nonspecific ef fects, a random DNA sequence conjugated with MNPs was used for nanosensor preparation instead of MNP Lys aptamer. To demonstrate that Lys cannot induce disassembly of pre existing clusters prepared between MNP random sequences and MNP Linker, seven bases a t the end of Lys aptamer and the extended part were preserved in random sequences in order to form the clusters. As expected, the nanosensor prepared by random sequences did not bind with the Lys target, resulting in only minimal change in T 2 (Figure 3 5 ). This result

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79 confirmed that the disassembly of nanosensors occurs because of interaction of the aptamer conjugated MNPs with target Lys to the exclusion of nonspecific interactions. Quantitative Analysis of Lysozyme The range of detection was determined b y measuring the change of T 2 for samples with different concentrations of Lys. The result showed a continuous increase 2 as the Lys concentration was raised from 0 to 500 nM (Figu re 3 6 ). The change of T 2 reached maximum when the Lys concentration inc reased to more than 500 nM, indicating that binding saturation had occurred between Lys aptamers and their targets. 2 was observed in the concentration range of 0.5 80 nM with a correlation coefficient (R 2 ) of 0.9914, as shown in the inset of Figure 3 6 Using this nanosensor, Lys could be detected at concentrations as low as 0.5nM without any separation or amplification step. The low detection limit of this nanosensor can be attributed to the high affin ity of the aptamer to Lys with a dissociation constant (K d ) of 30 nM, 134 as well as the low background noise inherent in MRSw detection. Detection in complex biologi cal media was also demonstrated by spiking Lys into 100% human serum. The result demonstrated that this nanosensor was able to detect 2 and Lys concentration from 1 to 80 nM was achie ved (Figure 3 7 ). It is interesting to note that magnetic nanosensors based on MRSw may not offer a very low detection limit compared to fluorescent techniques in buffer systems. 135 Nevertheless, because of the light independent property of MRSw, and the inherent low background, Lys was detectable in the nanomolar range in complex biological medium without any separation

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80 or amplification step. This result revealed the feasibility of using this magnetic nanosensor in clinical analysis. The detection of Lys in human serum was also confirmed using T 2 weighted MR imaging. An increase of Lys concentration led to more disassembly of nanosensor clusters, resulting in an increase in T 2 and an increase in brightness of the T 2 images, as shown in Figure 3 8 The change in contrast was also observed in the nanomolar range of Lys protein which corresponds to detection using a benchtop relaxometer. Analysis of Lys ozyme in Cell Lysates To validate the use of this Lys nanosensor for real clinical samples, an analysis of Lys in lysates from leukemia cells was performed. CEM, Ramos, and K562 cells w ere chosen to represent a variety of leukemia cell types. Within this set, normal white blood cells were used as a control. Lysates from one million cells of each cell type were incubated with the Lys nanosensor followed by T 2 relaxation time measurement s similar to those in previous assays The cell lines containing a large amount of Lys could induce a high degree of cluster disassembly, resulting in significant change in 2 Based on the MR response, t he three cancer cell lines showed a variation of 2 indicating that different amounts of Lys were contained in each cell type (Figure 3 9 ). Normal white blood cells showed only a small change in T 2 indicating a low concen tration of Lys. It was previously reported that a high concentration of Lys was detected in myeloid leukemia cells. 136 As expected, a significant increase in T 2 was observed in K562 cells, which b elong to a myeloid leukemia cell line, based on their high Lys expression. In contrast, CEM and Ramos, which are T and B lymphoid leukemia cells, showed small 2 similar to that of normal cells, indicating a low Lys expression, a result which agrees with the literature. 136 137 The result suggested that this

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81 magnetic nanosensor could be potentially used for dif ferentiation of Lys levels in specimens between healthy individuals and patients with leukemia. Conclusion In conclusion, we successfully demonstra ted an aptamer based biosensor for protein detect ion using MRSw and Lys as the model target protein. Good se lectivity for Lys compared to other proteins was demonstrated by minimal disassembly of pre existing clusters prepared by random DNA sequence s conjugated with MNPs A detection limit in the nanomolar range was achieved for Lys detection in both buffer and human serum. Detection was confirmed by t he T 2 weighted MR image of Lys in duced disassembly. An assay to determine the level of Lys in cell lysates also demonstrated the potential of this Lys nanosensor for real clinical sample analysis. Overall, this MRSw based nanosensor offers the advantages of high sensitivity and simplicity for the detection in turbid media and biological samples without protein purification or separation; consequently, the system is feasible for point of care diagnostics.

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82 Figure 3 1. Schematic representation of the magnetic nanosensor for Lys detection based on MRSw. The iron oxide nanoparticles are conjugated with either Lys aptamer or linker DNA which can hybridize with the extended part of the aptamer to form clusters. Upon th e addition of Lys, the aptamers bind with their targets, leading to disassembly of clusters and increased T 2 relaxation time of the adjacent protons. Figure 3 2 Effect of incubating MNP Lys aptamer with MNP Linker at high concentration overnight. The i ndividual conjugates showed good dispersion, corresponding to long T 2 However, the mixture of MNP Lys and MNP Linker showed precipitation at the bottom due to the hybridization between complementary strands, resulting in the formation of large clusters.

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83 Figure 3 2 upon the addition of Lys T he signal reached the maximum within 20 min, indicating the rapid disassembly of magnetic nanosensors. Figure 3 4 Selectivity of the Lys nanosensor. Disassembly of magnetic clusters upon the addition of 50 nM of Lys target was detected by significant change in T 2 relaxation time. At the same time, other proteins, as noted, showed only negligible changes, indicating no interaction with the nanosensor.

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84 Figure 3 5 Specificity of the Lys nanosensor. Random sequences were employed to test specificity; results showed no binding to target and only minimal c hange in T2 at a Lys concentration of 250 nM. Figure 3 6 Changes in T2 relaxation time with increasing concentrations of Lys. The det ection range was determined in PBS; inset: expanded linear region of the curve.

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85 Figure 3 7 The detection of Lys spiked human serum using relaxometry measurements. Figure 3 8. The detection of Lys spiked human serum using T 2 weighted MR images.

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86 Figure 3 9. Determination of Lys in cell lysates. Normal white blood cells were used as a standard to represent the normal Lys level. A significantly elevated amount of Lys was observed in K562 cells, while CEM and Ramos only showed small differences in Lys levels compared to normal white blood cells. Table 3 1. List of DNA sequences DNA sequences Lys Aptamer Library Linker AGA GA G ATT CAT TGA GAC GTG AGA AAT CGG GAC TA T TTT TT Biotin AGA GA G ATT CAT NNN NNN NNN NNN NNN NNN NNN NN T TTT TT Biotin ATG AAT CTC TCT TTT TTT Biotin The underlined bases demonstrate the complementary part, and the blue highlight demonstrates the extended part of Lysozyme aptamer. Table 3 2. The formation of magnetic clusters upon hybridizatio n between c omplementary strands. The clusters were detected by changes in T 2 at 5 minute intervals after mixing. Time (min) T 2 (ms) MNP Lys MNP Linker MNP Lys+MNP Linker 5 93.72.1 97.01.7 78.02.4 10 93.31.9 96.83.1 76.61.8 20 91.6 3.7 95.82.6 76.22.2

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87 CHAPTER 4 MAGNETICALLY DRIVEN SINGLE DNA NANOMOTOR Introduction In biological systems, nanoscale molecular motors can generate forces from spontaneous reactions of energy rich molecules, mostly adenosine triphosphate (ATP), to convert chemical energy to mechanical movement. These motors are roughly 10 nm in size, take steps of a few nanometers, and can exert forces in the piconewton range. 138 139 However, to efficiently design and construct nanoscale motor s in the laboratory, two key requirements must be satisfied: 1) the generation of forces sufficient to power nanomachines or nanodevices and 2) the exertion of precise control over the motion produced. Accomplishing these objectives begins with DNA, which is the ideal building block for nanostructures. In addition, DNA can also be used to create dynamic molecules that replicate machine functions including tweez ers, 10 79 140 gears, 11 walkers, 13 141 142 and motors. 84 143 144 The DNA nanomoto rs, which can be operated with high efficiency for several cycles, require exergonic reactions, such as hydrolysis of the DNA backbone, 141 142 144 hydrolysis of ATP, 13 and DNA hybridization. 14 79 However, operating such DNA nanomot ors requires the addition and removal of fuels and waste strands for motor function. This mode of operation, however, is accompanied by the accumulation of waste products, which results in decreased motor efficiency. Therefore, coupling the nanomotors to c lean alternative energy sources would both eliminate the accumulation of waste products and produce a practical high efficiency device. Single molecules can be manipulated through magnetic force by attaching them on magnetic particles and applying an exte rnal magnetic field. Using a magnetic field to

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88 control the movement of molecules can also avoid molecular damage from photonic flux. Therefore, the application of a magnetic field could be of great interest as an alternative energy source for DNA nanomotor s and could also satisfy the design requirements, as enumerated above. We have designed a DNA nanomotor which is fueled by an oscillating magnetic field gradient. The nanomotor consists of DNA hairpins that are immobilized on a glass surface inside of a mi cro channel and subsequently conjugated to magnetic particles. An external magnetic field gradient is then used to apply a force on the magnetic particles perpendicular to the glass surface, thereby opening the DNA hairpins. We interpret the separation of power stroke of our nanomotor and the hybridization of the hairpin as the recovery stroke. The movement of the hairpin molecule can be monitored by fluorescence resonance energy transfer (FRET) be tween a fluorophore and a quencher on the stem ends. 99 145 146 Compared with other DNA nanomotor systems, in which the cycles involve the addition of several DNA strands, the magnetic hairpin DNA nanomotor can be operated by the simple application of an external magnetic field gradient. As such, this magnetically d riven hairpin DNA nanomotor adds no DNA fuels and generates no DNA waste products after each cycle, in addition to which it can be operated at room temperature with low salt concentration. Experimental Section DNA Molecular Probes Synthesis All sequences as demonstrated in Table 4 1 were synthesized using standard phosphoramidite chemistry. The DNA reagents for the synthesis of DNA hairpins and biotinylated linker were purchased from Glen Research. All DNA hairpins were

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89 synthesized with an ABI3400 DNA/RNA s ynthesizer (Applied Biosystems, CA). Biotin core pore glass (CPG) was used for all DNA hairpins and DNA linker synthesis. Deprotection of the DNA was performed using overnight incubation with ammonium hydroxide at room temperature. The solution resulting f rom deprotection was precipitated in cold ethanol. Subsequently, the precipitates were dissolved in 0.5 mL of 0.1 M triethylammonium acetate (TEAA, pH 7.0) for further purification with reverse phase high pressure liquid chromatography (RP HPLC). RP HPLC w as performed on a ProStar HPLC Station (Varian, CA) equipped with a fluorescent and a photodiode array detector using a C 18 reverse phase column (Econosil, C18, 5 M, 250 x 4.6 mm) from Altech (Dearfield, IL). The product collected from the HPLC was vacuu m dried and then added with TEAA for a second round of HPLC. The final product was collected, vacuum dried and dissolved in 200 L of acetic acid (80%) for 30 min, follow by 200 L of cold ethanol and then vacuum dried once more. Quantification of all DNA was performed using a UV Vis Spectrometer (Cary Bio 300, Varian, CA). Molecular Probes Assays The maximum fluorescence signal enhancement of each DNA hairpin was observed in solution before and after incubation with excess DNase I in order to determine its 20 mM of Tris HCl with MgCl 2 and NaCl, pH 7.4 was loaded into a quartz cell for ex for FAM), the emission spectra were collected em for FAM). After scanning the emission of DNA hairpins for 5 min, 2000 Units/mL of DNase I (Deoxyribonuclease I) enzyme (New England BioLabs, Inc) was added to the solution of DNA hairpins. DNase I catalyzes cleavage of the phosphodiester lin kages in the DNA backbone separating the fluorophore from the

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90 quencher to generate the maximum fluorescence signal. It has been reported that the ionic strength, especially the concentration of divalent cations, affects the stability and reactivity of hair pin structured DNA. To optimize the buffer system, the DNA cleavage by DNase I was performed at pH 7.4 in 20 mM Tris HCl solution with different concentrations of MgCl 2 (5 and 10 mM) and NaCl (50, 100, and 200 mM). The fluorescence enhancement was calculat ed using the following equation: Fluorescence Enhancement = (S cleaved B buffer )/(S close B buffer ) ( 4 1) where S cleaved is the signal of DNA hairpins after cleavage by DNase I, S close is the signal of DNA hairpins prior to cleavage, and B buffer is the b ackground fluorescence intensity of buffer. Immobilization of DNA Molecular Probes on Glass Surface The surface experiments were performed using microscope slides and glass slips from Fisher (optical borosilicate glass with a size of 45 x 50 mm and 0.13 t o 0.17 mm in thickness). The surfaces were cleaned with piranha solution (3:1 ratio of conc. H 2 SO 4 :30% H 2 O 2 ), washed thoroughly with deionized water, and dried with nitrogen. Strips of double sided tape (3M) were placed on the cleaned microscope slide and a cover glass was placed on top to generate micro channels. The channels were filled with Tris HCl buffer with optimum concentrations of MgCl 2 and NaCl by capillary action, and the solution was withdrawn with filter paper (Fisher). After washing channels w ith buffer 3 times, 7 L of 1 mg/mL, avidin was incubated in the channel for 5 min. The excess avidin was removed by washing 3 times with buffer. Next, 7 L of 5 M biotinylated linker was incubated in the avidin treated channels for 10 min. After washing to remove excess linker, a solution of 5 M DNA hairpins was incubated in the channels to hybridize with the immobilized linker for 15 min, and the excess DNA

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91 hairpins were removed by buffer washes. The fluorescence of the DNA hairpins in the closed form w as observed by a confocal fluorescence microscope setup consisting of an Olympus IX 81 inverted microscope with an Olympus Fluoview 500 confocal scanning system and an Argon laser with a photomultiplier tube (PMT) for detection, at the wavelength given abo ve using 20x objective. To find the glass liquid interface, the microscope was first focused on the glass surface of the micro channel, and a z section was recorded after each step in the experiment. Each z section was then analyzed to find the position wh ere the highest signal had been recorded. After immobilization of DNA hairpin on the surface of the channel, DNase I was incubated with the immobilized DNA hairpins for 45 min Then the average fluorescence intensities of the image from avidin, immobilize d DNA hairpins, and DNase I addition were taken at the same position for that particula r channel, and the average fluorescence was monitored to determine the signal enhancement Construction of DNA Nanomotor The DNA hairpins were immobilized on the glass s urface and a z section was recorded following each step as described above. After immobilization of the DNA hairpins, the surface was passivated against nonspecific interactions between magnetic beads and the surface by incubating unreacted avidin with 2 m g/mL of biotinylated PEG solution for 15 min. The streptavidin coated iron oxide beads (either 1 m, 2.8 m from Dynabeads, Invitrogen, or 0.2 m from Chemicell, GmbH) were sonicated for 1 min to break up the bead clumps. A 2 mg/mL suspension of the beads was then incubated for 20 min in the DNA hairpins coated channel. After washing, a z section was recorded and the average fluorescence signal of the image (closed form) was observed by confocal microscope. A rare earth magnet, NdFeB from Applied Magnets, w as attached

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92 to an adjustable stage and positioned directly above the channel for 1 min and the average fluorescence intensity was measured at the same position to observe the open state of the DNA hairpins. Three minutes after removal of the magnet, the cl osed state of DNA hairpins was observed by fluorescence. The magnet was positioned and removed for several cycles to measure the change between shrinkage and extension of the DNA nanomotor. Results and Discussion The DNA hairpins and biotinylated linker a re summarized in Table 4 1. Specifically, DNA hairpin structures were selected because they can be switched from ) state. Each DNA hairpin has 20 thymidine (T) bases in the loop and 6, 9, or 12 base pairs (6ds, 9ds, or 12ds) in the stem part. In order to visualize movement between the two states, a fluorophore (FAM) is attached to one arm of the stem and a quencher (Dabcy l) on the other arm of the stem. Poly T (20 bases) was used as the spacer between DNA hairpins and magnetic DNA linker which was immobilized on a glass surface. It is necessary to point out that the fluorescence resonance energy transfer (FRET) pair i s not needed for motor function but gives a convenient way to monitor the motion of the motor. The fluorescence intensity is related to the distance between the FRET pair in the stem ends, which indicates the closed or open state of the DNA nanomotor movem ent 99 145 146 As shown in Figure 4 1, an external magnetic field attracts the end is tethered to a glass surface. In the absence of the magnet ic field, the DNA

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93 hairpins are in the contracted state and the fluorophore is quenched by FRET. When the external magnetic field is applied, the magnetic beads are attracted to the magnet and cause the DNA hairpins to extend, restoring the fluorescence sig nal. Signal Enhancemen t of the DNA Molecular Probes by Dnase I Cleavage The signal enhancement of three DNA hairpins in solution was tested and the results are shown in Figure 4 2 The DNA hairpins with longer stems exhibit lower background fluorescence, h owever, the DNA hairpins with fewer base pairs in the stem yielded higher signal enhancement after DNase I addition by the higher enzymatic cleavage efficiency. We further tested the signal enhancement of the DNA hairpins immobilized on glass surface by DN ase I. Since the DNA nanomotor will be immobilized and operated on a glass surface, the fluorescence signal enhancement of each DNA hairpin on the micro channel surface was also determined. Equation 4 1 was also used to calculate the signal enhancement for DNA hairpins immobilized on the glass slide, and the results are shown in Figure 4 3 On the glass surface, the signal enhancement of DNA hairpins decreases significantly compared to that of DNA hairpins in solution. Whereas DNA hairpins can typically ach ieve fluorescence enhancements above 25 fold in solution, these values can drop to 2 to 5 fold once immobilized. This behavior mainly results from the interaction between the DNA hairpins and the surface, which disrupts the loop and destabilizes the DNA ha reflected in higher background signal, thus results in lower signal enhancement.

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94 DNA Nanomotor Driven by Magnetic Field Generally, the mechanical response of a single DNA molecule to an a pplied force can be divided into entropic and elastic responses. 147 Entropic responses are driven by thermal energy, and elastic responses are generated by base pair interaction. Thermal energy corresponds to Brownian motion, which is proportional to small fluctu ation of flexible DNA molecules tethered to magnetic beads. Typically, the thermal fluctuation of a single DNA molecule is less than 5 pN. 148 Our system involves DNA base pair interaction, i.e., the unzipping of the DNA hairpins. By using atomic force microscopy (AFM), it was reported that the DNA unzipping force of 10 base pairs is about 10 pN depending on the l ocal G C content. 149 As expressed in equation 4 2 the force acting on a magnetic particle is proportional to the external field strength and the magnetic moment of the particle. 150 151 F m = ( 4 2 ) where m and B are magnetization of the particle and magneti c induction, respectively. Magnetic field gradient has been successfully used to manipulate tethered bead DNA molecules with generated force up to 20 pN where the maximum value of the force depends on the size of a particle. 150 151 When the magnet position is fixed, the force acting on the magnetic particle can be kept constant because the spatial region occupied by the particle is small enough for the magnetic field gradient to be considered uniform. In order to demonstrate a DNA nanomotor driven by magnetic field gradient DNA hairpin and magnetic beads were immobilized on a glass surface followed by the

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95 investi gation of magnetically controlled process To optimize the motor system, two design factors were investigated: the number of base pairs in the stem part of the DNA hairpins and the size of magnetic beads. In normal condition, the DNA hairpins were in the c ontracted (closed) conformation, and the hairpin structure was maintained. When the external field was applied to drive the movement of the DNA nanomotor, the magnetic particles experienced sufficient force to unzip and extend the DNA hairpins as the power stroke of the motor. After removal of the external field, the DNA molecular probes reformed to the hairpin structure as the recovery stroke. By repeated application and removal of the external magnetic field, the DNA hairpins can be regarded as a reversib le DNA nanomotor as demonstrated by the fluorescence signal changes shown in Figure 4 4 A It is important to point out that the extension of DNA molecular probes as the power stroke occurred within 1 minute and a few minutes were used to reform the hairpin s as the recovery stroke. Based on the assumption that the diameter of the DNA duplex is 2.2 nm and the distance between two bases is 0.34 nm, we estimated the distance variation between closed and open states to be 2.2 nm and 10.8 nm, respectively, for th e DNA hairpin with 6 base pairs in the stem part. 67 150 Accordingly, molecular motors moving with larger step s generate more forces. Therefore, to change distance and create a larger working stroke, the number of base pairs in the stem part of the DNA hairpins was varied. As such, longer distance in the open state, i.e., from 10.8 nm to 12.9 and 14.9 nm, was achi eved by changing from 6 to 9 and 12 base pairs in the stem part of the DNA hairpin, respectively. However, increasing the number of base pairs requires more force to unzip the DNA hybridization resulting in slightly less fluorescence intensity change for e ach

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96 cycle of the motors as shown in Figure 4 4B and 4 4C All DNA hairpins with 6, 9, and 12 base pairs show consistent recovery and the signal enhancement shows no tendency to decrease after 5 cycles using 1 m magnetic beads. This result confirms consist ent efficiency and no influence of fluorescence bleaching after several cycles of operation. Excess amount of DNase I enzyme was added at the end for each nanomotor to compare the fluorescence signal of each DNA hairpin with the maximum fluorescence signal In this way, we can calibrate the system to achieve consistency. By setting the fluorescence intensity when DNA hairpins were in the closed state as a baseline (0%) and the intensity after addition of excess DNase I as 100%, we can estimate the number of DNA hairpins in the open state for the magnetically controlled process. We use a fluorescence recovery parameter, recovery (%), to evaluate the opening efficiency of DNA molecular probes as follows: Recovery (%) = (I open I closed )/(I cleaved I closed ) ( 4 3 ) Where I open is the average fluorescence intensity of DNA hairpins after external magnetic field application, I closed is the average fluorescence intensity after removal of the external field, and I cleaved is the average fluorescence intensity after adding DNase I enzyme. The higher recovery value demonstrates the higher amount of DNA hairpins in the open state by external magnetic field application. When we used recovery (%) to compare the opening efficiency of all DNA hairpins under the same conditi ons, we found approximately 37.6% of recovery for DNA hairpins with 6 base pairs, 36.4 % for 9 base pairs, and 35.6% for 12 base pairs, respectively. This result supports our assumption that more force is needed to unzip the hybridization of hairpin struct ure,

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97 when more base pairs are added. However, the recovery of each DNA molecular probe is slightly different. Since the external magnetic field gradient is kept constant, the maximum force reacts to DNA nanomotor depends on the size of the magnetic parti cle. In order to generate different forces for the power stroke to drive the DNA nanomotor, the effect of different sizes of magnetic beads was also observed using 2.8 m and 0.2 m magnetic particles, as shown in Figure 4 5 Figure 4 6 and Figure 4 7 res pectively. Interestingly, all DNA hairpins show slightly lower signal enhancement for each cycle when the larger particles were used. This behavior of the 2.8 m system is mainly attributed to a lower signal in the opened state compared to the 1 m system. The larger particles have a larger radius of curvature and this allows more hairpins to form the link between the surface and the bead, thus increasing the force needed to pull the bead away from the surface. This increase in force to open more hairpins is balanced by the increase in force created by using a larger particle. However, when 0.2 m of magnetic particles were attached to the DNA hairpins, there was no change in fluorescence intensity after applyi ng the magnetic field (Figure 4 7 A ). The smalle r magnetic particles experienced smaller magnetic force because of the saturation of their magnetic susceptibility, which was insufficient to unzip the hairpin structure. The average signal enhancement of different DNA hairpins and magnetic particles is su mmarized in Table 4 2. In addition, a control experiment was performed by using 6ds hairpins attached to 5 m silica particles. The results demonstrated that the fluorescence signal did not change after cycling the e xternal magnetic field (Figure 4 7 D ), co nfirming that the

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98 fluorescence intensity change from the previous system was not an artificial signal and hairpins tethered to magnetic beads can be manipulated by the applied magnetic field. Conclusion In conclusion, a magnetically driven DNA nanomotor us ing DNA hairpins conjugated to magnetic particles has been successfully demonstrated. Specifically, DNA hairpins with different numbers of base pairs in the stem were synthesized. One end of the DNA hairpin was immobilized on the glass surface of a micro c hannel and the other end was attached to iron oxide particles. An external magnetic field was then applied to pull the iron oxide particles away from the glass surface. This pulling force opens the hairpin, as observed by an increased fluorescence signal, and is interpreted as the power stroke. Removal of the magnetic field reforms the closed state of DNA hairpins, as observed by decreased fluorescence signal, and represents the recovery stroke. Motorization of the nanodevice results from the cyclical shri nking and extending movements. This DNA nanomotor holds promise for a new class of nanomachines that can be operated by magnetic field without any additional DNA strands as fuels. In addition, using magnetic field to control the movement of molecules can a lso avoid molecular damage from photonic fluxes, which in contrast to previous photoregulated DNA nanomotors.

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99 Figure 4 1. Concept of magnetically driven DNA nanomotor 15 2 One end of DNA hairpins were immobilized on a glass surface and the other conjugated to magnetic particles. DNA hairpins molecules were opened by an external magnetic field gradient and subsequently closed through DNA hybridization after the removal of nanomotor and the hybridization of the hairpin as the recovery stroke.

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100 Figure 4 2 Signal enhancement of DNA hairpins in solution after the addition of DNase I. 152 Fluorescence emission at 520 nm was measured for the DNA hairpins after DNase I addition: [DNA hairpin] = 100nM, DNase I = 2000 U/mL, and buffer is 20 mM Tris HCl buffer pH 7.4, Na + = 100 nM, Mg 2+ = 10 mM

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101 Figure 4 3. Signal enhancement of DNA hairpins on the glass surface of micro channel after DNase I addition. 152 Fluorescence emission at 520 nm was measured for the DNA hairpins after DNase I addition: [DNA hairpin] = 5M, DNase I = 2000 U/mL, and buffer is 20 mM Tris HCl buffer pH 7.4, Na + = 100 nM, Mg 2+ = 10 mM Figure 4 4. Cycles of closed open state fr om DNA hairpins with 6 (A), 9 (B), and 12 (C) base pairs, respectively, conjugated with 1 .0 m magnetic beads 152 The cycles were demonstrated by repeated application (Bn) and re moval (Rn) of the magnetic field.

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102 Figure 4 5. Cycles of close d open state from DNA hairpins with 6 ( A), 9 (B ), and 12 ( C ) base pairs, respectively, conjugated with 2.8 m magnetic beads 152 The cycles w ere represented by repeated application (Bn) and removal (Rn) of the magnetic field. Figure 4 6 Cycles of close d open s tate from DNA hairpins with 6 (A ), 9 ( B ), and 12 ( C ) base pairs, r espectively, conjugated with 0.2 m magnet ic beads 152 The cycles were demonstrated by repeated application (Bn) and removal (Rn) of the magnetic field.

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103 Figure 4 7 Magnetic beads effect on the DNA nanomotor 152 Cycles of close d open state from 6 base pair DNA hairpins ; (a) 0.2 m ( b ) 1. 0 m and ( c ) 2.8 m magnetic particle; ( d ) a control experiment using 5 .0 m silica particle.

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104 Table 4 1. DNA hairpins and biotinylated linker. Stem bases are underlined. DNA hairpins Melting a Temperature (C) 6ds TGG TGT GGT TGG TGG TTT FAM CCA TCG TTT TTT TTT TTT TTT TTT TT C GAT GG DAB T TTT TTT TTT TTT TTT TTT T Biotin 66.5 9ds 5 TGG TGT GGT TGG TGG TTT FAM CCG CCA TCG TTT TTT TTT TTT TTT TTT TT C GAT GGC GG DAB T TTT TTT TTT TTT TTT TTT T Biotin 68.9 12ds TGG TGT GGT TGG TGG TTT FAM CCG CCG CCA TCG TTT TTT TTT TTT TTT TTT TT C GAT GGC GGC GG DAB T TTT TTT TTT TTT TT T TTT T Biotin 70.8 Linker CCA CCA ACC ACA CCA TTT TTT TTT T Biotin a Calculated melting temperature from IDT website Table 4 2. The average signal enhancement of nanomotor after repeating several c ycles MNP Signal Enhancement 6ds 9 ds 12ds 2.8 m 1.7 1.6 1.4 1.0 m 2.1 2.0 1.8 0.2 m No change No change No change

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105 CHAPTER 5 SUMMARY AND FUTURE D IRECTIONS The Development of DNA Conjugated Magnetic Nanoparticles for B io A nalytical and Biomedical A pplications Great efforts have been made toward the improvement of high ly sensitive and selective medical diagnostic tools to meet the demand for rapid and accurate disease screening, especially for c ancer. Nanomaterials, whose properties can be tuned by size and shape are used not onl y in mat erial science but also in the translation of nanotechnology to medical practice. Specifically, magnetic nanoparticles (MNPs) have attracted the attention of scientists as multimodal nanoplatforms with both diagnostic and therapeutic functionalities Owing to their large surface to volume ratios and magnetic properties, selective recognition elements including oligonucleotides, can be loaded on s for targeting, separating, and imaging. Besides carrying genetic information DNA is reno wned for the specifici ty of Watson Crick base pairing, which provides highly specific molecular recognition and enables programmed sequences and structures. I ntegration of MNPs and DNA t o construct hybrid materials has led to the development of advanced b ioanalysis tools and fabrication of novel DNA based nanomachines. In this research, t hree key projects have been demonstrated: 1) m olecular profiling of cancer cells using aptamer conjugated magnetic nanoparticles; 2) d etection of lysozyme using magnetic r elaxation switches based on aptamer functionalized superparamagnetic nanoparticles; 3) m agnetically driven single DNA nanomotor. We first applied DNA aptamers conjugated to MNPs as magnetic nanosensors based on the magnetic relaxation switch (MRSw) mechani sm. Aptamer conjugated

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106 MNPs (ACMNPs) switched from their original dispersed to aggregated states upon binding with target cells. The aggregation induced the formation of magnetic clusters generating the coupling of magne tic moments and resulting in a decre ase in the spin spin relaxation times (T 2 ) of the surrounding water protons The change in T 2 2 ) indicated the binding of ACMNPs to their target cells. The synergism between the large surface area of MNPs and the high specificity of aptamers played a key role in high selectivity and sensitivity. The magnetic n anosensors were able to detect as fe w as 10 target cancer cells in both buffer and bio logical media, as well as in a mix ture of cells. Additionally, a molecular profiling of cancer cells was designed by using an array of ACMNPs. Due to different affinity of aptamers to each cell type, distin ct recognition patterns were generated allowing the accurate identification of cancer cells. The expansion of MRSw based nanosensors was demonstrated for protein detection. Lys ozyme which has high affinity for its aptamer was selected as a model protein I n this system, MNPs were conjugated with either aptamers or linker DNA that could hybridize to the extended part of aptamers leading to magnetic cluster formation. The presence of targets induced the base pair disruption of aptamers resulting in the disass embly of pre existing clusters and an increase in T 2. The high selectivity and sensitivity of detection was achieved in both buffer and serum. The successful identification of lysozyme in cancer cell lysates was also demonstrated. These studies validated t he potential of using DNA conjugated MNPs as magnetic nanosensors for a wide range of molecular targets. With low magnetic background, high sensitivity was achieved in biological samples without separation, purification, amplification and washing step s Fu rthermore, the detection could be performed using a

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107 bench top relaxometer which provides rapid results, and easy to operate. Considering theses advantages, this method is feasible for clinical diagnostics. Besides using DNA conjugated MNPs as bioanalysis tools, a DNA based nanomachine driven by magnetic fields was also demonstrated. DNA hairpins were selected to serve as a dynamic part of the molecular motor due to their ability to shrink and extend by external stimuli. DNA hairpins were immobilized onto a glass surface and then conjugated to magnetic particles. The application of an external magnetic field triggered the switch of molecular conformation between closed and open states. The movement of a nanomotor could be tracked by the change of fluorescen ce from the intercalated fluorophore and quencher in DNA hairpin structures. By repetitive shrinking and extending movements, DNA hairpins were considered as a nanometer scale motor. Overa ll, this research mainly focused on the development of DNA conjugate d MNPs for cancer detection and the fabrication of DNA based nanomotor. A successful outcome from these studies will lead to advanced hybrid nanomaterials for bioanalysis, biomedical applications, and bionanomaterial research. Future Directions Although ma gnetic nanosensors have shown advantages in simple and sensitive detection, they still have some limitations. In chapter 3, we demonstrated that the presence of targets triggered the disassembly of magnetic nanoclusters and the T 2 increase was observed. Ho wever, a small numbers of targets may not be sufficient to induce the disassembly of pre existing clusters. Typically, a certain number of analyte molecules is always needed to disrupt magnetic clusters and generate significant change s in T 2 thus limiting sensitivity of detection. In order to improve the efficiency of

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108 magnetic nanosensor s for rare target detection, an enzymatic reaction could be incorporated into magnetic nanosensors for signal amplification. It has been found that DNA also has catalytic activity and can catalyze a number of biochemical reactions, including RNA or DNA cleavage, ligation, and DNA self modification. 126 153 Catalytic ally active DNA molecules are known as deoxyribozymes o r DNAzymes and can be obtained by the SELEX process. 154 155 Typically, catalytic activities of DNAzymes are dependent on metal ions, especially transition metal ions DNAzymes that are specific for Pb 2+ 156 Cu 2+ 157 Zn 2+ 158 and Co 2+ 159 have been reported Compared to RNA m olecules or proteins, DNAzymes are much more stable and the synthesis, as well as modification is relatively easy. Consequently, DNAzymes are used as analytical tools for metal ion detections. 160 and their ease of s ynthesis and modification, we are attempt ing to apply DNAzymes into magnetic nanosensors system. The Pb 2+ dependent DNAzyme is selected due to its rel atively high catalytic efficiency and structural stability 156 and is designed to have 7 extended bases in order to hybridize with a part of the lysozyme aptamer (as shown in Figure 7 1). The twelve base pairs between DNAzyme and aptamer can maintain stable duplex at room temperature. To assemble magnetic nanoclusters, the DNAzyme s ubstrate contain ing a single RNA linkage (rA) as the cleavage site is extended on both ends by 12 bases to be complementary to the linker DNA attached to MNPs. In the presence of lysozyme targets, aptamers undergo structural change and DNAzymes are release d. By the addition of pre existing magnetic nanoclusters having substrates as linkers, the released DNAzymes recognize and cleave the substrates inducing cluster disassembly.

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109 After cleavage, DNAzymes can leave the cleaved substrates and catalyze the cleava ge of other nearby substrates. In this way, the catalytic reaction can cycle several time s with only a few target molecules. Consequently, disassembly of magnetic nanoclusters can be triggered resulting in an increase in T 2 With ity, ease of synthesis, and sequence modification combined with the flexibility of the MRSw to sense various types of targets, it is very likely that DNAzyme based magnetic nanosensors will become high ly efficient bioanalysis tools for clinical applicatio ns.

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110 Figure 7 1. Schematic representation of DNAzyme based magnetic nanosensor for Lys detection. The sequences of DNAzyme, substrate, linker, and Lys aptame r are shown The underlined based are the extended parts of either DNAzyme or aptamer. The highlight demonstrates the complementary parts between DNAzyme and aptamer.

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120 BIOGRAPHICAL SK ETCH Suwussa Bamrungsap was born i n Samutsongkhram, in 1981 to Somkuan and Wasana Bamrungsap. She obtained the scholarship from the Development and Promotion of Science and Technology Talent Projects (DPST) to complete her high school at Sriboonyanon Schoo l in 1999 and attended Kasetsart University to study Chemist ry. After Suwussa received her b achelor degree with the second class honors in 2003, she continued her m aster hemistry at Kasetsart University under the supervision of Dr. Jumras Limtrakul. She obtained the scholarship under the National Nanotechnology Center, Thailand from the Royal Thai government and began her Doctorate study under the supervision of Dr. Weihong Tan at the University of Florida in fall 2006. She received her Doctor of Philosophy degree in c hemistry in August 2011.